WorldWideScience

Sample records for stromal cells fibroblasts

  1. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

    Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  2. Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

    Science.gov (United States)

    Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

    2011-01-01

    Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

  3. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhangyong1956@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China); Gao, Ming-Qing, E-mail: gaomingqing@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China)

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  4. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

    Science.gov (United States)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    International Nuclear Information System (INIS)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  6. CD117 expression in fibroblasts-like stromal cells indicates unfavorable clinical outcomes in ovarian carcinoma patients.

    Directory of Open Access Journals (Sweden)

    Ruixia Huang

    Full Text Available The stem cell factor (SCF receptor CD117 (c-kit, is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05, and it was significantly associated with poor overall survival (OS and progression free survival (PFS (Kaplan-Meier analysis; p<0.05, log-rank test. CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP and α smooth muscle actin (α-SMA, indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.

  7. Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Jason A Neidleman

    2017-02-01

    Full Text Available Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

  8. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

  9. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    Science.gov (United States)

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  10. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

    OpenAIRE

    Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

    2014-01-01

    STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductiv...

  11. Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

    Science.gov (United States)

    Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy

    2015-01-01

    Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. PMID:26100252

  12. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    International Nuclear Information System (INIS)

    Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

    2014-01-01

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  13. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2014-09-05

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  14. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

    2015-01-01

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

  15. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

    2015-02-27

    During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

  16. Breast cancer cells induce stromal fibroblasts to secrete ADAMTS1 for cancer invasion through an epigenetic change.

    Directory of Open Access Journals (Sweden)

    Shiaw-Wei Tyan

    Full Text Available Microenvironment plays an important role in cancer development. We have reported that the cancer-associated stromal cells exhibit phenotypic and functional changes compared to stromal cells neighboring to normal tissues. However, the molecular mechanisms as well as the maintenance of these changes remain elusive. Here we showed that through co-culture with breast cancer cells for at least three to four passages, breast normal tissue-associated fibroblasts (NAFs gained persistent activity for promoting cancer cell invasion, partly via up-regulating ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1. Furthermore, we demonstrated that the DNA methylation pattern in the ADAMTS1 promoter has no alteration. Instead, the loss of EZH2 binding to the ADAMTS1 promoter and the resulting decrease of promoter-associated histone H3K27 methylation may account for the up-regulation of ADAMTS1. Importantly, the lack of EZH2 binding and the H3K27 methylation on the ADAMTS1 promoter were sustained in cancer cell-precocultured NAFs after removal of cancer cells. These results suggest that cancer cells are capable of inducing stromal fibroblasts to secrete ADAMTS1 persistently for their invasion and the effect is epigenetically inheritable.

  17. Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

    Science.gov (United States)

    Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

    2017-10-01

    The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  18. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  19. Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

    Science.gov (United States)

    Van Eijk, F; Saris, D B F; Riesle, J; Willems, W J; Van Blitterswijk, C A; Verbout, A J; Dhert, W J A

    2004-01-01

    Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p engineered ligament.

  20. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been...... by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite...... and differentiation to osteoblasts, adipocytes and chondrocytes....

  1. Effects of human umbilical cord blood-derived mesenchymal stromal cells and dermal fibroblasts on diabetic wound healing.

    Science.gov (United States)

    Moon, Kyung-Chul; Lee, Jong-Seok; Han, Seung-Kyu; Lee, Hyup-Woo; Dhong, Eun-Sang

    2017-07-01

    A previous study demonstrated that human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have superior wound-healing activity compared with fibroblasts in vitro. However, wound healing in vivo is a complex process that involves multiple factors. The purpose of this study was to compare the effects of hUCB-MSCs and fibroblasts on diabetic wound healing in vivo. This study especially focused on collagen synthesis and angiogenesis, which are considered to be the important factors affecting diabetic wound healing. Porous polyethylene discs were loaded with either fibroblasts or hUCB-MSCs, and a third group, which served as a control, was not loaded with cells. The discs were then implanted in the back of diabetic mice. During the first and the second week after implantation, the discs were harvested, and collagen level and microvascular density were compared. In terms of collagen synthesis, the hUCB-MSC group showed the highest collagen level (117.7 ± 8.9 ng/mL), followed by the fibroblast group (83.2 ± 5.2 ng/mL) and the no-cell group (60.0 ± 4.7 ng/mL) in the second week after implantation. In terms of angiogenesis, the microvascular density in the hUCB-MSC group was 56.8 ± 16.4, which was much higher than that in the fibroblast group (14.3 ± 4.0) and the no-cell group (5.7 ± 2.1) in the second week after implantation. These results demonstrate that hUCB-MSCs are superior to fibroblasts in terms of their effect on diabetic wound healing in vivo. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

    Directory of Open Access Journals (Sweden)

    Jung-Seok Lee

    2018-04-01

    Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

  3. Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Rattigan, Yanique I.; Patel, Brijesh B. [Graduate School of Biomedical Sciences, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901 (United States); Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901 (United States); Ackerstaff, Ellen [Department of Medical Physics, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 (United States); Sukenick, George [Molecular Pharmacology and Chemistry Research Program, Sloan-Kettering Institute, 415 E 68th Street, New York, NY 10065 (United States); Koutcher, Jason A. [Department of Medical Physics, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 (United States); Glod, John W. [Graduate School of Biomedical Sciences, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901 (United States); Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901 (United States); Department of Pediatric Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901 (United States); and others

    2012-02-15

    Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O{sub 2}) than under 20% O{sub 2} and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our {sup 13}C NMR spectroscopic measurements indicate that {sup 13}C-lactate is converted to {sup 13}C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.

  4. Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

    International Nuclear Information System (INIS)

    Rattigan, Yanique I.; Patel, Brijesh B.; Ackerstaff, Ellen; Sukenick, George; Koutcher, Jason A.; Glod, John W.

    2012-01-01

    Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O 2 ) than under 20% O 2 and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our 13 C NMR spectroscopic measurements indicate that 13 C-lactate is converted to 13 C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.

  5. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  6. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts.

    Science.gov (United States)

    Chen, Joseph C; Johnson, Brittni A; Erikson, David W; Piltonen, Terhi T; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C; Greene, Warner C; Giudice, Linda C; Roan, Nadia R

    2014-06-01

    How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11

  7. Teaming Up for Trouble: Cancer Cells, Transforming Growth Factor-β1 Signaling and the Epigenetic Corruption of Stromal Naïve Fibroblasts.

    Science.gov (United States)

    Lamprecht, Sergio; Sigal-Batikoff, Ina; Shany, Shraga; Abu-Freha, Naim; Ling, Eduard; Delinasios, George J; Moyal-Atias, Keren; Delinasios, John G; Fich, Alexander

    2018-02-27

    It is well recognized that cancer cells subvert the phenotype of stromal naïve fibroblasts and instruct the neighboring cells to sustain their growth agenda. The mechanisms underpinning the switch of fibroblasts to cancer-associated fibroblasts (CAFs) are the focus of intense investigation. One of the most significant hallmarks of the biological identity of CAFs is that their tumor-promoting phenotype is stably maintained during in vitro and ex vivo propagation without the continual interaction with the adjacent cancer cells. In this review, we discuss robust evidence showing that the master cytokine Transforming Growth Factor-β1 (TGFβ-1) is a prime mover in reshaping, via epigenetic switches, the phenotype of stromal fibroblasts to a durable state. We also examine, in detail, the pervasive involvement of TGFβ-1 signaling from both cancer cells and CAFs in fostering cancer development, taking colorectal cancer (CRC) as a paradigm of human neoplasia. Finally, we review the stroma-centric anticancer therapeutic approach focused on CAFs-the most abundant cell population of the tumor microenvironment (TME)-as target cells.

  8. Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

    International Nuclear Information System (INIS)

    Hirata, Eri; Takita, Hiroko; Watari, Fumio; Yokoyama, Atsuro; Ménard-Moyon, Cécilia; Venturelli, Enrica; Bianco, Alberto

    2013-01-01

    Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF–CNT) showed the same effect as FGF alone. In addition, FGF–CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF–CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF–CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications. (paper)

  9. Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

    Science.gov (United States)

    Hirata, Eri; Ménard-Moyon, Cécilia; Venturelli, Enrica; Takita, Hiroko; Watari, Fumio; Bianco, Alberto; Yokoyama, Atsuro

    2013-11-01

    Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

  10. Comparisons of phenotype and immunomodulatory capacity among rhesus bone-marrow-derived mesenchymal stem/stromal cells, multipotent adult progenitor cells, and dermal fibroblasts

    Science.gov (United States)

    Wang, Qi; Clarkson, Christina; Graham, Melanie; Donahue, Robert; Hering, Bernhard J.; Verfaillie, Catherine M.; Bansal-Pakala, Pratima; O'Brien, Timothy D.

    2015-01-01

    Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable. PMID:24825538

  11. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  12. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  13. Fibroblast Growth Factor-2 Enhances Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells without Diminishing Their Immunosuppressive Potential

    OpenAIRE

    Auletta, Jeffery J.; Zale, Elizabeth A.; Welter, Jean F.; Solchaga, Luis A.

    2011-01-01

    Allogeneic hematopoietic stem cell transplantation is the main curative therapy for many hematologic malignancies. Its potential relies on graft-versus-tumor effects which associate with graft-versus-host disease. Mesenchymal stromal cells (MSCs) possess immunomodulatory properties that make them attractive therapeutic alternatives. We evaluated the in vitro immunosuppressive activity of medium conditioned by human MSCs from 5 donors expanded 13 passages with or without FGF-2. FGF-2 supplemen...

  14. Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus.

    Directory of Open Access Journals (Sweden)

    Masuma Khatun

    Full Text Available Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs and endometrial fibroblasts (eSFs.The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS-induced state.Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF-A, stromal cell-derived factor-1 alpha (SDF-1α, interleukin-1 receptor antagonist (IL-1RA, IL-6, interferon-gamma inducible protein (IP-10, monocyte chemoattractant protein (MCP-1, macrophage inflammatory protein (MIP1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs.Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed

  15. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes

    OpenAIRE

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation...

  16. Bone marrow stromal elements in murine leukemia; Decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishay, Z [Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School (Israel); Barak, V [Laboratory of Immunology, Department of Oncology, Hadassah University Hospital (Israel); Shoshan, S [Faculty of Dental Medicine, Connective Tissue Research Laboratory, Hebrew University, Jerusalem (Israel); Prindull, G [Department of Pediatrics, University of Gottingen, Gottingen (Germany, F.R.)

    1990-01-01

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF{alpha} in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author).

  17. Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

    Science.gov (United States)

    Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

    2016-06-07

    Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.

  18. Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche

    OpenAIRE

    Hoffman, Robert M

    2013-01-01

    Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggestin...

  19. High Numbers of Stromal Cancer-Associated Fibroblasts Are Associated With a Shorter Survival Time in Cats With Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Klobukowska, H J; Munday, J S

    2016-11-01

    Cancer-associated fibroblasts (CAFs) are fibroblastic cells that express α-smooth muscle actin and have been identified in the stroma of numerous epithelial tumors. The presence of CAFs within the tumor stroma has been associated with a poorer prognosis in some human cancers, including oral squamous cell carcinomas (SCCs). Cats frequently develop oral SCCs, and although these are generally highly aggressive neoplasms, there is currently a lack of prognostic markers for these tumors. The authors investigated the prognostic value of the presence of CAFs within the stroma of oral SCC biopsy specimens from 47 cats. In addition, several epidemiologic, clinical, and histologic variables were also assessed for prognostic significance. A CAF-positive stroma was identified in 35 of 47 SCCs (74.5%), and the median survival time (ST) of cats with CAF-positive SCCs (35 days) was significantly shorter than that of cats with CAF-negative SCCs (48.5 days) (P = .031). ST was also associated with the location of the primary tumor (P = .0018): the median ST for oropharyngeal SCCs (179 days) was significantly longer than for maxillary (43.5 days; P = .047), mandibular (42 days; P = .022), and sublingual SCCs (22.5 days; P = .0005). The median ST of sublingual SCCs was also shorter compared with maxillary SCCs (P = .0017). Furthermore, a significant association was identified between site and the presence of stromal CAFs (P = .025). On the basis of this retrospective study, evaluating the tumor stroma for CAFs in feline oral SCC biopsy specimens may be of potential prognostic value. © The Author(s) 2016.

  20. Tissue engineering of ligaments : A comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source

    NARCIS (Netherlands)

    Van Eijk, F; Riesle, J; Willems, WJ; Van Blitterswijk, CA; Verbout, AJ; Dhert, WJA

    Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell

  1. Skeletal (stromal) stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Kermani, Abbas Jafari; Zaher, Walid

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy....../preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone....

  2. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

    Directory of Open Access Journals (Sweden)

    Ramiro Olivera

    Full Text Available The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647 on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I induced pluripotent stem cells (iPSCs vs. adult fibroblasts (AF fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II umbilical cord-derived mesenchymal stromal cells (UC-MSCs vs. fetal fibroblasts derived from an unborn cloned foetus (FF vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively, respect to <1h (5.2% and 2%, respectively and 1-2h (5.6% and 4.7%, respectively groups (P<0.05. However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2% or 1-2h (35.7% were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6% than using FF (8.9% or AF (9.3%, (P<0.05. Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively, viable foals (two were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

  3. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  4. Modeling triple-negative breast cancer heterogeneity: effects of stromal macrophages, fibroblasts and tumor vasculature.

    Science.gov (United States)

    Norton, Kerri-Ann; Jin, Kideok; Popel, Aleksander S

    2018-05-08

    A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates. Copyright © 2018. Published by Elsevier Ltd.

  5. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  6. Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

    Directory of Open Access Journals (Sweden)

    Lilian Chuaire-Noack

    2011-04-01

    Full Text Available A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, withmalignant transformation. Given the high incidence of ovarian cancerand its main origin from the ovarian surface epithelium, as well asthe possibility that an epithelial-mesenchymal transition occurs, weevaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6,enzyme whose expression is considered as a marker of replicativesenescence. Methods: 48 samples of ovarian cortical fibroblasts fromdonors without a history of cancer were serially cultured untilthe end of their replicative life. β-galactosidase activity at pH 6was quantified in each passage by the chemiluminiscent method. Ascontrol, we used ovarian epithelial cell cultures from the samedonors. The enzyme activity was also evaluated in fibroblastspreviously induced to senescence by exposure to hydrogen peroxide.Results: The analysis of the enzyme activity and the replicativecapacity taken together showed that the fibroblast cultures reachedthe senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions:The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to theepithelialmesenchymal transition that has been proposed to explaintheir behavior in the genesis of cancer arising from ovarian surfaceepithelium. Low β-galactosidase activity values at pH 6 would suggestpossible inactivation of the response pathways to oxidative stress.

  7. Metabolically conditioned media derived from bone marrow stromal cells or human skin fibroblasts act as effective chemoattractants for mesenchymal stem cells

    OpenAIRE

    Gabrielyan, Anastasia; Neumann, Elena; Gelinsky, Michael; Rösen-Wolff, Angela

    2017-01-01

    Background The main goal of bone tissue engineering has been the generation of healthy bone in order to replace affected tissue. Therefore, optimized biomaterials are needed which allow the survival and growth of mesenchymal stem cells. Until now the key challenge in the clinical application of cell-based tissue engineering bone implants was poor diffusion of oxygen into the tissue, making functional blood vessel networks a necessity. With their ability to evolve into different cell types, to...

  8. Radiosensitivity of marrow stromal cells and the effect of some radioprotective agents

    International Nuclear Information System (INIS)

    Liu Shuhua

    1992-01-01

    The results showed that marrow stromal cells include fibroblasts, reticular cells, macrophages and adipocytes. The capability of the adherent layer derived from marrow cells of 2 mouse femurs to support hematopoietic stem cells was stronger than those of layers derived from 0.5 or 1 mouse femurs. The radiosensitivity of bone marrow stromal cells was lower than that of hematopoietic stem cells. The radioprotective effect of AET and PLP (polysaccharide of Lobaria Pulmonaria Hoffm) on the bone marrow stromal cells and their capability to support hematopoietic stem cells was clearly demonstrated

  9. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Science.gov (United States)

    Yu, Yue; Lee, Jennifer Suehyun; Xie, Ning; Li, Estelle; Hurtado-Coll, Antonio; Fazli, Ladan; Cox, Michael; Plymate, Stephen; Gleave, Martin; Dong, Xuesen

    2014-01-01

    Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  10. Overexpression of COL11A1 by cancer-associated fibroblasts: clinical relevance of a stromal marker in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Carmen García-Pravia

    Full Text Available BACKGROUND: The collagen11A1 (COL11A1 gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available. METHODS AND FINDINGS: A total of 54 pancreatic ductal adenocarcinomas (PDAC, 23 chronic pancreatitis (CP samples, and cultured peritumoral stromal cells of PDAC (passages 3-6 were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1 Validation of COL11A1 gene overexpression by q-RT-PCR. FINDINGS: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2 Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. FINDINGS: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs of PDAC but it does not stain chronic benign condition (chronic pancreatitis stromal cells, epithelial cells, or normal fibroblasts. 3 Evaluation of the discrimination ability of the antibody. FINDINGS: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981. 4 Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. FINDINGS: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype in different proportions. CONCLUSIONS/SIGNIFICANCE: Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for

  11. Evaluation of median survival patients with acute lymphoblastic leukemia exposed ionizing radiation by the Chornobyl accident, depending on the aminoacid composition of stromal fibroblasts bone marrow and indicators of myelogram

    International Nuclear Information System (INIS)

    Bebeshko, V.G.; Bruslova, K.M.; Tsvjetkova, N.M.; Pushkar'ova, T.Yi.; Panchenko, L.M.; Yatsemirs'kij, S.N.; Volodyina, T.T.; Lyashenko, L.O.

    2015-01-01

    The aminoacid composition in the stromal fibroblasts of bone marrow and indexes of hemogram in children with acute lymphoblastic leukemias, which exposed to radiation from the Chornobyl accident, for evaluation of the median survival was investigated. In the study 54 patients with acute lymphoblastic leukemia, residents of Kyiv, Zhytomyr and Chernihiv regions of Ukraine were involved. Effectiveness of stromal fibroblasts colony forming units of bone marrow and aminoacid composition of fibroblasts were studied. Bone marrow examination was conducted on day 33 and after stop of chemotherapy, the median survival of patients and exposure dose were evaluated. In children with acute leukemias effectiveness of stromal fibroblasts of bone marrow was 2-2.3 times lower than normative. Correlation (r_s = +0.83) between the number of erythroid progenitor cells in the bone marrow and proline content in stromal fibroblasts established . The correlation between patients median survival and number of granulocytes in myelogram (r_s = + 0.82) was proven. Mean exposure doses in patients was (5.86 ±1.11) mSv and it did not correlate with myelogram results, effectiveness of stromal fibroblasts of bone marrow and median survival. The median survival of patients with acute leukemias depends on the content of amino acids in stromal fibroblasts of bone marrow and recover of hematopoiesis after chemotherapy

  12. Targeting Stromal Recruitment by Prostate Cancer Cells

    Science.gov (United States)

    2006-03-01

    Ensinger, C., Tumer , Z., Tommerup, N. et al.: Hedgehog signaling in small-cell lung cancer : frequent in vivo but a rare event in vitro. Lung Cancer , 52...W81XWH-04-1-0157 TITLE: Targeting Stromal Recruitment by Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Jingxian Zhang, Ph.D...DATES COVERED (From - To) 15 Feb 2004 – 14 Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Stromal Recruitment by Prostate Cancer

  13. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  14. The Bone Marrow-Derived Stromal Cells

    DEFF Research Database (Denmark)

    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

    Bone marrow (BM) microenvironment represents an important compartment of bone that regulates bone homeostasis and the balance between bone formation and bone resorption depending on the physiological needs of the organism. Abnormalities of BM microenvironmental dynamics can lead to metabolic bone...... diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage...

  15. Stromal Activation Associated with Development of Prostate Cancer in Prostate-Targeted Fibroblast Growth Factor 8b Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Teresa D. Elo

    2010-11-01

    Full Text Available Expression of fibroblast growth factor 8 (FGF-8 is commonly increased in prostate cancer. Experimental studies have provided evidence that it plays a role in prostate tumorigenesis and tumor progression. To study how increased FGF-8 affects the prostate, we generated and analyzed transgenic (TG mice expressing FGF-8b under the probasin promoter that targets expression to prostate epithelium. Prostates of the TG mice showed an increased size and changes in stromal and epithelialmorphology progressing fromatypia and prostatic intraepithelial neoplasia (mouse PIN, mPIN lesions to tumors with highly variable phenotype bearing features of adenocarcinoma, carcinosarcoma, and sarcoma. The development of mPIN lesions was preceded by formation of activated stroma containing increased proportion of fibroblastic cells, rich vasculature, and inflammation. The association between advancing stromal and epithelial alterations was statistically significant. Microarray analysis and validation with quantitative polymerase chain reaction revealed that expression of osteopontin and connective tissue growth factor was markedly upregulated in TG mouse prostates compared with wild type prostates. Androgen receptor staining was decreased in transformed epithelium and in hypercellular stroma but strongly increased in the sarcoma-like lesions. In conclusion, our data demonstrate that disruption of FGF signaling pathways by increased epithelial production of FGF-8b leads to strongly activated and atypical stroma, which precedes development of mPIN lesions and prostate cancer with mixed features of adenocarcinoma and sarcoma in the prostates of TG mice. The results suggest that increased FGF-8 in human prostate may also contribute to prostate tumorigenesis by stromal activation.

  16. Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

    Science.gov (United States)

    Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

    2018-01-01

    Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

  17. Innate lymphoid cells and their stromal microenvironments.

    Science.gov (United States)

    Kellermayer, Zoltán; Vojkovics, Dóra; Balogh, Péter

    2017-09-01

    In addition to the interaction between antigen presenting cells, T and B lymphocytes, recent studies have revealed important roles for a diverse set of auxiliary cells that profoundly influence the induction and regulation of immune responses against pathogens. Of these the stromal cells composed of various non-hematopoietic constituents are crucial for the creation and maintenance of specialized semi-static three-dimensional lymphoid tissue microenvironment, whereas the more recently described innate lymphoid cells are generated by the diversification of committed lymphoid precursor cells independently from clonally rearranged antigen receptor genes. Recent findings have revealed important contributions by innate lymphoid cells in inflammation and protection against pathogens in a tissue-specific manner. Importantly, lymphoid stromal cells also influence the onset of immune responses in tissue-specific fashion, raising the possibility of tissue-specific stromal - innate lymphoid cell collaboration. In this review we summarize the main features and interactions between these two cells types, with particular emphasis on ILC type 3 cells and their microenvironmental partners. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  18. Mesenchymal Stem Cell-Induced DDR2 Mediates Stromal-Breast Cancer Interactions and Metastasis Growth

    Directory of Open Access Journals (Sweden)

    Maria E. Gonzalez

    2017-01-01

    Full Text Available Increased collagen deposition by breast cancer (BC-associated mesenchymal stem/multipotent stromal cells (MSC promotes metastasis, but the mechanisms are unknown. Here, we report that the collagen receptor discoidin domain receptor 2 (DDR2 is essential for stromal-BC communication. In human BC metastasis, DDR2 is concordantly upregulated in metastatic cancer and multipotent mesenchymal stromal cells. In MSCs isolated from human BC metastasis, DDR2 maintains a fibroblastic phenotype with collagen deposition and induces pathological activation of DDR2 signaling in BC cells. Loss of DDR2 in MSCs impairs their ability to promote DDR2 phosphorylation in BC cells, as well as BC cell alignment, migration, and metastasis. Female ddr2-deficient mice homozygous for the slie mutation show inefficient spontaneous BC metastasis. These results point to a role for mesenchymal stem cell DDR2 in metastasis and suggest a therapeutic approach for metastatic BC.

  19. Functional Imaging of Proteolysis: Stromal and Inflammatory Cells Increase Tumor Proteolysis

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2003-07-01

    Full Text Available The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4–17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes.

  20. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

    2011-08-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

  1. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

    2011-01-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

  2. Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology.

    Science.gov (United States)

    Zhan, Han-Xiang; Zhou, Bin; Cheng, Yu-Gang; Xu, Jian-Wei; Wang, Lei; Zhang, Guang-Yong; Hu, San-Yuan

    2017-04-28

    Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide. Increasing evidence has confirmed the pivotal role of stromal components in the regulation of carcinogenesis, invasion, metastasis, and therapeutic resistance in PC. Interaction between neoplastic cells and stromal cells builds a specific microenvironment, which further modulates the malignant properties of cancer cells. Instead of being a "passive bystander", stroma may play a role as a "partner in crime" in PC. However, the role of stromal components in PC is complex and requires further investigation. In this article, we review recent advances regarding the regulatory roles and mechanisms of stroma biology, especially the cellular components such as pancreatic stellate cells, macrophages, neutrophils, adipocytes, epithelial cells, pericytes, mast cells, and lymphocytes, in PC. Crosstalk between stromal cells and cancer cells is thoroughly investigated. We also review the prognostic value and molecular therapeutic targets of stroma in PC. This review may help us further understand the molecular mechanisms of stromal biology and its role in PC development and therapeutic resistance. Moreover, targeting stroma components may provide new therapeutic strategies for this stubborn disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Effect of chemotherapy and irradiation on interactions between stromal and hemopoietic cells in vitro

    International Nuclear Information System (INIS)

    Cohen, G.I.; Greenberger, J.S.; Canellos, G.P.

    1982-01-01

    We examined the interactions between stromal and hemopoietic cells in mouse long-term bone marrow cultures. The adherent stroma is formed by several layers of cells consisting of macrophage, fibroblasts, and adventitial cells which accumulate lipid to become adipocytes. Stromal cells become closely apposed to loosely adherent hemopoietic cells but gap junctions occur only among cells in the adherent layer. The hemopoietic cells form tightly packed structures resembling cobblestones which contain granulocytes in all stages of differentiation. Using an in vitro model for bone marrow transplantation (BMT), we treated pure mouse stromal cell cultures with irradiation (1000 R) or chemotherapy (BCNU) prior to engraftment with hemopoietic stem cells. After two weeks, engrafted cultures were indistinguishable from the long-term bone marrow cultures previously described by Dexter. The adipocytes in irradiated cultures developed numerous submembrane pinocytotic vesicles but stromal-hemopoietic cell interactions remained unchanged compared to unirradiated controls. By contrast, granulocytes grafted onto chemotherapy treated stroma showed swelling of endoplasmic reticulum suggesting early toxic injury. These findings are consistent with functional studies of hemopoiesis after engraftment onto treated stroma and confirm an important role for stromal cells in the support of hemopoiesis

  4. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    International Nuclear Information System (INIS)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

    2013-01-01

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1 +/+ MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1 −/− MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1 +/+ and Med1 −/− MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells

  5. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Nagai, Mami [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Matsui, Keiji; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Asano, Shigetaka [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan)

    2013-10-11

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

  6. Origin of hemopoietic stromal progenitor cells in chimeras

    International Nuclear Information System (INIS)

    Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.; Samoylova, R.S.

    1985-01-01

    Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice

  7. Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

    Science.gov (United States)

    You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-11-01

    Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Mammary fibroadenoma with pleomorphic stromal cells.

    Science.gov (United States)

    Abid, Najla; Kallel, Rim; Ellouze, Sameh; Mellouli, Manel; Gouiaa, Naourez; Mnif, Héla; Boudawara, Tahia

    2015-01-01

    The presence of enlarged and pleomorphic nuclei is usually regarded as a feature of malignancy, but it may on occasion be seen in benign lesions such as mammary fibroadenomas. We present such a case of fibroadenoma occurring in a 37-year-old woman presenting with a self-palpable right breast mass. Histological examination of the tumor revealed the presence of multi and mononucleated giant cells with pleomorphic nuclei. The recognition of the benign nature of these cells is necessary for differential diagnosis from malignant lesions of the breast. fibroadenoma - pleomorphic stromal cells - atypia - breast.

  9. Fibrocytes: A Novel Stromal Cells to Regulate Resistance to Anti-Angiogenic Therapy and Cancer Progression.

    Science.gov (United States)

    Goto, Hisatsugu; Nishioka, Yasuhiko

    2017-12-29

    An adequate blood supply is essential for cancer cells to survive and grow; thus, the concept of inhibiting tumor angiogenesis has been applied to cancer therapy, and several drugs are already in clinical use. It has been shown that treatment with those anti-angiogenic drugs improved the response rate and prolonged the survival of patients with various types of cancer; however, it is also true that the effect was mostly limited. Currently, the disappointing clinical results are explained by the existence of intrinsic or acquired resistance to the therapy mediated by both tumor cells and stromal cells. This article reviews the mechanisms of resistance mediated by stromal cells such as endothelial cells, pericytes, fibroblasts and myeloid cells, with an emphasis on fibrocytes, which were recently identified as the cell type responsible for regulating acquired resistance to anti-angiogenic therapy. In addition, the other emerging role of fibrocytes as mediator-producing cells in tumor progression is discussed.

  10. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs......Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been......) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation...

  11. Tumor and Stromal-Based Contributions to Head and Neck Squamous Cell Carcinoma Invasion

    Energy Technology Data Exchange (ETDEWEB)

    Markwell, Steven M.; Weed, Scott A., E-mail: scweed@hsc.wvu.edu [Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506 (United States)

    2015-02-27

    Head and neck squamous cell carcinoma (HNSCC) is typically diagnosed at advanced stages with evident loco-regional and/or distal metastases. The prevalence of metastatic lesions directly correlates with poor patient outcome, resulting in high patient mortality rates following metastatic development. The progression to metastatic disease requires changes not only in the carcinoma cells, but also in the surrounding stromal cells and tumor microenvironment. Within the microenvironment, acellular contributions from the surrounding extracellular matrix, along with contributions from various infiltrating immune cells, tumor associated fibroblasts, and endothelial cells facilitate the spread of tumor cells from the primary site to the rest of the body. Thus far, most attempts to limit metastatic spread through therapeutic intervention have failed to show patient benefit in clinic trails. The goal of this review is highlight the complexity of invasion-promoting interactions in the HNSCC tumor microenvironment, focusing on contributions from tumor and stromal cells in order to assist future therapeutic development and patient treatment.

  12. Adipose-derived mesenchymal stromal cells for chronic myocardial ischemia (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Haack-Sørensen, Mandana; Mathiasen, Anders Bruun

    2012-01-01

    Adipose tissue represents an abundant, accessible source of multipotent adipose-derived stromal cells (ADSCs). Animal studies have suggested that ADSCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes. This makes ADSCs a promising new cell source...... for regenerative therapy to replace injured tissue by creating new blood vessels and cardiomyocytes in patients with chronic ischemic heart disease. The aim of this special report is to review the present preclinical data leading to clinical stem cell therapy using ADSCs in patients with ischemic heart disease....... In addition, we give an introduction to the first-in-man clinical trial, MyStromalCell Trial, which is a prospective, randomized, double-blind, placebo-controlled study using culture-expanded ADSCs obtained from adipose-derived cells from abdominal adipose tissue and stimulated with VEGF-A(165) the week...

  13. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

    Directory of Open Access Journals (Sweden)

    P.B. Soares

    2013-01-01

    Full Text Available Imatinib mesylate (IM is used to treat chronic myeloid leukemia (CML because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM. The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM, using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells increased. At higher concentrations (15 µM, the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control. Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

  14. PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

    Science.gov (United States)

    Ko, Sae Hee; Nauta, Allison C; Morrison, Shane D; Hu, Michael S; Zimmermann, Andrew S; Chung, Michael T; Glotzbach, Jason P; Wong, Victor W; Walmsley, Graham G; Peter Lorenz, H; Chan, Denise A; Gurtner, Geoffrey C; Giaccia, Amato J; Longaker, Michael T

    2018-01-01

    Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p cells (p cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

  15. Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment

    DEFF Research Database (Denmark)

    De Neergaard, Michala; Kim, Jiyoung; Villadsen, René

    2010-01-01

    Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures...... of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general...... cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation-a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor...

  16. Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin

    DEFF Research Database (Denmark)

    Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mollerup, Sarah

    2017-01-01

    of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.......BACKGROUND: Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety...... of neoplasms but their role in BCC is poorly understood. METHODS: Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical...

  17. Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin

    DEFF Research Database (Denmark)

    Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mourier, Tobias

    2017-01-01

    of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.......Background: Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety...... of neoplasms but their role in BCC is poorly understood. Methods: Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical...

  18. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    2011-02-01

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  19. Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

    Science.gov (United States)

    Jurco, S; Nadji, M; Harvey, D G; Parker, J C; Font, R L; Morales, A R

    1982-01-01

    Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

  20. Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

    Science.gov (United States)

    Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

    2016-04-01

    The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  1. Cancer-associated fibroblasts as another polarized cell type of the tumor microenvironment

    Directory of Open Access Journals (Sweden)

    Martin eAugsten

    2014-03-01

    Full Text Available Tumor- or cancer-associated fibroblasts (CAFs are one of the most abundant stromal cell types in different carcinomas and comprise a heterogeneous cell population. Classically, CAFs are assigned with pro-tumorigenic effects stimulating tumor growth and progression. More recent studies demonstrated also tumor-inhibitory effects of CAFs suggesting that tumor-residing fibroblasts exhibit a similar degree of plasticity as other stromal cell types. Reciprocal interactions with the tumor milieu and different sources of origin are emerging as two important factors underlying CAF heterogeneity. This review highlights recent advances in our understanding of CAF biology and proposes to expand the term of cellular ´polarization´, previously introduced to describe different activation states of various immune cells, onto CAFs to reflect their phenotypic diversity.

  2. Cryopreservation and revival of human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens

    2016-01-01

    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities. The establishment ...

  3. Modulation of hemopoiesis by novel stromal cell factors

    International Nuclear Information System (INIS)

    Zipori, D.

    1988-01-01

    The microenvironment of the bone marrow in mammals is a crucial site for the maintenance of a pluripotent hemopoietic stem cell pool. Our previous studies and present findings support the notion that both this function and the fine architecture of hemopoietic organs, i.e., the spatial arrangement of blood cells within the tissue, may be directed by stromal cells. Despite the ability of cloned stromal cells to support prolonged hempoiesis and maintenance in vitro of stem cells with high radioprotective ability, they are a poor source of colony stimulating factor-1 (CSF-1) and do not secrete the other species of CSF. Furthermore, cultured stromal cells antagonize the activity of CSF. It is proposed that stromal cell factors distinct from known CSFs, regulate stem cell renewal. An additional phenomenon that is mediated by stromal cells and can not be attributed to CSF, is their ability to specifically inhibit the accumulation of cells of particular lineage and stage of differentiation. A glycoprotein that inhibits the growth of plasmacytomas but not a variety of other cell types was isolated from one type of cloned stromal cells. Such specific inhibitors may account for the control of cell localization in the hemopoietic system

  4. Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

    Science.gov (United States)

    Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

    2017-11-01

    Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

  5. Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

    Science.gov (United States)

    Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

    2015-07-01

    The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

  6. Designed Surface Topographies Control ICAM-1 Expression in Tonsil-Derived Human Stromal Cells

    Directory of Open Access Journals (Sweden)

    Aliaksei S. Vasilevich

    2018-06-01

    Full Text Available Fibroblastic reticular cells (FRCs, the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs. Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

  7. Fibroblast-mediated in vivo and in vitro growth promotion of tumorigenic rat thyroid carcinoma cells but not normal Fisher rat thyroid follicular cells.

    Science.gov (United States)

    Saitoh, Ohki; Mitsutake, Norisato; Nakayama, Toshiyuki; Nagayama, Yuji

    2009-07-01

    It is known that genetic abnormalities in oncogenes and/or tumor suppressor genes promote carcinogenesis. Numerous recent articles, however, have demonstrated that epithelial-stromal interaction also plays a critical role for initiation and progression of carcinoma cells. Furthermore, ionizing radiation induces alterations in the tissue microenvironments that promote carcinogenesis. There is little or no information on epithelial-stromal interaction in thyroid carcinoma cells. The objective of this study was to determine if epithelial-stromal interaction influenced the growth of thyroid carcinoma cells in vivo and in vitro and to determine if radiation had added or interacting effects. Normal Fisher rat thyroid follicular cells (FRTL5 cells) and tumorigenic rat thyroid carcinoma cells (FRTL-Tc cells) derived from FRTL5 cells were employed. The cells were injected into thyroids or subcutaneously into left flanks of rats alone or in combination with skin-derived fibroblasts. In groups of rats, fibroblasts were irradiated with 0.1 or 4 Gy x-ray 3 days before inoculation. In vitro growth of FRTL-Tc and FRTL-5 cells were evaluated using the fibroblast-conditioned medium and in a co-culture system with fibroblasts. The in vivo experiments demonstrated that FRTL-Tc cells injected intrathyroidally grew faster than those injected subcutaneously, and that admixed fibroblasts enhanced growth of subcutaneous FRTL-Tc tumors, indicating that the intrathyroidal milieu, particularly in the presence of fibroblasts, confer growth-promoting advantage to thyroid carcinoma cells. This in vivo growth-promoting effect of fibroblasts on FRTL-Tc cells was duplicated in the in vitro experiments using the fibroblast-conditioned medium. Thus, our data demonstrate that this effect is mediated by soluble factor(s), is reversible, and is comparable to that of 10% fetal bovine serum. However, normal FRTL5 cells did not respond to the fibroblast-conditioned medium. Furthermore, high- and low

  8. Intestinal stromal cells in mucosal immunity and homeostasis.

    Science.gov (United States)

    Owens, B M J; Simmons, A

    2013-03-01

    A growing body of evidence suggests that non-hematopoietic stromal cells of the intestine have multiple roles in immune responses and inflammation at this mucosal site. Despite this, many still consider gut stromal cells as passive structural entities, with past research focused heavily on their roles in fibrosis, tumor progression, and wound healing, rather than their contributions to immune function. In this review, we discuss our current knowledge of stromal cells in intestinal immunity, highlighting the many immunological axes in which stromal cells have a functional role. We also consider emerging data that broaden the potential scope of their contribution to immunity in the gut and argue that these so-called "non-immune" cells are reclassified in light of their diverse contributions to intestinal innate immunity and the maintenance of mucosal homeostasis.

  9. Gut Mesenchymal Stromal Cells in Immunity

    Directory of Open Access Journals (Sweden)

    Valeria Messina

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs, first found in bone marrow (BM, are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal or interspersed within intestinal submucosa (intercryptal. In Crohn’s disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC. The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn’s disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

  10. Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

    Directory of Open Access Journals (Sweden)

    Yoon Se Lee

    2013-04-01

    Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

  11. Mesenchymal Stromal Cells for Antineoplastic Drug Loading and Delivery.

    Science.gov (United States)

    Petrella, Francesco; Rimoldi, Isabella; Rizzo, Stefania; Spaggiari, Lorenzo

    2017-11-23

    Mesenchymal stromal cells are a population of undifferentiated multipotent adult cells possessing extensive self-renewal properties and the potential to differentiate into a variety of mesenchymal lineage cells. They express broad anti-inflammatory and immunomodulatory activity on the immune system and after transplantation can interact with the surrounding microenvironment, promoting tissue healing and regeneration. For this reason, mesenchymal stromal cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Another clinical application of mesenchymal stromal cells is the targeted delivery of chemotherapeutic agents to neoplastic cells, maximizing the cytotoxic activity against cancer cells and minimizing collateral damage to non-neoplastic tissues. Mesenchymal stem cells are home to the stroma of several primary and metastatic neoplasms and hence can be used as vectors for targeted delivery of antineoplastic drugs to the tumour microenvironment, thereby reducing systemic toxicity and maximizing antitumour effects. Paclitaxel and gemcitabine are the chemotherapeutic drugs best loaded by mesenchymal stromal cells and delivered to neoplastic cells, whereas other agents, like pemetrexed, are not internalized by mesenchymal stromal cells and therefore are not suitable for advanced antineoplastic therapy. This review focuses on the state of the art of advanced antineoplastic cell therapy and its future perspectives, emphasizing in vitro and in vivo preclinical results and future clinical applications.

  12. CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

    Science.gov (United States)

    Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

    2010-01-01

    Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

  13. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  14. SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

    Science.gov (United States)

    He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

    2017-01-01

    Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

  15. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

  16. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    David M Harris

    Full Text Available Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza and the growth factors (GF granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  17. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  18. Targeting Stromal-Cancer Cell Crosstalk Networks in Ovarian Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Tsz-Lun Yeung

    2016-01-01

    Full Text Available Ovarian cancer is a histologically, clinically, and molecularly diverse disease with a five-year survival rate of less than 30%. It has been estimated that approximately 21,980 new cases of epithelial ovarian cancer will be diagnosed and 14,270 deaths will occur in the United States in 2015, making it the most lethal gynecologic malignancy. Ovarian tumor tissue is composed of cancer cells and a collection of different stromal cells. There is increasing evidence that demonstrates that stromal involvement is important in ovarian cancer pathogenesis. Therefore, stroma-specific signaling pathways, stroma-derived factors, and genetic changes in the tumor stroma present unique opportunities for improving the diagnosis and treatment of ovarian cancer. Cancer-associated fibroblasts (CAFs are one of the major components of the tumor stroma that have demonstrated supportive roles in tumor progression. In this review, we highlight various types of signaling crosstalk between ovarian cancer cells and stromal cells, particularly with CAFs. In addition to evaluating the importance of signaling crosstalk in ovarian cancer progression, we discuss approaches that can be used to target tumor-promoting signaling crosstalk and how these approaches can be translated into potential ovarian cancer treatment.

  19. Nonviral transfection of adipose tissue stromal cells: an experimental study.

    Science.gov (United States)

    Lopatina, T V; Kalinina, N I; Parfyonova, E V

    2009-04-01

    Delivery of plasmid DNA and interfering RNA into adipose tissue stromal cells was carried out by the methods of lipofection, calcium phosphate method, and by electroporation. The percent of transfected cells after delivery of plasmid DNA by the calcium phosphate method and lipofection was 0 and 15%, respectively, vs. more than 50% after electroporation. Similar results were obtained for delivery of short-strand RNA into cells. These data indicate that electroporation is the most effective method of nonviral transfection of adipose tissue stromal cells.

  20. Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

    Directory of Open Access Journals (Sweden)

    Bhavani S. Kowtharapu

    2018-02-01

    Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

  1. Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation.

    Science.gov (United States)

    Ahirwar, Dinesh K; Nasser, Mohd W; Ouseph, Madhu M; Elbaz, Mohamad; Cuitiño, Maria C; Kladney, Raleigh D; Varikuti, Sanjay; Kaul, Kirti; Satoskar, Abhay R; Ramaswamy, Bhuvaneswari; Zhang, Xiaoli; Ostrowski, Michael C; Leone, Gustavo; Ganju, Ramesh K

    2018-05-03

    The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

  2. Metabolic cooperation between cancer and non-cancerous stromal cells is pivotal in cancer progression.

    Science.gov (United States)

    Lopes-Coelho, Filipa; Gouveia-Fernandes, Sofia; Serpa, Jacinta

    2018-02-01

    The way cancer cells adapt to microenvironment is crucial for the success of carcinogenesis, and metabolic fitness is essential for a cancer cell to survive and proliferate in a certain organ/tissue. The metabolic remodeling in a tumor niche is endured not only by cancer cells but also by non-cancerous cells that share the same microenvironment. For this reason, tumor cells and stromal cells constitute a complex network of signal and organic compound transfer that supports cellular viability and proliferation. The intensive dual-address cooperation of all components of a tumor sustains disease progression and metastasis. Herein, we will detail the role of cancer-associated fibroblasts, cancer-associated adipocytes, and inflammatory cells, mainly monocytes/macrophages (tumor-associated macrophages), in the remodeling and metabolic adaptation of tumors.

  3. A stromal myoid cell line provokes thymic erythropoiesis between ...

    African Journals Online (AJOL)

    Background: The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular ...

  4. Senescence and quiescence in adipose-derived stromal cells

    DEFF Research Database (Denmark)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd

    2017-01-01

    Background aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine...

  5. Stimulated human fibroblast cell survival

    International Nuclear Information System (INIS)

    Smith, B.P.; Gale, K.L.; Einspenner, M.; Greenstock, C.L.; Gentner, N.E.

    1992-01-01

    Techniques for cloning cultured mammalian cells have supported the most universally-accepted method for measuring the induction of lethality by geno-toxicants such as ionizing radiation: the 'survival of colony-forming ability (CFA)' assay. Since most cultured human cell lines exhibit plating efficiency (i.e. the percentage of cells that are capable of reproductively surviving and dividing to form visible colonies) well below 100%, such assays are in essence 'survival of plating efficiency' assays, since they are referred to the plating (or cloning) efficiency of control (i.e. unirradiated) cells. (author). 8 refs., 2 figs

  6. The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro.

    Science.gov (United States)

    Kamishina, Hiroaki; Cheeseman, Jennifer A; Clemmons, Roger M

    2009-10-01

    The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media. DRG neurons grown on BMSCs extended longer neurites and developed a much more elaborate conformation of branching neurites compared to those on fibroblasts or laminin. Quantitative analysis revealed that these effects were associated with the emergence of increased numbers of primary and branching neurites. The effect appears to be dependent upon cell-cell interactions rather than by elaboration of diffusible molecules. With more extensive investigations into the basic biology of canine BMSCs, their ability for promoting neurite outgrowth may be translated into a novel therapeutic strategy for dogs with a variety of neurological disorders.

  7. Stromal cell regulation of homeostatic and inflammatory lymphoid organogenesis

    Science.gov (United States)

    Kain, Matthew J W; Owens, Benjamin M J

    2013-01-01

    Summary Secondary lymphoid organs function to increase the efficiency of interactions between rare, antigen-specific lymphocytes and antigen presenting cells, concentrating antigen and lymphocytes in a supportive environment that facilitates the initiation of an adaptive immune response. Homeostatic lymphoid tissue organogenesis proceeds via exquisitely controlled spatiotemporal interactions between haematopoietic lymphoid tissue inducer populations and multiple subsets of non-haematopoietic stromal cells. However, it is becoming clear that in a range of inflammatory contexts, ectopic or tertiary lymphoid tissues can develop inappropriately under pathological stress. Here we summarize the role of stromal cells in the development of homeostatic lymphoid tissue, and assess emerging evidence that suggests a critical role for stromal involvement in the tertiary lymphoid tissue development associated with chronic infections and inflammation. PMID:23621403

  8. Stromal cell-derived factor 1α (SDF-1α)

    DEFF Research Database (Denmark)

    Li, Dana; Bjørnager, Louise; Langkilde, Anne

    2016-01-01

    OBJECTIVES: Stromal cell-derived factor 1a (SDF-1α), is a chemokine and is able to home hematopoietic progenitor cells to injured areas of heart tissue for structural repair. Previous studies have found increased levels of SDF-1α in several cardiac diseases, but only few studies have investigated...

  9. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  10. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  11. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  12. Expression of tyrosine kinase gene in mouse thymic stromal cells

    NARCIS (Netherlands)

    Rinke de Wit, T. F.; Izon, D. J.; Revilla, C.; Oosterwegel, M.; Bakker, A. Q.; van Ewijk, W.; Kruisbeek, A. M.

    1996-01-01

    Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both

  13. Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

    NARCIS (Netherlands)

    Ritfeld, Gaby Jane

    2014-01-01

    Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic

  14. IL-33 and Thymic Stromal Lymphopoietin in mast cell functions

    NARCIS (Netherlands)

    Saluja, Rohit; Zoltowska, Anna; Ketelaar, Maria Elizabeth; Nilsson, Gunnar

    2016-01-01

    Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL-33) are two cytokines released by cells that are in proximity to our environment, e.g., keratinocytes of the skin and epithelial cells of the airways. Pathogens, allergens, chemicals and other agents induce the release of TSLP and IL-33,

  15. Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells

    Science.gov (United States)

    Chen, Weiwei; Tang, Tracy; Eastham-Anderson, Jeff; Dunlap, Debra; Alicke, Bruno; Nannini, Michelle; Gould, Stephen; Yauch, Robert; Modrusan, Zora; DuPree, Kelly J.; Darbonne, Walter C.; Plowman, Greg; de Sauvage, Frederic J.; Callahan, Christopher A.

    2011-01-01

    Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh–expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo. PMID:21597001

  16. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

    Science.gov (United States)

    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  17. File list: DNS.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Utr.10.AllAg.Endometrial_stromal_cells hg19 DNase-seq Uterus Endometrial stroma...l cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  18. File list: Pol.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Utr.05.AllAg.Endometrial_stromal_cells hg19 RNA polymerase Uterus Endometrial s...tromal cells SRX1048949 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  19. File list: Unc.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.05.AllAg.Endometrial_stromal_cells hg19 Unclassified Uterus Endometrial str...omal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  20. File list: Unc.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.10.AllAg.Endometrial_stromal_cells hg19 Unclassified Uterus Endometrial str...omal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  1. File list: DNS.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Utr.50.AllAg.Endometrial_stromal_cells hg19 DNase-seq Uterus Endometrial stroma...l cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  2. File list: Unc.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.50.AllAg.Endometrial_stromal_cells hg19 Unclassified Uterus Endometrial str...omal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  3. File list: Pol.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Utr.50.AllAg.Endometrial_stromal_cells hg19 RNA polymerase Uterus Endometrial s...tromal cells SRX1048949 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  4. File list: Oth.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.20.AllAg.Endometrial_stromal_cells hg19 TFs and others Uterus Endometrial s...tromal cells SRX1048945,SRX372174,SRX1048948,SRX1048946,SRX735140,SRX735139 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  5. File list: DNS.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Utr.20.AllAg.Endometrial_stromal_cells hg19 DNase-seq Uterus Endometrial stroma...l cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  6. File list: Oth.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.50.AllAg.Endometrial_stromal_cells hg19 TFs and others Uterus Endometrial s...tromal cells SRX372174,SRX1048948,SRX735140,SRX735139,SRX1048946,SRX1048945 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  7. File list: Pol.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Utr.10.AllAg.Endometrial_stromal_cells hg19 RNA polymerase Uterus Endometrial s...tromal cells SRX1048949 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  8. File list: Unc.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Utr.20.AllAg.Endometrial_stromal_cells hg19 Unclassified Uterus Endometrial str...omal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  9. File list: DNS.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Utr.05.AllAg.Endometrial_stromal_cells hg19 DNase-seq Uterus Endometrial stroma...l cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  10. File list: Oth.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.05.AllAg.Endometrial_stromal_cells hg19 TFs and others Uterus Endometrial s...tromal cells SRX1048945,SRX1048948,SRX1048946,SRX372174,SRX735140,SRX735139 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  11. File list: Oth.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.10.AllAg.Endometrial_stromal_cells hg19 TFs and others Uterus Endometrial s...tromal cells SRX1048945,SRX1048948,SRX1048946,SRX372174,SRX735140,SRX735139 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  12. File list: Pol.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Utr.20.AllAg.Endometrial_stromal_cells hg19 RNA polymerase Uterus Endometrial s...tromal cells SRX1048949 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  13. Adult Stromal (Skeletal, Mesenchymal) Stem Cells: Advances Towards Clinical Applications

    DEFF Research Database (Denmark)

    Kermani, Abbas Jafari; Harkness, Linda; Zaher, Walid

    2014-01-01

    Mesenchymal Stem Cells (MSC) are non-hematopoietic adult stromal cells that reside in a perivascular niche in close association with pericytes and endothelial cells and possess self-renewal and multi-lineage differentiation capacity. The origin, unique properties, and therapeutic benefits of MSC ...... the translation of MSC into clinic: Generation of MSC-like cells from human pluripotent stem cells, strategies to enhance homing of MSC to injured tissues, and targeting of MSC in vivo.......Mesenchymal Stem Cells (MSC) are non-hematopoietic adult stromal cells that reside in a perivascular niche in close association with pericytes and endothelial cells and possess self-renewal and multi-lineage differentiation capacity. The origin, unique properties, and therapeutic benefits of MSC...

  14. Immunocytochemical characterization of explant cultures of human prostatic stromal cells

    NARCIS (Netherlands)

    A. Kooistra (Anko); A.M.J. Elissen (Arianne ); J.J. Konig (Josee); M. Vermey; Th.H. van der Kwast (Theo); J.C. Romijn (Johannes); F.H. Schröder (Fritz)

    1995-01-01

    textabstractThe study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human

  15. Human mesenchymal stromal cells : biological characterization and clinical application

    NARCIS (Netherlands)

    Bernardo, Maria Ester

    2010-01-01

    This thesis focuses on the characterization of the biological and functional properties of human mesenchymal stromal cells (MSCs), isolated from different tissue sources. The differentiation capacity of MSCs from fetal and adult tissues has been tested and compared. Umbilical cord blood (UCB) has

  16. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2016-11-01

    Full Text Available The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells’ growth and expansion can influence neighboring cells’ behavior, leading to a modulation of mesenchymal stromal cell (MSC activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT, a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity.

  17. File list: His.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Utr.50.AllAg.Endometrial_stromal_cells hg19 Histone Uterus Endometrial stromal ...X524966,SRX524979,SRX524974,SRX524968,SRX524964,SRX524973 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  18. File list: His.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Utr.20.AllAg.Endometrial_stromal_cells hg19 Histone Uterus Endometrial stromal ...X524966,SRX524964,SRX524963,SRX524979,SRX524962,SRX524974 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  19. File list: His.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Utr.10.AllAg.Endometrial_stromal_cells hg19 Histone Uterus Endometrial stromal ...X524966,SRX524963,SRX524979,SRX524969,SRX524974,SRX524967 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  20. File list: His.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Utr.05.AllAg.Endometrial_stromal_cells hg19 Histone Uterus Endometrial stromal ...X524964,SRX524979,SRX524974,SRX524967,SRX524969,SRX524963 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  1. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  2. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells.

    Science.gov (United States)

    Geyh, S; Oz, S; Cadeddu, R-P; Fröbel, J; Brückner, B; Kündgen, A; Fenk, R; Bruns, I; Zilkens, C; Hermsen, D; Gattermann, N; Kobbe, G; Germing, U; Lyko, F; Haas, R; Schroeder, T

    2013-09-01

    Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

  3. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation.

    Science.gov (United States)

    Tarte, Karin; Gaillard, Julien; Lataillade, Jean-Jacques; Fouillard, Loic; Becker, Martine; Mossafa, Hossein; Tchirkov, Andrei; Rouard, Hélène; Henry, Catherine; Splingard, Marie; Dulong, Joelle; Monnier, Delphine; Gourmelon, Patrick; Gorin, Norbert-Claude; Sensebé, Luc

    2010-02-25

    Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

  4. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Takahara, Kiyoshi [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Ii, Masaaki, E-mail: masaii@art.osaka-med.ac.jp [Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Asahi, Michio [Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Azuma, Haruhito [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan)

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

  5. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    International Nuclear Information System (INIS)

    Takahara, Kiyoshi; Ii, Masaaki; Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi; Asahi, Michio; Azuma, Haruhito

    2014-01-01

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa

  6. Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts.

    Science.gov (United States)

    Anitua, E; de la Fuente, M; Muruzabal, F; Riestra, A; Merayo-Lloves, J; Orive, G

    2015-06-01

    Autologous serum (AS) eye drops was the first blood-derived product used for the treatment of corneal pathologies but nowadays PRGF arises as a novel interesting alternative to this type of diseases. The purpose of this study was to evaluate and compare the biological outcomes of autologous serum eye drops or Plasma rich in growth factors (PRGF) eye drops on corneal stromal keratocytes (HK) and conjunctival fibroblasts (HConF). To address this, blood from healthy donors was collected and processed to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Blood-derivates were aliquoted and stored at -80°C until use. PDGF-AB, VEGF, EGF, FGFb and TGF-β1 were quantified. The potential of PRGF and AS in promoting wound healing was evaluated by means of proliferation and migration assays in HK and HConF. Fibroblast cells were induced to myofibroblast differentiation after treatment with 2.5ng/mL of TGF-β1. The capability of PRGF and AS to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results showed significant higher levels of all growth factors analyzed in PRGF eye drops compared to AS. Moreover, PRGF eye drops enhanced significantly the biological outcomes of both HK and HConF, and reduced TGF-β1-induced myofibroblast differentiation in contrast to autologous serum eye drops (AS). In summary, these results suggest that PRGF exerts enhanced biological outcomes than AS. PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to AS. Results obtained herein suggest that PRGF protects and reverses the myofibroblast phenotype while promotes cell proliferation and migration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.

    Science.gov (United States)

    Wu, Xiuwen; Ren, Jianan; Li, Jieshou

    2012-05-01

    The use of tissue-engineering techniques such as stem-cell therapy to renew injured tissues is a promising strategy in regenerative medicine. As a cell-delivery vehicle, fibrin glues (FG) facilitate cell attachment, growth and differentiation and, ultimately, tissue formation and organization by its three-dimensional structure. Numerous studies have provided evidence that stromal cells derived from bone marrow (bone marrow stromal cells; BMSC) and adipose tissue (adipose-derived stromal cells; ADSC) contain a population of adult multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells that can differentiate into several lineages. By combining MSC with FG, the implantation could take advantage of the mutual benefits. Researchers and physicians have pinned their hopes on stem cells for developing novel approaches in regenerative medicine. This review focuses on the therapeutic potential of MSC with FG in bone defect reconstruction, cartilage and tendon injury repair, ligament, heart and nerve regeneration, and, furthermore, wound healing.

  8. Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

    Czech Academy of Sciences Publication Activity Database

    Strnad, Hynek; Lacina, L.; Kolář, Michal; Čada, Z.; Vlček, Čestmír; Dvořánková, B.; Betka, J.; Plzák, J.; Chovanec, M.; Šáchová, Jana; Valach, Jaroslav; Urbanová, Markéta; Smetana, K. Jr.

    2010-01-01

    Roč. 133, č. 2 (2010), s. 201-211 ISSN 0948-6143 R&D Projects: GA MŠk 2B06106 Grant - others:GA ČR(CZ) GP304/08/P175 Institutional research plan: CEZ:AV0Z50520514 Keywords : Cancer microenvironment * Epithelial–mesenchymal interaction * Cancer-associated fibroblasts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.727, year: 2010

  9. Degenerative Suspensory Ligament Desmitis (DSLD in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Wei Luo

    Full Text Available Equine degenerative suspensory ligament desmitis (DSLD in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05 in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1, SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2 represent master-regulators in a wide range of cellular metabolic responses.

  10. Podoplanin-positive Cancer-associated Stromal Fibroblasts in Primary Tumor and Synchronous Lymph Node Metastases of HER2-overexpressing Breast Carcinomas.

    Science.gov (United States)

    Niemiec, Joanna; Adamczyk, Agnieszka; Harazin-Lechowska, Agnieszka; Ambicka, Aleksandra; Grela-Wojewoda, Aleksandra; Majchrzyk, Kaja; Kruczak, Anna; Sas-Korczyńska, Beata; Ryś, Janusz

    2018-04-01

    We compared the status of stromal podoplanin-positive cancer-associated fibroblasts (ppCAFs) between primary tumors and paired synchronous lymph node metastases (LNMs) and analyzed the prognostic significance of tumoral ppCAFs in 203 patients with human epidermal growth factor receptor 2-positive breast carcinoma. ppCAFs were found in 167/203 and in 35/87 tumors and LNM, respectively. ppCAFs were most frequently found in tumors and corresponding LNM (n=52, 59.8%; p=0.001). However, for all LNMs (n=12) without ppCAFs, their paired tumors also lacked ppCAFs. In both tumors and LNMs, ppCAFs were α-smooth muscle actin-positive and cluster of differentiation 21 protein-negative, suggesting them not to be resident lymph node cells. Moreover, in our series, the presence of ppCAFs in tumors was borderline related to poor disease-free survival (p=0.058). These results speak in favor of a hypothesis suggesting ppCAFs accompany metastatic cancer cells migrating from tumor to LNMs. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  11. Co-Culturing of Multipotent Mesenchymal Stromal Cells with Autological and Allogenic Lymphocytes.

    Science.gov (United States)

    Kapranov, N M; Davydova, Yu O; Gal'tseva, I V; Petinati, N A; Bakshinskaitė, M V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

    2018-03-01

    We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

  12. Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

    International Nuclear Information System (INIS)

    Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

    2005-01-01

    Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

  13. Mesenchymal stem cells induce dermal fibroblast responses to injury

    International Nuclear Information System (INIS)

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  14. Fetal liver stromal cells promote hematopoietic cell expansion

    International Nuclear Information System (INIS)

    Zhou, Kun; Hu, Caihong; Zhou, Zhigang; Huang, Lifang; Liu, Wenli; Sun, Hanying

    2009-01-01

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  15. Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

    Science.gov (United States)

    Kawada, Manabu; Inoue, Hiroyuki; Ohba, Shun-ichi; Yoshida, Junjiro; Masuda, Tohru; Yamasaki, Manabu; Usami, Ihomi; Sakamoto, Shuichi; Abe, Hikaru; Watanabe, Takumi; Yamori, Takao; Shibasaki, Masakatsu; Nomoto, Akio

    2015-01-01

    Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy. PMID:25785838

  16. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

    International Nuclear Information System (INIS)

    Lee, Hyung-Ok; Mullins, Stefanie R; Franco-Barraza, Janusz; Valianou, Matthildi; Cukierman, Edna; Cheng, Jonathan D

    2011-01-01

    Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP + matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP + matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP + matrix-induced tumor invasion phenotype is β 1 -integrin/FAK mediated. Cancer cell invasiveness can be affected by alterations in the tumor

  17. Attenuation of radiation-induced DNA damage due to paracrine interactions between normal human epithelial and stromal cells

    International Nuclear Information System (INIS)

    Saenko, V.A.; Nakazawa, Yu.; Rogounovitch, T.I.; Suzuki, K.; Mitsutake, N.; Matsuse, M.; Yamashita, S.

    2007-01-01

    Complete text of publication follows. Objective: Developmentally, every tissue accommodates different types of cells, such as epitheliocytes and stromal cells in parenchymal organs. To better understand the complexity of radiation response, it is necessary to evaluate possible cross-talk between different tissue components. This work was set out to investigate reciprocal influence of normal human epithelial cells and fibroblasts on the extent of radiation-induced DNA damage. Methods: Model cultures of primary human thyrocytes (PT), normal diploid fibroblasts (BJ), PT/BJ cell co-culture and conditioned medium transfer were used to examine DNA damage in terms of γ-H2AX foci number per cell or by Comet assay after exposure to different doses of γ-rays. Results: In co-cultures, the kinetics of γ-H2AX foci number change was dose-dependent and similar to that in individual PT and BJ cultures. The number of γ-H2AX foci in co-cultures was significantly lower (∼25%) in both types of cells comparing to individual cultures. Reciprocal conditioned medium transfer to individual counterpart cells prior to irradiation resulted in approximately 35% reduction in the number γ-H2AX foci at 1 Gy and lower doses in both PT and BJ demonstrating the role of paracrine soluble factors. Comet assay corroborated the results of γ-H2AX foci counting in conditioned medium transfer experiments. In contrast to medium conditioned on PT cells, conditioned medium collected from several human thyroid cancer cell lines failed to establish DNA-protected state in BJ fibroblasts. In its turn, medium conditioned on BJ cells did not change the extent of radiation-induced DNA damage in cancer cell lines tested. Conclusion: The results imply the existence of a network of soluble factor-mediated paracrine interactions between normal epithelial and stromal cells that could be a part of natural mechanism by which cells protect DNA from genotoxic stress.

  18. Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Honglei Chen

    2012-10-01

    Full Text Available Caveolin-1 (Cav-1 expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV-associated GC (EBVaGC is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029 that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032 and was positively associated with Cav-1 expression (r = 0.432, p < 0.001. EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

  19. Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer.

    Science.gov (United States)

    Hirayama, Kazuyoshi; Kono, Hiroshi; Nakata, Yuuki; Akazawa, Yoshihiro; Wakana, Hiroyuki; Fukushima, Hisataka; Fujii, Hideki

    2018-01-01

    To investigate the role of podoplanin (PDPN) expression in invasive ductal carcinoma of the pancreas (IDCP) in humans. Tumor samples were obtained from 95 patients with IDCP. Immunohistochemical staining was done to evaluate the expression of PDPN in cancer tissues. PDPN was detected predominantly in stromal fibroblasts, stained with α-smooth muscle actin. The cutoff value of PDPN-positive areas was calculated according to a histogram. There was no significant difference in clinicopathologic factors between patients with high vs. those with low PDPN expression. The high PDPN group showed significantly poorer disease-free and disease-specific survival rates than the low PDPN group. Among patients from the high PDPN group, those with lymph node metastases and those with a tumor larger than 20 cm in diameter had significantly poorer prognoses than similar patients from the low PDPN group. Multivariate Cox proportional hazards analysis indicated that a high expression of PDPN was an independent risk factor for disease-specific survival. PDPN expression in cancer-related fibrotic tissues is associated with a poor prognosis, especially in patients with large tumors or lymph node metastases.

  20. Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

    Science.gov (United States)

    Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

    2015-04-11

    The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising

  1. Adipose-Derived Stromal Cells for Treatment of Patients with Chronic Ischemic Heart Disease (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Mathiasen, Anders Bruun; Mygind, Naja Dam

    2017-01-01

    We aimed to evaluate the effect of intramyocardial injections of autologous VEGF-A165-stimulated adipose-derived stromal cells (ASCs) in patients with refractory angina. MyStromalCell trial is a randomized double-blind placebo-controlled study including sixty patients with CCS/NYHA class II...... to 54) (P= 0.41), and in METs 0.1 (95%CI -1.7 to 1.9) (P= 0.757). The difference between the groups was not significant (P= 0.680,P= 0.608, andP= 0.720 for time duration, watt, and METs, resp.). Intramyocardial delivered VEGF-A165-stimulated ASC treatment was safe but did not improve exercise capacity...

  2. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte...

  3. Mesenchymal stromal cell therapy in ischemic heart disease

    DEFF Research Database (Denmark)

    Kastrup, Jens; Mygind, Naja Dam; Ali Qayyum, Abbas

    2016-01-01

    is very costly for the health care system. Therefore, new treatment options and strategies are being researched intensely. Stem cell therapy to improve myocardial perfusion and stimulate growth of new cardiomyocytes could be a new way to go. Nevertheless, the results from clinical studies have varied...... considerably, probably due to the use of many different cell lines obtained from different tissues and the different patient populations. The present review will focus on treatment with the mesenchymal stromal cell from bone marrow and adipose tissue in animal and patients with acute and chronic IHD (CIHD)....

  4. Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

    DEFF Research Database (Denmark)

    Madsen, Chris D; Pedersen, Jesper Thorhauge; Venning, Freja A

    2015-01-01

    , which can be prevented by simultaneous depletion of HIF-1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co......-injected with tumour cells similarly prevents CAF-induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications....

  5. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

    Science.gov (United States)

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-01-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  6. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have be...

  7. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    International Nuclear Information System (INIS)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia; Mazzanti, Benedetta; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2014-01-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7 + satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration

  8. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Mazzanti, Benedetta [Dept. of Experimental and Clinical Medicine—Section of Haematology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Quercioli, Franco [CNR-National Institute of Optics (INO), Largo Enrico Fermi 6, 50125 Arcetri-Florence (Italy); Zecchi-Orlandini, Sandra [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Formigli, Lucia, E-mail: formigli@unifi.it [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy)

    2014-05-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

  9. Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

    Science.gov (United States)

    Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

    2018-03-01

    Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

  10. Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

    Science.gov (United States)

    Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

    2013-11-01

    Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

  11. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    International Nuclear Information System (INIS)

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34 + cells, CD34 + blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34 + acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO + leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO + leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells

  12. Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

    Science.gov (United States)

    Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

    2017-08-15

    Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

  13. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  14. Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells.

    Science.gov (United States)

    Zhao, Qinjun; Ren, Hongying; Li, Xiyuan; Chen, Zhong; Zhang, Xiangyu; Gong, Wei; Liu, Yongjun; Pang, Tianxiang; Han, Zhong Chao

    2009-01-01

    Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

  15. Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Nakamura, Ryosuke; Kayamori, Kou; Oue, Erika; Sakamoto, Kei; Harada, Kiyoshi; Yamaguchi, Akira

    2015-01-01

    Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC

  16. Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Ryosuke [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Kayamori, Kou [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Oue, Erika [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Sakamoto, Kei [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Harada, Kiyoshi [Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-20

    Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC

  17. Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

    Science.gov (United States)

    Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Whitaker-Menezes, Diana; Daumer, Kristin M; Milliman, Janet N; Chiavarina, Barbara; Migneco, Gemma; Witkiewicz, Agnieszka K; Martinez-Cantarin, Maria P; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Lisanti, Michael P; Sotgia, Federica

    2010-06-15

    Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti

  18. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    DEFF Research Database (Denmark)

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra

    2014-01-01

    Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show...... exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key...... that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells...

  19. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2015-07-01

    Full Text Available The Low-Affinity Nerve Growth Factor Receptor (LNGFR, also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271− mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  20. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation.

    Science.gov (United States)

    Calabrese, Giovanna; Giuffrida, Raffaella; Lo Furno, Debora; Parrinello, Nunziatina Laura; Forte, Stefano; Gulino, Rosario; Colarossi, Cristina; Schinocca, Luciana Rita; Giuffrida, Rosario; Cardile, Venera; Memeo, Lorenzo

    2015-07-09

    The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271- mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  1. In vitro effects of direct current electric fields on adipose-derived stromal cells.

    Science.gov (United States)

    Hammerick, Kyle E; Longaker, Michael T; Prinz, Fritz B

    2010-06-18

    Endogenous electric fields play an important role in embryogenesis, regeneration, and wound repair and previous studies have shown that many populations of cells, leukocytes, fibroblasts, epithelial cells, and endothelial cells, exhibit directed migration in response to electric fields. As regenerative therapies continue to explore ways to control mesenchymal progenitor cells to recreate desirable tissues, it is increasingly necessary to characterize the vast nature of biological responses imposed by physical phenomena. Murine adipose-derived stromal cells (mASCs) migrated toward the cathode in direct current (DC) fields of physiologic strength and show a dose dependence of migration rate to stronger fields. Electric fields also caused mASCs to orient perpendicularly to the field vector and elicited a transient increase in cytosolic calcium. Additionally, their galvanotactic response appears to share classic chemotactic signaling pathways that are involved in the migration of other cell types. Galvanotaxis is one predominant result of electric fields on mASCs and it may be exploited to engineer adult stem cell concentrations and locations within implanted grafts or toward sites of wound repair. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  2. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  3. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; hide

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pcell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  4. A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

    Science.gov (United States)

    Caires, Hugo R; Barros da Silva, Patrícia; Barbosa, Mário A; Almeida, Catarina R

    2018-03-01

    The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Experimental Models for Investigating Intra-Stromal Migration of Corneal Keratocytes, Fibroblasts and Myofibroblasts

    Directory of Open Access Journals (Sweden)

    Lisha Ma

    2012-03-01

    Full Text Available Following laser vision correction, corneal keratocytes must repopulate areas of cell loss by migrating through the intact corneal stroma, and this can impact corneal shape and transparency. In this study, we evaluate 3D culture models for simulating this process in vitro. Buttons (8 mm diameter were first punched out of keratocyte populated compressed collagen matrices, exposed to a 3 mm diameter freeze injury, and cultured in serum-free media (basal media or media supplemented with 10% FBS, TGFb1 or PDGF BB. Following freeze injury, a region of cell death was observed in the center of the constructs. Although cells readily migrated on top of the matrices to cover the wound area, a limited amount of cell migration was observed within the constructs. We next developed a novel “sandwich” model, which better mimics the native lamellar architecture of the cornea. Using this model, significant migration was observed under all conditions studied. In both models, cells in TGFb and 10% FBS developed stress fibers; whereas cells in PDGF were more dendritic. PDGF stimulated the most inter-lamellar migration in the sandwich construct. Overall, these models provide insights into the complex interplay between growth factors, cell mechanical phenotypes and the structural properties of the ECM.

  6. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  7. Bone marrow stromal cells spontaneously produce Flt3-ligand: influence of ionizing radiations and cytokine stimulation.

    Science.gov (United States)

    Bertho, Jean Marc; Demarquay, Christelle; Mouiseddine, Moubarak; Douenat, Noémie; Stefani, Johanna; Prat, Marie; Paquet, François

    2008-08-01

    To define the ability of human bone marrow (BM) stromal cells to produce fms-like tyrosine kinase 3 (Flt3)-ligand (FL), and the effect of irradiation, tumour necrosis factor-alpha (TNFalpha) or tumour growth factor beta (TGFbeta) on FL production. Primary BM stromal cell cultures were irradiated at 2-10 Gy or were stimulated with TNFalpha or TGFbeta1. The presence of FL was tested in culture supernatants and in cell lysate. The presence of a membrane-bound form of FL and the level of gene expression were also tested. Primary BM stromal cells spontaneously released FL. This production was increased by TNFalpha but not by TGFbeta1 or by irradiation. Chemical induction of osteoblastic differentiation from BM stromal cells also induced an increase in FL release. Our results suggest that the observed increase in FL concentration after in vivo irradiation is an indirect effect. The possible implication of BM stromal cells in these mechanisms is discussed.

  8. File list: ALL.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.10.AllAg.Endometrial_stromal_cells hg19 All antigens Uterus Endometrial str...X1048949,SRX524965 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  9. File list: ALL.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.50.AllAg.Endometrial_stromal_cells hg19 All antigens Uterus Endometrial str...RX524970,SRX524973 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  10. File list: ALL.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.05.AllAg.Endometrial_stromal_cells hg19 All antigens Uterus Endometrial str...RX735139,SRX735141 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  11. File list: ALL.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Utr.20.AllAg.Endometrial_stromal_cells hg19 All antigens Uterus Endometrial str...RX524962,SRX524974 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  12. Sequential growth factor application in bone marrow stromal cell ligament engineering.

    Science.gov (United States)

    Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

    2005-01-01

    In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

  13. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  14. The mandibular ridge oral mucosa model of stromal influences on the endothelial tip cells: an immunohistochemical and TEM study.

    Science.gov (United States)

    Rusu, Mugurel Constantin; Didilescu, Andreea Cristiana; Stănescu, Ruxandra; Pop, Florinel; Mănoiu, Valentina Mariana; Jianu, Adelina Maria; Vâlcu, Marek

    2013-02-01

    This study aimed to evaluate by immunohistochemistry and transmission electron microscopy (TEM) the morphological features of the oral mucosa endothelial tip cells (ETCs) and to determine the immune and ultrastructural patterns of the stromal nonimmune cells which could influence healing processes. Immune labeling was performed on bioptic samples obtained from six edentulous patients undergoing surgery for dental implants placement; three normal samples were collected from patients prior to the extraction of the third mandibular molar. The antibodies were tested for CD34, CD117(c-kit), platelet derived growth factor receptor-alpha (PDGFR-α), Mast Cell Tryptase, CD44, vimentin, CD45, CD105, alpha-smooth muscle actin, FGF2, Ki67. In light microscopy, while stromal cells (StrCs) of the reparatory and normal oral mucosa, with a fibroblastic appearance, were found positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype, the CD117/c-kit labeling led to a positive stromal reaction only in the reparatory mucosa. In TEM, non-immune StrCs presenting particular ultrastructural features were identified as circulating fibrocytes (CFCs). Within the lamina propria CFCs were in close contact with ETCs. Long processes of the ETCs were moniliform, and hook-like collaterals were arising from the dilated segments, suggestive for a different stage migration. Maintenance and healing of oral mucosa are so supported by extensive processes of angiogenesis, guided by ETCs that, in turn, are influenced by the CFCs that populate the stromal compartment both in normal and reparatory states. Therefore, CFCs could be targeted by specific therapies, with pro- or anti-angiogenic purposes. Copyright © 2012 Wiley Periodicals, Inc.

  15. Regenerative Potential of Mesenchymal Stromal Cells: Age-Related Changes

    Directory of Open Access Journals (Sweden)

    Flavia Bruna

    2016-01-01

    Full Text Available Preclinical and clinical studies have shown that a therapeutic effect results from mesenchymal stromal cells (MSCs transplant. No systematic information is currently available regarding whether donor age modifies MSC regenerative potential on cutaneous wound healing. Here, we evaluate whether donor age influences this potential. Two different doses of bone marrow MSCs (BM-MSCs from young, adult, or old mouse donors or two doses of their acellular derivatives mesenchymal stromal cells (acd-MSCs were intradermally injected around wounds in the midline of C57BL/6 mice. Every two days, wound healing was macroscopically assessed (wound closure and microscopically assessed (reepithelialization, dermal-epidermal junction, skin appendage regeneration, granulation tissue, leukocyte infiltration, and density dermal collagen fibers after 12 days from MSC transplant. Significant differences in the wound closure kinetic, quality, and healing of skin regenerated were observed in lesions which received BM-MSCs from different ages or their acd-MSCs compared to lesions which received vehicle. Nevertheless, our data shows that adult’s BM-MSCs or their acd-MSCs were the most efficient for recovery of most parameters analyzed. Our data suggest that MSC efficacy was negatively affected by donor age, where the treatment with adult’s BM-MSCs or their acd-MSCs in cutaneous wound promotes a better tissue repair/regeneration. This is due to their paracrine factors secretion.

  16. Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin.

    Science.gov (United States)

    Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mollerup, Sarah; Asplund, Maria; Mourier, Tobias; Hansen, Anders Johannes; Gniadecki, Robert

    2017-10-07

    Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety of neoplasms but their role in BCC is poorly understood. Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical and immunofluorescent staining were performed to validate the NGS results and to investigate CAF-related cyto-and chemokines. NGS revealed upregulation of 65 genes in BCC coding for extracellular matrix components pointing at CAF-related matrix remodeling. qRT-PCR showed increased mRNA expression of CAF markers FAP-α, PDGFR-β and prolyl-4-hydroxylase in BCC. Peritumoural skin (but not buttock skin) also exhibited high expression of PDGFR-β and prolyl-4-hydroxylase but not FAP-α. We found a similar pattern for the CAF-associated chemokines CCL17, CCL18, CCL22, CCL25, CXCL12 and IL6 with high expression in BCC and peritumoural skin but absence in buttock skin. Immunofluorescence revealed correlation between FAP-α and PDGFR-β and CXCL12 and CCL17. Matrix remodeling is the most prominent molecular feature of BCC. CAFs are present within BCC stroma and associated with increased expression of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.

  17. File list: NoD.Utr.05.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.05.AllAg.Endometrial_stromal_cells hg19 No description Uterus Endometrial s...tromal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.05.AllAg.Endometrial_stromal_cells.bed ...

  18. File list: NoD.Utr.50.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.50.AllAg.Endometrial_stromal_cells hg19 No description Uterus Endometrial s...tromal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.50.AllAg.Endometrial_stromal_cells.bed ...

  19. File list: NoD.Utr.10.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.10.AllAg.Endometrial_stromal_cells hg19 No description Uterus Endometrial s...tromal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.10.AllAg.Endometrial_stromal_cells.bed ...

  20. File list: NoD.Utr.20.AllAg.Endometrial_stromal_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Utr.20.AllAg.Endometrial_stromal_cells hg19 No description Uterus Endometrial s...tromal cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Utr.20.AllAg.Endometrial_stromal_cells.bed ...

  1. Accelerated Development of Supramolecular Corneal Stromal-Like Assemblies from Corneal Fibroblasts in the Presence of Macromolecular Crowders.

    Science.gov (United States)

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

    2015-07-01

    Tissue engineering by self-assembly uses the cells' secretome as a regeneration template and biological factory of trophic factors. Despite the several advantages that have been witnessed in preclinical and clinical settings, the major obstacle for wide acceptance of this technology remains the tardy extracellular matrix formation. In this study, we assessed the influence of macromolecular crowding (MMC)/excluding volume effect, a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude, in human corneal fibroblast (HCF) culture. Our data indicate that the addition of negatively charged galactose derivative (carrageenan) in HCF culture, even at 0.5% serum, increases by 12-fold tissue-specific matrix deposition, while maintaining physiological cell morphology and protein/gene expression. Gene analysis indicates that a glucose derivative (dextran sulfate) may drive corneal fibroblasts toward a myofibroblast lineage. Collectively, these results indicate that MMC may be suitable not only for clinical translation and commercialization of tissue engineering by self-assembly therapies, but also for the development of in vitro pathophysiology models.

  2. CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Al-Shammary, Asma; Skagen, Peter

    2015-01-01

    Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and their progenitors have been identified based on retrospective functional criteria. CD markers are employed to define cell populations with distinct functional characteristics. However, defining and pro...

  3. Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells.

    Science.gov (United States)

    Topoluk, Natasha; Hawkins, Richard; Tokish, John; Mercuri, Jeremy

    2017-09-01

    Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Controlled laboratory study. MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 10 6 ± 3.7 × 10 5 and 1.6 × 10 6 ± 4.7 × 10 5 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P

  4. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    International Nuclear Information System (INIS)

    Kramer, Brian C.; Woodbury, Dale; Black, Ira B.

    2006-01-01

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRα1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease

  5. Effects of conditioned medium from LL-37 treated adipose stem cells on human fibroblast migration.

    Science.gov (United States)

    Yang, Eun-Jung; Bang, Sa-Ik

    2017-07-01

    Adipose stem cell-conditioned medium may promote human dermal fibroblast (HDF) proliferation and migration by activating paracrine peptides during the re-epithelization phase of wound healing. Human antimicrobial peptide LL-37 is upregulated in the skin epithelium as part of the normal response to injury. The effects of conditioned medium (CM) from LL-37 treated adipose stem cells (ASCs) on cutaneous wound healing, including the mediation of fibroblast migration, remain to be elucidated, therefore the aim of the present study was to determine how ASCs would react to an LL-37-rich microenvironment and if CM from LL-37 treated ASCs may influence the migration of HDFs. The present study conducted migration assays with HDFs treated with CM from LL-37 treated ASCs. Expression of CXC chemokine receptor 4 (CXCR4), which controls the recruitment of HDFs, was analyzed at the mRNA and protein levels. To further characterize the stimulatory effects of LL-37 on ASCs, the expression of stromal cell-derived factor-1α (SDF-1α), a CXC chemokine, was investigated. CM from LL-37-treated ASCs induced migration of HDFs in a time- and dose-dependent manner, with a maximum difference in migration observed 24 h following stimulation with LL-37 at a concentration of 10 µg/ml. The HDF migration and the expression of CXCR4 in fibroblasts was markedly increased upon treatment with CM from LL-37-treated ASCs compared with CM from untreated ASCs. SDF-1α expression was markedly increased in CM from LL-37 treated ASCs. It was additionally observed that SDF-1α blockade significantly reduced HDF migration. These findings suggest the feasibility of CM from LL-37-treated ASCs as a potential therapeutic for human dermal fibroblast migration.

  6. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  7. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  8. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    Directory of Open Access Journals (Sweden)

    Li Chen

    2018-05-01

    Full Text Available Human stromal stem cells (hMSCs differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs: Cofilin 1 (CFL1 and Destrin (DSTN or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4. In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1 which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cells

  9. Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

    Directory of Open Access Journals (Sweden)

    Xu Qian

    2017-01-01

    Full Text Available Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ on day 5 and secondary decidual zone (SDZ on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA and estradiol-17β (E2. RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

  10. The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2

    NARCIS (Netherlands)

    Terlizzi, Vincenzo; Kolibabka, Matthias; Burgess, Janette Kay; Hammes, Hans Peter; Harmsen, Martin Conrad

    Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to

  11. A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells.

    Directory of Open Access Journals (Sweden)

    Dimitrios Karamichos

    Full Text Available Human corneal fibroblasts (HCF and corneal stromal stem cells (CSSC each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7. Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

  12. Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells.

    Science.gov (United States)

    Liu, Xunxian; Allen, Jeffrey D; Arnold, Julia T; Blackman, Marc R

    2008-04-01

    Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

  13. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  14. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  15. Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

    Science.gov (United States)

    VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

    2014-01-01

    The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

  16. Cell death induced by hydroxyapatite on L929 fibroblast cells.

    Science.gov (United States)

    Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

    2004-05-01

    Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

  17. The effects of acoustic vibration on fibroblast cell migration.

    Science.gov (United States)

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

    Science.gov (United States)

    Hoorweg, Kerim; Narang, Priyanka; Li, Zhi; Thuery, Anne; Papazian, Natalie; Withers, David R; Coles, Mark C; Cupedo, Tom

    2015-11-01

    Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs. Copyright © 2015 by The American Association of Immunologists, Inc.

  19. Developing a Continuous Bioprocessing Approach to Stromal Cell Manufacture.

    Science.gov (United States)

    Miotto, Martina; Gouveia, Ricardo; Abidin, Fadhilah Zainal; Figueiredo, Francisco; Connon, Che J

    2017-11-29

    To this day, the concept of continuous bioprocessing has been applied mostly to the manufacture of molecular biologics such as proteins, growth factors, and secondary metabolites with biopharmaceutical uses. The present work now sets to explore the potential application of continuous bioprocess methods to source large numbers of human adherent cells with potential therapeutic value. To this purpose, we developed a smart multifunctional surface coating capable of controlling the attachment, proliferation, and subsequent self-detachment of human corneal stromal cells. This system allowed the maintenance of cell cultures under steady-state growth conditions, where self-detaching cells were continuously replenished by the proliferation of those remaining attached. This facilitated a closed, continuous bioprocessing platform with recovery of approximately 1% of the total adherent cells per hour, a yield rate that was maintained for 1 month. Moreover, both attached and self-detached cells were shown to retain their original phenotype. Together, these results represent the proof-of-concept for a new high-throughput, high-standard, and low-cost biomanufacturing strategy with multiple potentials and important downstream applications.

  20. Bone Marrow Regeneration Promoted by Biophysically Sorted Osteoprogenitors From Mesenchymal Stromal Cells

    Science.gov (United States)

    Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon

    2015-01-01

    Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as “cell factories” that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies. PMID:25411477

  1. The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Adriana Borriello

    Full Text Available BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs, the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs. RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

  2. Human umbilical cord mesenchymal stromal cells in regenerative medicine.

    Science.gov (United States)

    Detamore, Michael S

    2013-11-25

    Cells of the human umbilical cord offer tremendous potential for improving human health. Cells from the Wharton’s jelly (umbilical cord stroma) in particular, referred to as human umbilical cord mesenchymal stromal cells (HUCMSCs), hold several advantages that make them appealing for translational research. In the previous issue of Stem Cell Research & Therapy, Chon and colleagues made an important contribution to the HUCMSC literature not only by presenting HUCMSCs as an emerging cell source for intervertebral disc regeneration in general and the nucleus pulposus in particular, but also by demonstrating that an extracellular matrix-based strategy might be preferred over the use of growth factors. By culturing HUCMSCs under hypoxia in serum-free conditions in the presence of Matrigel with laminin-111, they were able to achieve intense collagen II staining by 21 days without the addition of exogenous growth factors. There is tremendous translational significance here in that such raw materials may alleviate the need for the use of growth factors in some instances, and this may have important ramifications in reducing product cost and streamlining regulatory approval. Chon and colleagues provide a promising example of the potential of HUCMSCs, demonstrating the ability to guide HUCMSC differentiation even in the absence of serum and growth factors and supporting the use of HUCMSCs as a viable alternative in intervertebral disc regeneration.

  3. Human amniotic mesenchymal stromal cell transplantation improves endometrial regeneration in rodent models of intrauterine adhesions.

    Science.gov (United States)

    Gan, Lu; Duan, Hua; Xu, Qian; Tang, Yi-Qun; Li, Jin-Jiao; Sun, Fu-Qing; Wang, Sha

    2017-05-01

    Intrauterine adhesion (IUA) is a common uterine cavity disease characterized by the unsatisfactory regeneration of damaged endometria. Recently, stem cell transplantation has been proposed to promote the recovery process. Here we investigated whether human amniotic mesenchymal stromal cells (hAMSCs), a valuable resource for transplantation therapy, could improve endometrial regeneration in rodent IUA models. Forty female Sprague-Dawley rats were randomly assigned to five groups: normal, sham-operated, mechanical injury, hAMSC transplantation, and negative control group. One week after intervention and transplantation, histological analyses were performed, and immunofluorescent and immunohistochemical expression of cell-specific markers and messenger RNA expression of cytokines were measured. Thicker endometria, increased gland numbers and fewer fibrotic areas were found in the hAMSC transplantation group compared with the mechanical injury group. Engraftment of hAMSCs was detected by the presence of anti-human nuclear antigen-positive cells in the endometrial glands of the transplantation uteri. Transplantation of hAMSCs significantly decreased messenger RNA levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), and increased those of anti-inflammatory cytokines (basic fibroblast growth factor, and interleukin-6) compared with the injured uterine horns. Immunohistochemical expression of endometrial epithelial cells was revealed in specimens after hAMSC transplantation, whereas it was absent in the mechanically injured uteri. hAMSC transplantation promotes endometrial regeneration after injury in IUA rat models, possibly due to immunomodulatory properties. These cells provide a more easily accessible source of stem cells for future research into the impact of cell transplantation on damaged endometria. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Radiation effects on haematopoietic stem cells in vitro: possible role of stromal niches in the stem cell hierarchy

    International Nuclear Information System (INIS)

    Sharp, J.G.; Crouse, D.A.; Jackson, J.D.; Schmidt, C.M.; Ritter, E.K.; Udeaja, G.C.; Mann, S.L.

    1986-01-01

    The authors describe experiments which attempt to elucidate the nature of haemopoietic stem cell and microenvironmental stromal cell interactions which might explain anomalies in explanations of the differential effects of radiation on HSC versus MSC. In particular, there is an attempt to demonstrate the existence of stromal niches. (UK)

  5. Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

    Science.gov (United States)

    Lee, Yoon; Ahn, Jin-Soo; Yi, Young-Ah; Chung, Shin-Hye; Yoo, Yeon-Jee; Ju, Sung-Won; Hwang, Ji-Yun; Seo, Deog-Gyu

    2015-02-01

    The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

  6. Lewis lung carcinoma progression is facilitated by TIG-3 fibroblast cells.

    Science.gov (United States)

    Yamauchi, Yoshikane; Izumi, Yotaro; Asakura, Keisuke; Kawai, Kenji; Wakui, Masatoshi; Ohmura, Mitsuyo; Suematsu, Makoto; Nomori, Hiroaki

    2013-09-01

    The interactions of tumor cells with stromal fibroblasts influence tumor biology, but the exact mechanisms involved are still unclear. In the present study, we evaluated the effects of a human lung fibroblast cell line, TIG-3, on Lewis lung carcinoma (LLC) cells both in vitro and in vivo. LLC and TIG-3 cells were co-cultured/co-implanted in vitro and in vivo. Cell invasion was assayed. Local tumor growth, as well as lung metastasis, were evaluated after subcutaneous cell co-implantation into NOD/SCID/γ-null (NOG) mice. LLC, and TIG-3 cells were pre-treated with either SB431542, a small molecule TGF-β receptor antagonist, or siRNA for transforming growth factor (TGF)-β before co-culture or co-implantation, and the effects of pre-treatments were compared both in cell culture and in mice. Subcutaneous LLC tumor growth (L group) in NOG mice was significantly increased by co-implantation of TIG-3 cells (L+T group) at four weeks. The number of macroscopic lung metastases was also significantly increased in the L+T group in comparison to the L group. In vitro cell invasion was significantly increased in the L+T group in comparison to the L group. In vitro expression of phosphorylated-SMAD3 was significantly increased in the L+T group in comparison to the L group. Furthermore, pre-treatment with either SB431542 or siRNA for TGF-β reduced the invasiveness both in culture and in mice. This study suggested that in vitro as well as in vivo progression of LLC was facilitated by co-culture/co-implantation with TIG-3 cells, and that this process was at least in part dependent on TGF-β-mediated interactions.

  7. HOX and TALE signatures specify human stromal stem cell populations from different sources.

    Science.gov (United States)

    Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

    2013-04-01

    Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.

  8. The Stromal Microenvironment Modulates Mitochondrial Oxidative Phosphorylation in Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Hima V. Vangapandu

    2017-10-01

    Full Text Available Peripheral blood chronic lymphocytic leukemia (CLL cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow–derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively. Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.

  9. Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection.

    Science.gov (United States)

    Real, Fernando; Sennepin, Alexis; Ganor, Yonatan; Schmitt, Alain; Bomsel, Morgane

    2018-05-08

    During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa. Copyright © 2018. Published by Elsevier Inc.

  10. Translating Research into Clinical Scale Manufacturing of Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Karen Bieback

    2010-01-01

    Full Text Available It sounds simple to obtain sufficient numbers of cells derived from fetal or adult human tissues, isolate and/or expand the stem cells, and then transplant an appropriate number of these cells into the patient at the correct location. However, translating basic research into routine therapies is a complex multistep process which necessitates product regulation. The challenge relates to managing the expected therapeutic benefits with the potential risks and to balance the fast move to clinical trials with time-consuming cautious risk assessment. This paper will focus on the definition of mesenchymal stromal cells (MSCs, and challenges and achievements in the manufacturing process enabling their use in clinical studies. It will allude to different cellular sources, special capacities of MSCs, but also to current regulations, with a special focus on accessory material of human or animal origin, like media supplements. As cellular integrity and purity, formulation and lot release testing of the final product, validation of all procedures, and quality assurance are of utmost necessity, these topics will be addressed.

  11. Gold nanoparticles cellular toxicity and recovery: adipose Derived Stromal cells.

    Science.gov (United States)

    Mironava, Tatsiana; Hadjiargyrou, Michael; Simon, Marcia; Rafailovich, Miriam H

    2014-03-01

    Gold nanoparticles (AuNPs) are currently used in numerous medical applications. Herein, we describe their in vitro impact on human adipose-derived stromal cells (ADSCs) using 13 nm and 45 nm citrate-coated AuNPs. In their non-differentiated state, ADSCs were penetrated by the AuNPs and stored in vacuoles. The presence of the AuNPs in ADSCs resulted in increased population doubling times, decreased cell motility and cell-mediated collagen contraction. The degree to which the cells were impacted was a function of particle concentration, where the smaller particles required a sevenfold higher concentration to have the same effect as the larger ones. Furthermore, AuNPs reduced adipogenesis as measured by lipid droplet accumulation and adiponectin secretion. These effects correlated with transient increases in DLK1 and with relative reductions in fibronectin. Upon removal of exogenous AuNPs, cellular NP levels decreased and normal ADSC functions were restored. As adiponectin helps regulate energy metabolism, local fluctuations triggered by AuNPs can lead to systemic changes. Hence, careful choice of size, concentration and clinical application duration of AuNPs is warranted.

  12. Mesenchymal Stromal Cell Therapy for Pancreatitis: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Sara M. Ahmed

    2018-01-01

    Full Text Available Background. Based on animal studies, adult mesenchymal stromal cells (MSCs are promising for the treatment of pancreatitis. However, the best type of this form of cell therapy and its mechanism of action remain unclear. Methods. We searched the PubMed, Web of Science, Scopus, Google Scholar, and Clinical Trials.gov websites for studies using MSCs as a therapy for both acute and chronic pancreatitis published until September 2017. Results. We identified 276 publications; of these publications, 18 met our inclusion criteria. In animal studies, stem cell therapy was applied more frequently for acute pancreatitis than for chronic pancreatitis. No clinical trials were identified. MSC therapy ameliorated pancreatic inflammation in acute pancreatitis and pancreatic fibrosis in chronic pancreatitis. Bone marrow and umbilical cord MSCs were the most frequently administered cell types. Due to the substantial heterogeneity among the studies regarding the type, source, and dose of MSCs used, conducting a meta-analysis was not feasible to determine the best type of MSCs. Conclusion. The available data were insufficient for determining the best type of MSCs for the treatment of acute or chronic pancreatitis; therefore, clinical trials investigating the use of MSCs as therapy for pancreatitis are not warranted.

  13. Stromal cell migration precedes hemopoietic repopulation of the bone marrow after irradiation

    International Nuclear Information System (INIS)

    Werts, E.D.; Gibson, D.P.; Knapp, S.A.; DeGowin, R.L.

    1980-01-01

    Circulation of hemopoietic stem cells into an irradiated site has been thoroughly documented, but migration of stromal cells to repair radiation damage has not. We determined the radiosensitivity of mouse bone marrow stroma and evaluated stromal and hemopoietic repopulation in x-irradiated marrow. The D 0 for growth of colonies of marrow stromal cells (MSC) was 215 to 230 rad. Total-body irradiation (TB) obliterated marrow stromal and hemopoietic cells within 3 days. In contrast, 1 day after 1000 rad leg irradiation (LI), MSC rose to 80% of normal, but fell to 34% by 3 days and recovered to 72% by 30 days. However, femoral nucleated cells diminished to 20% by 3 days and recovered to 74% of normal by 30 days. Likewise, differentiated marrow cells and hemopoietic stem cells were initially depleted. With 1000 rad LI followed 3 h later by 1000 rad to the body while shielding the leg, MSC and femoral nucleated cells recovered to values intermediate between 1000 rad TB and 1000 rad LI. We concluded that: (1) the D 0 for MSC was 215 to 230 rad, (2) stromal repopulation preceded hemopoietic recovery, and (3) immigration of stromal cells from an unirradiated sanctuary facilitated hemopoietic repopulation of a heavily irradiated site

  14. Activation of NK Cells in Mixed Cultures of Wharton's Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes.

    Science.gov (United States)

    Svirshchevskaya, E V; Poltavtsev, A M; Os'mak, G Zh; Poltavtseva, R A

    2018-01-01

    Mesenchymal stromal cells possess immunosuppressive properties that might be used for the therapy of inflammatory diseases of various geneses. The effects of mesenchymal stromal cells depend on their lifetime in the recipient tissues. During heterologous transplantation, mesenchymal stromal cells are eliminated by NK cells. We studied NK cell formation in mixed cultures of Wharton's jelly mesenchymal stromal cells and peripheral blood lymphocytes from an autologous donor. Lymphocytes were activated by a mitogen or IL-2. The lifetime of mesenchymal stromal cells was estimated by MTT test. Cytotoxic activity and phenotype of NK cells were evaluated by flow cytometry. It was found that activation of NK cells depended on IL-2 and was registered on day 2 of incubation with IL-2. In cultures with mitogen-activated lymphocytes, cytotoxicity was observed after 5-6 days. Cytotoxicity of NK correlated with significant decrease in CD16+ and increase in CD56+ NK and with reduction of mesenchymal stromal cell viability. Thus, the main mechanism of elimination of mesenchymal stromal cells is cytotoxicity of NK cells that depended on IL-2 production.

  15. CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

    Directory of Open Access Journals (Sweden)

    Yu. В. Basok

    2018-01-01

    Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

  16. Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

    International Nuclear Information System (INIS)

    Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

    2003-01-01

    Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

  17. Fibroadenoma With Pleomorphic Stromal Giant Cells: It's Not as Bad as It Looks!

    Science.gov (United States)

    Wawire, Jonathan; Singh, Kamaljeet; Steinhoff, Margaret M

    2017-08-01

    Clinically relevant histological categorization of fibroepithelial lesions can be a daunting task, especially in a core needle biopsy. Assessment of stromal nuclear atypia, including nuclear pleomorphism and mitotic activity, is a key morphological feature employed to classify fibroepithelial lesions. We describe a case of fibroadenoma with markedly atypical nuclear features in the stromal cells that led to misclassification as phyllodes tumor in the core needle biopsy. Excision showed a fibroadenoma containing pleomorphic stromal giant cells, with occasional mitotic figures, including atypical forms. Aforementioned nuclear findings in a fibroepithelial lesion raise a legitimate question of phyllodes tumor. Knowledge of this pitfall may help avoid overtreatment of an otherwise benign fibroepithelial lesion.

  18. Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis.

    Science.gov (United States)

    Hirota, Keiji; Hashimoto, Motomu; Ito, Yoshinaga; Matsuura, Mayumi; Ito, Hiromu; Tanaka, Masao; Watanabe, Hitomi; Kondoh, Gen; Tanaka, Atsushi; Yasuda, Keiko; Kopf, Manfred; Potocnik, Alexandre J; Stockinger, Brigitta; Sakaguchi, Noriko; Sakaguchi, Shimon

    2018-06-19

    Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis

    NARCIS (Netherlands)

    Hähnlein, Janine S.; Nadafi, Reza; de Jong, Tineke; Ramwadhdoebe, Tamara H.; Semmelink, Johanna F.; Maijer, Karen I.; Zijlstra, Ijsbrand A.; Maas, Mario; Gerlag, Danielle M.; Geijtenbeek, Teunis B. H.; Tak, Paul P.; Mebius, Reina E.; van Baarsen, Lisa G. M.

    2018-01-01

    Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of

  20. Expanded cryopreserved mesenchymal stromal cells as an optimal source for graft-versus-host disease treatment

    Czech Academy of Sciences Publication Activity Database

    Holubová, M.; Lysák, D.; Vlas, T.; Vannucci, Luca; Jindra, P.

    2014-01-01

    Roč. 42, č. 3 (2014), s. 139-144 ISSN 1045-1056 Institutional support: RVO:61388971 Keywords : Mesenchymal stromal cells * Cryopreservation * Immunomodulation Subject RIV: EC - Immunology Impact factor: 1.209, year: 2014

  1. Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators

    DEFF Research Database (Denmark)

    Hess, AP; Hamilton, AE; Talbi, S

    2007-01-01

    During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important...... a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and were further treated with conditioned media (CM) from human trophoblasts (TCM) or, as a control, with conditioned media (CCM) from non-decidualized stromal cells...... regulated groups. The data demonstrate a significant induction of pro-inflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune...

  2. Reducing macrophages to improve bone marrow stromal cell survival in the contused spinal cord.

    NARCIS (Netherlands)

    Ritfeld, G.J.; Nandoe Tewarie, R.D.S.; Rahiem, S.T.; Hurtado, A.; Roos, R.A.; Grotenhuis, A.; Oudega, M.

    2010-01-01

    We tested whether reducing macrophage infiltration would improve the survival of allogeneic bone marrow stromal cells (BMSC) transplanted in the contused adult rat thoracic spinal cord. Treatment with cyclosporine, minocycline, or methylprednisolone all resulted in a significant decrease in

  3. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-05-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

  4. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

    Science.gov (United States)

    Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

    2018-05-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate

    NARCIS (Netherlands)

    Leijten, Jeroen Christianus Hermanus; Georgi, Nicole; Moreira Teixeira, Liliana; van Blitterswijk, Clemens; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2014-01-01

    Actively steering the chondrogenic differentiation of mesenchymal stromal cells (MSCs) into either permanent cartilage or hypertrophic cartilage destined to be replaced by bone has not yet been possible. During limb development, the developing long bone is exposed to a concentration gradient of

  6. Isolation and differentiation of stromal vascular cells to beige/brite cells

    DEFF Research Database (Denmark)

    Aune, Ulrike Liisberg; Ruiz, Lauren; Kajimura, Shingo

    2013-01-01

    cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction...

  7. Controlled Inhibition of the Mesenchymal Stromal Cell Pro-inflammatory Secretome via Microparticle Engineering

    Directory of Open Access Journals (Sweden)

    Sudhir H. Ranganath

    2016-06-01

    Full Text Available Mesenchymal stromal cells (MSCs are promising therapeutic candidates given their potent immunomodulatory and anti-inflammatory secretome. However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. To address this, we used a microparticle-based engineering approach to non-genetically modulate pro-inflammatory pathways in human MSCs (hMSCs under simulated inflammatory conditions. Here we show that microparticles loaded with TPCA-1, a small-molecule NF-κB inhibitor, when delivered to hMSCs can attenuate secretion of pro-inflammatory factors for at least 6 days in vitro. Conditioned medium (CM derived from TPCA-1-loaded hMSCs also showed reduced ability to attract human monocytes and prevented differentiation of human cardiac fibroblasts to myofibroblasts, compared with CM from untreated or TPCA-1-preconditioned hMSCs. Thus, we provide a broadly applicable bioengineering solution to facilitate intracellular sustained release of agents that modulate signaling. We propose that this approach could be harnessed to improve control over MSC secretome post-transplantation, especially to prevent adverse remodeling post-myocardial infarction.

  8. Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells.

    Czech Academy of Sciences Publication Activity Database

    Forostyak, Oksana; Butenko, Olena; Anděrová, Miroslava; Forostyak, Serhiy; Syková, Eva; Verkhratsky, A.; Dayanithi, Govindan

    2016-01-01

    Roč. 16, č. 3 (2016), s. 622-634 ISSN 1873-5061 R&D Projects: GA ČR(CZ) GA14-34077S; GA ČR(CZ) GAP304/11/2373; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : adipose derived stromal cells * bone marrow stromal cell * Ca(2+) signaling * Ion channels Subject RIV: FH - Neurology Impact factor: 3.494, year: 2016

  9. The clinical application of mesenchymal stromal cells in hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Ke Zhao

    2016-05-01

    Full Text Available Abstract Mesenchymal stromal cells (MSCs are multipotent stem cells well known for repairing tissue, supporting hematopoiesis, and modulating immune and inflammation response. These outstanding properties make MSCs as an attractive candidate for cellular therapy in immune-based disorders, especially hematopoietic stem cell transplantation (HSCT. In this review, we outline the progress of MSCs in preventing and treating engraftment failure (EF, graft-versus-host disease (GVHD following HSCT and critically discuss unsolved issues in clinical applications.

  10. Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment.

    Science.gov (United States)

    Salvatore, Viviana; Focaroli, Stefano; Teti, Gabriella; Mazzotti, Antonio; Falconi, Mirella

    2015-10-06

    The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. These findings demonstrated that the tumor microenvironment has an influence on the

  11. Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

    Directory of Open Access Journals (Sweden)

    Aroem Naruni

    2016-01-01

    Full Text Available AbstrakLatar belakang: Lingkungan mikro yaitu sel stromal dam matriks ekstraseluler saat ini dinyatakansebagai kontributor dalam perkembangan tumor. Beberapa penelitian telah mengembangkan matriksekstraseluler yang mendukung perkembangan sel in vitro. Matriks ekstraseluler adalah suatu komplekssusunan supramolekuler dari berbagai macam glycoprotein dan proteoglycan. Matriks ekstraselulermenyediakan integritas jaringan, bertindak sebagai scaffold alami tempat sel melekat dan berinteraksiserta berperan sebagai reservoir pertumbuhan sel. Penelitian ini bertujuan untuk mendapatkan deposisidan deselularisasi yang optimal pada matriks ekstraseluler.Metode: Dalam penelitian ini, kami mengembangkan cells crowder untuk meningkatkan deposit matriksekstraseluler dari kultur sel primer fibroblast payudara yang diperoleh dari spesimen hasil operasimammoplasty. Dextran 500 kDa ditambahkan dalam media kultur DMEM lengkap yang telah ditambahkan0.5% FBS dan 100μM L-ascorbic acid 2-phosphate. Setelah tujuh hari, sel dilisis dengan menggunakanSodium Deoxycolate (DOC.Hasil: Deposisi matriks ekstraseluler dan proses deselulerisasi dari sel primer fibroblas payudara dapatterdeteksi dengan menggunakan antibodi Rabbit anti human fibronectin yang selanjutnya ditambahkandengan anti rabbit IgG yang telah dikonjugasi dengan Alexa Fluor 488.Kesimpulan: Penambahan dextran sulfat dan prosesing lysis dengan sodium deoxycolate dapatmeningkatkan deposisi dan menghasilkan deselularisasi matriks ekstraseluler. (Health Science Journalof Indonesia 2015;6:43-7Kata kunci: matriks ekstra selular, kanker mammae, stem cell, sel fibroblast AbstractBackground: The microenvironment including stromal cells and extracellular matrix (ECM is now consideredan active contributor to tumor progression. Certain studies have developed ECM which supports a suitable cellulargrowth in vitro. The ECM is a complex supramolecular assembly of a variety of glycoproteins and proteoglycans

  12. Isolation, culture and intraportal transplantation of rat marrow stromal cell

    International Nuclear Information System (INIS)

    Wang Ping; Wang Jianhua; Yan Zhiping; Li Wentao; Lin Genlai; Hu Meiyu; Wang Yanhong

    2004-01-01

    Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 10 5 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

  13. Ultrastructural and radiobiological characterization of stromal cells in continuous, long-term marrow culture

    International Nuclear Information System (INIS)

    Tavassoli, M.

    1982-01-01

    Hemopoietic stromal cells were studied in continuous, long-term marrow culture. A correlative study was carried out involving cytochemistry as well as scanning (SEM), and transmission electron microscopy (TEM) with sections cut either perpendicular or parallel to the substratum. Only two stromal cell types were identified: epithelioid cells and macrophages. The appearance of these cells, however, varied according to their topography in the culture and the method of observation; a finding that may explain the multiplicity of the cell types reported in these cultures. The two cell types displayed considerable interconnections and interactions which may be essential in their support function for the proliferation and maintenance of hemopoietic stem cells. They also demonstrated numerous coated pits and vesicles suggestive of extensive receptor-mediated endocytosis. Stromal cells, generally thought to be relatively radioresistant, demonstrated hitherto unrecognized radiosensitivity in culture. Doses of radiation as low as 500 rads interfered with their support function for the maintenance of the hemopoietic stem cell

  14. Mesenchymal stromal cell therapy in COPD: from bench to bedside

    Directory of Open Access Journals (Sweden)

    Antunes MA

    2017-10-01

    Full Text Available Mariana A Antunes,1,2 José Roberto Lapa e Silva,3 Patricia RM Rocco1,2 1Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro (UFRJ, RJ, Brazil; 2National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, RJ, Brazil; 3Institute of Thoracic Medicine, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (UFRJ, Rio de Janeiro, RJ, Brazil Abstract: COPD is the most frequent chronic respiratory disease and a leading cause of morbidity and mortality. The major risk factor for COPD development is cigarette smoke, and the most efficient treatment for COPD is smoking cessation. However, even after smoking cessation, inflammation, apoptosis, and oxidative stress may persist and continue contributing to disease progression. Although current therapies for COPD (primarily based on anti-inflammatory agents contribute to the reduction of airway obstruction and minimize COPD exacerbations, none can avoid disease progression or reduce mortality. Within this context, recent advances in mesenchymal stromal cell (MSC therapy have made this approach a strong candidate for clinical use in the treatment of several pulmonary diseases. MSCs can be readily harvested from diverse tissues and expanded with high efficiency, and have strong immunosuppressive properties. Preclinical studies have demonstrated encouraging outcomes of MSCs therapy for lung disorders, including emphysema. These findings instigated research groups to assess the impact of MSCs in human COPD/emphysema, but clinical results have fallen short of expectations. However, MSCs have demonstrated a good adjuvant role in the clinical scenario. Trials that used MSCs combined with another, primary treatment (eg, endobronchial valves found that patients derived greater benefit in pulmonary function tests and/or quality of life reports, as well as reductions in systemic

  15. Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

    Science.gov (United States)

    Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

    2017-07-01

    Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

  16. Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

    Science.gov (United States)

    Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

    2018-04-01

    Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  17. The cannabinoid receptor type 2 as mediator of mesenchymal stromal cell immunosuppressive properties.

    Directory of Open Access Journals (Sweden)

    Francesca Rossi

    Full Text Available Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians. Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes. In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells. We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.

  18. Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration

    Science.gov (United States)

    Dezawa, Mari; Ishikawa, Hiroto; Itokazu, Yutaka; Yoshihara, Tomoyuki; Hoshino, Mikio; Takeda, Shin-ichi; Ide, Chizuka; Nabeshima, Yo-ichi

    2005-07-01

    Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.

  19. In vitro cytotoxicity of chemical preservatives on human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Daniel Gonsales Spindola

    2018-05-01

    Full Text Available ABSTRACT Preservatives are widely used substances that are commonly added to various cosmetic and pharmaceutical products to prevent or inhibit microbial growth. In this study, we compared the in vitro cytotoxicity of different types of currently used preservatives, including methylparaben, imidazolidinyl urea (IMU, and sodium benzoate, using the human newborn fibroblast cell line CCD1072Sk. Of the tested preservatives, only IMU induced a reduction in cell viability, as shown using the MTT assay and propidium iodide staining (IMU>methylparaben>sodium benzoate. IMU was shown to promote homeostatic alterations potentially related to the initiation of programed cell death, such as decreased mitochondrial membrane potential and caspase-3 activation, in the treated cells. Methylparaben and sodium benzoate were shown to have a very low cytotoxic activity. Taken together, our results suggest that IMU induces programed cell death in human fibroblasts by a canonical intrinsic pathway via mitochondrial perturbation and subsequent release of proapoptotic factors.

  20. Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro.

    Science.gov (United States)

    Ying, Xiaozhou; Zhang, Wei; Cheng, Shaowen; Nie, Pengfei; Cheng, Xiaojie; Shen, Yue; Wang, Wei; Xue, Enxing; Chen, Qingyu; Kou, Dongquan; Peng, Lei; Zhang, Yu; Lu, Chuanzhu

    2012-11-01

    Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5) M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5) M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6) M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P < 0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

  1. Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.

    Science.gov (United States)

    Centola, Matteo; Tonnarelli, Beatrice; Schären, Stefan; Glaser, Nicolas; Barbero, Andrea; Martin, Ivan

    2013-11-01

    The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

  2. Oncologic trogocytosis of an original stromal cells induces chemoresistance of ovarian tumours.

    Directory of Open Access Journals (Sweden)

    Arash Rafii

    Full Text Available The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours.We isolated an original type of stromal cells, referred to as "Hospicells" from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance.This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient's tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.

  3. Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia 1

    OpenAIRE

    Pitarresi, Jason R.; Liu, Xin; Sharma, Sudarshana M.; Cuiti?o, Maria C.; Kladney, Raleigh D.; Mace, Thomas A.; Donohue, Sydney; Nayak, Sunayana G.; Qu, Chunjing; Lee, James; Woelke, Sarah A.; Trela, Stefan; LaPak, Kyle; Yu, Lianbo; McElroy, Joseph

    2016-01-01

    Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin?positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue...

  4. A lactate shuttle system between tumour and stromal cells is associated with poor prognosis in prostate cancer

    International Nuclear Information System (INIS)

    Pértega-Gomes, Nelma; Vizcaíno, José R; Attig, Jan; Jurmeister, Sarah; Lopes, Carlos; Baltazar, Fátima

    2014-01-01

    In a malignant tumour, cancer cells are embedded in stromal cells, namely cancer-associated fibroblasts (CAFs). These CAFs are now accepted as important players in cancer dynamics, being involved in tumour growth and progression. Although there are various reports on the interaction between tumour and stromal cells, the clinico-pathological significance of this cross-talk is still largely unknown. In this study, we aimed to characterise the expression of key metabolic proteins involved in glucose transport, pyruvate/lactate shuttle system, glycolytic metabolism and fatty acid oxidation in CAFs and tumour cells in different stages of malignant transformation. We further aimed to contextualise the clinico-pathological significance of these protein expression profiles with reference to known prognostic indicators, including biochemical recurrence in pT stage. Prostate tissues were obtained from 480 patients with a median age of 64 years following radical prostatectomy with no previous hormonal therapy. Tissues were analysed for the expression of several key metabolism-related proteins in glands and surrounding fibroblasts by immunohistochemistry. Reliable markers of prognosis such as pT stage and biochemical recurrence were assessed for each case. We observed that prostate cancer cells did not rely mainly on glycolytic metabolism, while there was a high expression of MCT4 and CAIX - in CAFs. This corroborates the hypothesis of the “Reverse Warburg effect” in prostate cancer, in which fibroblasts are under oxidative stress and express CAIX, an established hypoxia marker. We found that alterations in the expression of metabolism-related proteins were already evident in the early stages of malignant transformation, suggesting the continuing alteration of CAFs from an early stage. Additionally, and for the first time, we show that cases showing high MCT4 expression in CAFs with concomitant strong MCT1 expression in prostate cancer (PCa) cells are associated with poor

  5. Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM

    International Nuclear Information System (INIS)

    Knospe, W.H.; Husseini, S.G.

    1986-01-01

    Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow

  6. A relativity concept in mesenchymal stromal cell manufacturing.

    Science.gov (United States)

    Martin, Ivan; De Boer, Jan; Sensebe, Luc

    2016-05-01

    Mesenchymal stromal cells (MSCs) are being experimentally tested in several biological systems and clinical settings with the aim of verifying possible therapeutic effects for a variety of indications. MSCs are also known to be heterogeneous populations, with phenotypic and functional features that depend heavily on the individual donor, the harvest site, and the culture conditions. In the context of this multidimensional complexity, a recurrent question is whether it is feasible to produce MSC batches as "standard" therapeutics, possibly within scalable manufacturing systems. Here, we provide a short overview of the literature on different culture methods for MSCs, including those employing innovative technologies, and of some typically assessed functional features (e.g., growth, senescence, genomic stability, clonogenicity, etc.). We then offer our perspective of a roadmap on how to identify and refine manufacturing systems for MSCs intended for specific clinical indications. We submit that the vision of producing MSCs according to a unique standard, although commercially attractive, cannot yet be scientifically substantiated. Instead, efforts should be concentrated on standardizing methods for characterization of MSCs generated by different groups, possibly covering a vast gamut of functionalities. Such assessments, combined with hypotheses on the therapeutic mode of action and associated clinical data, should ultimately allow definition of in-process controls and measurable release criteria for MSC manufacturing. These will have to be validated as predictive of potency in suitable pre-clinical models and of therapeutic efficacy in patients. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Responses of fibroblasts and glial cells to nanostructured platinum surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

    2009-09-23

    The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

  8. Pulmonary stromal cells induce the generation of regulatory DC attenuating T-cell-mediated lung inflammation.

    Science.gov (United States)

    Li, Qian; Guo, Zhenhong; Xu, Xiongfei; Xia, Sheng; Cao, Xuetao

    2008-10-01

    The tissue microenvironment may affect the development and function of immune cells such as DC. Whether and how the pulmonary stromal microenvironment can affect the development and function of lung DC need to be investigated. Regulatory DC (DCreg) can regulate T-cell response. We wondered whether such regulatory DC exist in the lung and what is the effect of the pulmonary stromal microenvironment on the generation of DCreg. Here we demonstrate that murine pulmonary stromal cells can drive immature DC, which are regarded as being widely distributed in the lung, to proliferate and differentiate into a distinct subset of DCreg, which express high levels of CD11b but low levels of MHC class II (I-A), CD11c, secrete high amounts of IL-10, NO and prostaglandin E2 (PGE2) and suppress T-cell proliferation. The natural counterpart of DCreg in the lung with similar phenotype and regulatory function has been identified. Pulmonary stroma-derived TGF-beta is responsible for the differentiation of immature DC to DCreg, and DCreg-derived PGE2 contributes to their suppression of T-cell proliferation. Moreover, DCreg can induce the generation of CD4+CD25+Foxp3+ Treg. Importantly, infusion with DCreg attenuates T-cell-mediated eosinophilic airway inflammation in vivo. Therefore, the pulmonary microenvironment may drive the generation of DCreg, thus contributing to the maintenance of immune homoeostasis and the control of inflammation in the lung.

  9. Analysis of primary cilia in directional cell migration in fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht

    2013-01-01

    summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration....... In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We...

  10. Stem and stromal cell reconstitution of lethally irradiated mice following transplantation of hematopoietic tissue from donors of various ages

    International Nuclear Information System (INIS)

    Schmidt, C.M.; Doran, G.A.; Crouse, D.A.; Sharp, J.G.

    1987-01-01

    If the limited life span of hematopoietic tissues in vitro is due to a finite proliferative capacity of individual stem cells, one might expect tissues of young donors to possess a greater proliferative capacity and to contain a larger population of primitive stem cells than those of older donors. To test this hypothesis, we used 12- and 8-day spleen colony formation (CFU-s) to assay more and less primitive stem cell subpopulations of three murine hematopoietic tissues: fetal liver (FL) and weanling (WBM) and adult (ABM) bone marrow. Subsequently, the same assays and a stromal cell assay were performed on the bone marrow from groups of lethally irradiated mice reconstituted with these tissues. Comparison of the CFU-s content of the donor tissues revealed that FL contained a significantly greater proportion of primitive stem cells as evidenced by a (Day 12):(Day 8) CFU-s ratio of 3.0 +/- 1.0 as compared to 0.9 +/- 0.1 for WBM and ABM. In addition, at 21 weeks post-transplantation the CFU-s/femur values of the FL reconstituted group were significantly greater than those of the ABM and WBM reconstituted groups. These results suggest that fetal hematopoietic tissue contains a greater proportion of primitive stem cells and has a greater proliferative potential than hematopoietic tissue from older donors. No differences were seen in stromal cell reconstitution of the three experimental groups. In all cases, assayable fibroblast colony forming cells (CFU-f) remained at 20-40% of control values, even at 21 weeks postreconstitution

  11. Regulatory perspective on in vitro potency assays for human mesenchymal stromal cells used in immunotherapy

    NARCIS (Netherlands)

    de Wolf, Charlotte; van de Bovenkamp, Marja; Hoefnagel, Marcel C

    Mesenchymal stromal cells (MSCs) are multipotent cells derived from various tissues that can differentiate into several cell types. MSCs are able to modulate the response of immune cells of the innate and adaptive immune system. Because of these multimodal properties, the potential use of MSCs for

  12. Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    M Mumme

    2012-09-01

    Full Text Available Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC, namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL enhanced colony-forming units-fibroblastic (CFU-f and -osteoblastic (CFU-o number (up to 1.5-fold and size (1.2-fold in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas, resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

  13. Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells

    Directory of Open Access Journals (Sweden)

    Peng CW

    2017-12-01

    Full Text Available Chunwei Peng,1 Jiuyang Liu,1 Guifang Yang,2 Yan Li3 1Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center, 2Department of Pathology, Zhongnan Hospital of Wuhan University, Wuchang District, Wuhan, 3Department of Peritoneal Cancer Surgery, Cancer Center of Beijing Shijitan Hospital Affiliated to the Capital Medical University, Yangfangdian, Beijing, People’s Republic of China Purpose: Semiconductor quantum dots (QDs are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methods: A QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs including 184 gastric cancer (GC tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX. The stromal status including infiltrating of immune cells (high density of macrophages, angiogenesis (high density of microvessel density [MVD], low neovessel maturation and extracellular matrix (ECM remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9] was evaluated.Results: This study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe

  14. Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie A Wallace

    Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

  15. Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

    Science.gov (United States)

    Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

    2018-02-15

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

  16. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Science.gov (United States)

    Rosu-Myles, Michael; She, Yi-Min; Fair, Joel; Muradia, Gauri; Mehic, Jelica; Menendez, Pablo; Prasad, Shiv S; Cyr, Terry D

    2012-01-01

    Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  17. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  18. Inflammatory response of a prostate stromal cell line induced by Trichomonas vaginalis.

    Science.gov (United States)

    Im, S J; Han, I H; Kim, J H; Gu, N Y; Seo, M Y; Chung, Y H; Ryu, J S

    2016-04-01

    While Trichomonas vaginalis, a cause of sexually transmitted infection, is known as a surface-dwelling protozoa, trichomonads have been detected in prostatic tissue from benign prostatic hyperplasia and prostatitis by immunoperoxidase assay or PCR. However, the immune response of prostate stromal cells infected with T. vaginalis has not been investigated. Our objective was to investigate whether T. vaginalis could induce an inflammatory response in prostate stromal cells. Incubation of a human prostate stromal myofibroblast cells (WPMY-1) with live T. vaginalis T016 increased expression of the inflammatory chemokines CXCL8 and CCL2. In addition, TLR4, ROS, MAPK and NF-κB expression increased, while inhibitors of TLR4, ROS, MAPKs and NF-κB reduced CXCL8 and CCL2 production. Medium conditioned by incubation of WPMY-1 cells with T. vaginalis stimulated the migration of human neutrophils and monocytes (THP-1 cells). We conclude that T. vaginalis increases CXCL8 and CCL2 production by human prostate stromal cells by activating TLR4, ROS, MAPKs and NF-κB, and this in turn attracts neutrophils and monocytes and leads to an inflammatory response. This study is the first attempt to demonstrate an inflammatory reaction in prostate stromal cells caused by T. vaginalis. © 2016 John Wiley & Sons Ltd.

  19. Changes in compartments of hemospoietic and stromal marrow progenitor cells after continuous low dose gamma-irradiation

    Science.gov (United States)

    Domaratskaya, E.; Starostin, V.

    The low dose continuous gamma-irradiation chosen corresponded with that affected the organisms onboard a spacecraft (Mitrikas, Tsetlin, 2000). F1 (CBAxC57Bl/6) male and female mice were used at 3 4 months of age. Experimental mice were- irradiated during 10 days to a total dose of 15 mGy (Co60 gamma-sources, mean dose rate of 1.5-2.0 mGy/day). Another group of intact mice served as control. Younger and advanced hemopoietic progenitors measured at day 11 (i.e. CFU -S-11) and day 7 (i.e. CFU-S-7), respectively, after transplantation of test donor cells were assayed by the method of Till and McCulloch (1961). Stromal changes were evaluated by estimation of in vitro fibroblastic colony-forming units (CFU -F ) content and by the ability of ectopically grafted (under renal capsule) stroma to regenerate the new bone marrow organ. CFU-S-11 number increased of 40% as compared with control and almost 2-fold higher than that of CFU-S-7. The CFU-F content increased almost of 3-fold. Size of ectopic marrow transplants was estimated at day 70 following grafting by counting myelokariocyte and CFU -S number that repopulated the newly formed bone marrow organ. It was found more than 2-fold increase of myelokariocytes in transplants produced by marrow stroma of irradiated donors. CFU -S contents in transplants increased strikingly in comparison to control level. CFU-S-7 and CFU-S-11 increased of 7.5- and of 3.7-fold, respectively, i.e. the rate of advanced CFU - S predominated. It should be noted a good correlation between number of stromal progenitor cells (CFU-F) and ectopic transplant sizes evaluated as myelokaryocyte counts when irradiated donors used. In the same time, if sizes of transplants was measured as CFU-S-7 and CFU - S-11 numbers, their increases were more pronounced. Therefore, continuous low dose gamma- irradiation augments significantly both hemopoietic and stromal progenitor cell number in bone marrow. Additionally, the ratio of distinct CFU -S subpopulations

  20. Intrathymic lymphopoiesis: stromal cell-associated proliferation of T cells is independent of lymphocyte genotype.

    Science.gov (United States)

    Kyewski, B A; Travis, M; Kaplan, H S

    1984-09-01

    We analyzed the genetic restriction of direct cell-cell interactions between thymocytes and a) cortical epithelial cells, b) macrophages, and c) medullary dendritic cells in the mouse thymus. Thymectomized (C3H X C57BL/Ka)F1 hybrid mice were doubly grafted with P1 and P2 neonatal thymus grafts, were lethally irradiated, and were reconstituted with a mixture of P1 and P2 bone marrow cells which differed in the Thy-1 locus. The contributions of both parental inocula to the composition of the free and stromal cell-associated T cell compartments were analyzed separately in thymic grafts of each parental strain. The lymphoid composition in both compartments essentially reflected the peripheral T cell-chimerism in the host. The development of lymphostromal complexes was not restricted by the genotype of the partner cells. Statistical analysis of the distributions of P1 and P2 T cells among free thymocytes and within individual lymphostromal complexes, however, suggests that the T cells of an individual complex are the progeny of oligoclonal proliferation. Thus, both epithelial cells and bone marrow-derived stromal cells seem to be involved in different stages of intrathymic lymphopoiesis.

  1. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

    Science.gov (United States)

    Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

    2015-03-01

    A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  3. Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells: Are mesenchymal stromal cells involved in scar formation?

    NARCIS (Netherlands)

    Bogaerdt, van den A.J.; Veen, van der A.G.; Zuijlen, van P.P.; Reijnen, L.; Verkerk, M.; Bank, R.A.; Middelkoop, E.; Ulrich, M.

    2009-01-01

    In this work, different fibroblast-like (mesenchymal) cell populations that might be involved in wound healing were characterized and their involvement in scar formation was studied by determining collagen synthesis and processing. Depending on the physical and mechanical properties of the tissues,

  4. Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells : Are mesenchymal stromal cells involved in scar formation?

    NARCIS (Netherlands)

    van den Bogaerdt, Antoon J.; van der Veen, Vincent C.; van Zuijlen, Paul P. M.; Reijnen, Linda; Verkerk, Michelle; Bank, Ruud A.; Middelkoop, Esther; Ulrich, Magda M. W.

    2009-01-01

    In this work, different fibroblast-like (mesenchymal) cell populations that might be involved in wound healing were characterized and their involvement in scar formation was studied by determining collagen synthesis and processing. Depending on the physical and mechanical properties of the tissues,

  5. Adipose-derived mesenchymal stromal cells prevented rat vocal fold scarring.

    Science.gov (United States)

    Morisaki, Tsuyoshi; Kishimoto, Yo; Tateya, Ichiro; Kawai, Yoshitaka; Suzuki, Ryo; Tsuji, Takuya; Hiwatashi, Nao; Nakamura, Tatsuo; Omori, Koichi; Kitano, Hiroya; Takeuchi, Hiromi; Hirano, Shigeru

    2018-01-01

    This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation. Prospective animal experiments with controls. ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection. Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14. ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring. NA. Laryngoscope, 128:E33-E40, 2018. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  6. Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

    Science.gov (United States)

    Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

    2018-03-01

    Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    Directory of Open Access Journals (Sweden)

    Hongzhe Li

    2014-12-01

    Full Text Available Human bone marrow (BM contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs, which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show that low/negative expression of CD140a (PDGFR-α on lin−/CD45−/CD271+ BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin−/CD45−/CD271+/CD140alow/− cells effectively mediated the ex vivo expansion of transplantable CD34+ hematopoietic stem cells. Taken together, these data indicate that CD140a is a key negative selection marker for adult human BM-MSCs, which enables to prospectively isolate a close to pure population of candidate human adult stroma stem/progenitor cells with potent hematopoiesis-supporting capacity.

  8. Osteogenic Differentiation of Mesenchymal Stromal Cells: A Comparative Analysis Between Human Subcutaneous Adipose Tissue and Dental Pulp.

    Science.gov (United States)

    D'Alimonte, Iolanda; Mastrangelo, Filiberto; Giuliani, Patricia; Pierdomenico, Laura; Marchisio, Marco; Zuccarini, Mariachiara; Di Iorio, Patrizia; Quaresima, Raimondo; Caciagli, Francesco; Ciccarelli, Renata

    2017-06-01

    White adipose tissue is a source of mesenchymal stromal/stem cells (MSCs) that are actively studied for their possible therapeutic use in bone tissue repair/remodeling. To better appreciate the osteogenic potential of these cells, we compared some properties of MSCs from human subcutaneous adipose tissue [subcutaneous-adipose stromal cells (S-ASCs)] and dental pulp stem cell (DPSCs) of third-impacted molars, the latter representing a well-established MSC source. Both undifferentiated cell types showed similar fibroblast-like morphology and mesenchymal marker expression. However, undifferentiated S-ASCs displayed a faster doubling time coupled to greater proliferation and colony-forming ability than DPSCs. Also, the osteogenic differentiation of S-ASCs was greater than that of DPSCs, as evaluated by the higher levels of expression of early osteogenic markers Runt-related transcription factor-2 (RUNX2) and alkaline phosphatase at days 3-14 and of extracellular matrix mineralization at days 14-21. Moreover, S-ASCs showed a better colonization of the titanium scaffold. In addition, we investigated whether S-ASC osteogenic commitment was enhanced by adenosine A1 receptor (A1R) stimulation, as previously shown for DPSCs. Although A1R expression was constant during DPSC differentiation, it increased in S-ASC at day 21 from osteogenesis induction. Accordingly, A1R stimulation by the agonist 2-chloro-N 6 -cyclopentyl-adenosine, added to the cultures at each medium change, stimulated proliferation only in differentiating DPSC and enhanced the osteogenic differentiation earlier in DPSCs than in S-ASCs. These effects were counteracted by cell pretreatment with a selective A1R antagonist. Thus, our findings suggest that S-ASCs could be advantageously used in regenerative orthopedics/dentistry, and locally released or exogenously added purines may play a role in bone repair/remodeling, even though this aspect should be more thoroughly evaluated.

  9. The Origin of Human Mesenchymal Stromal Cells Dictates Their Reparative Properties

    DEFF Research Database (Denmark)

    Naftali-Shani, Nili; Itzhaki-Alfia, Ayelet; Landa-Rouben, Natalie

    2013-01-01

    Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair....

  10. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide an un...

  11. Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel

    DEFF Research Database (Denmark)

    Larsen, Bjarke Follin; Juhl, Morten; Cohen, Smadar

    2015-01-01

    BACKGROUND AIMS: Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to in...

  12. Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

    Science.gov (United States)

    Gawlitta, Debby; van Rijen, Mattie H P; Schrijver, Edmée J M; Alblas, Jacqueline; Dhert, Wouter J A

    2012-10-01

    Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well

  13. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    Science.gov (United States)

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

  14. Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis : a systematic review including our case series

    NARCIS (Netherlands)

    Azevedo Koike Folgueira, Maria Aparecida; Maistro, Simone; Hirata Katayama, Maria Lucia; Roela, Rosimeire Aparecida; Lopes Mundim, Fiorita Gonzales; Nanogaki, Suely; de Bock, Geertruida H.; Brentani, M. Mitzi

    2013-01-01

    CAFs (cancer-associated fibroblasts), the most abundant cell type in breast cancer stroma, produce a plethora of chemokines, growth factors and ECM (extracellular matrix) proteins, that may contribute to dissemination and metastasis. Axillary nodes are the first metastatic site in breast cancer;

  15. Induction of pluripotent stem cells from fibroblast cultures.

    Science.gov (United States)

    Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

    2007-01-01

    Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

  16. Pseudoangiomatous stromal hyperplasia with multinucleated stromal giant cells is neither exceptional in gynecomastia nor characteristic of neurofibromatosis type 1.

    Science.gov (United States)

    Pižem, Jože; Velikonja, Mojca; Matjašič, Alenka; Jerše, Maja; Glavač, Damjan

    2015-04-01

    Six cases of gynecomastia with pseudoangiomatous stromal hyperplasia (PASH) and multinucleated stromal giant cells (MSGC) associated with neurofibromatosis type 1 (NF1) have been reported, and finding MSGC within PASH in gynecomastia has been suggested as being a characteristic of NF1. The frequency of PASH with MSGC in gynecomastia and its specificity for NF1 have not, however, been systematically studied. A total of 337 gynecomastia specimens from 215 patients, aged from 8 to 78 years (median, 22 years) were reevaluated for the presence of PASH with MSGC. Breast tissue samples of 25 patients were analyzed for the presence of an NF1 gene mutation using next generation sequencing. Rare MSGC, usually in the background of PASH, were noted at least unilaterally in 27 (13 %) patients; and prominent MSGC, always in the background of PASH, were noted in 8 (4 %) patients. The NF1 gene was mutated in only 1 (an 8-year-old boy with known NF1 and prominent MSGC) of the 25 tested patients, including 6 patients with prominent MSGC and 19 patients with rare MSGC. MSGC, usually in the background of PASH, are not characteristic of NF1.

  17. Effects of ultraviolet irradiation on prostaglandin-E2 production by cultured corneal stromal cells

    International Nuclear Information System (INIS)

    Weinreb, R.N.; Yue, B.Y.J.T.; Peyman, G.A.

    1990-01-01

    The authors examined the effects of ultraviolet (UV) irradiation on the release of prostaglandin E 2 (PGE 2 ) by rabbit corneal stromal cells in culture. Considerable amounts of PGE 2 were present in the media of control corneal cultures following 1, 2, 4, 8 and 24 hr of incubation. Irradiation with UV-A (320-400 nm) for 30 min resulted in more than a 50% increase in PGE 2 release. Dexamethasone inhibited PGE 2 release by corneal stromal cells. It was, however, ineffective in protecting the cells from the UV-induced release of PGE 2 . (author)

  18. Biochemical changes to fibroblast cells subjected to ionizing radiation.

    Science.gov (United States)

    Jones, Pamala; Benghuzzi, Hamed; Tucci, Michelle; Richards, Latoya; Harrison, George; Patel, Ramesh

    2008-01-01

    High energy X-rays are capable of interacting with biological membranes to cause both functional and structural modifications. The goal of the present study was to investigate the effects human fibroblast cells exposed multiple times to 10 Gy over time. Following exposures of 2, 3, or 4 times to 10 Gy/10min the cells were evaluated for cell number changes, membrane damage, and intracellular glutathione content after 24, 48 and 72 hours. Twenty-four hours following exposure the cell numbers were reduced and increased levels of cellular membrane damage was evident. This trend was observed for the duration of the study. Interestingly, there was not an exposure dependent increase in cell damage or cell loss with time. Intracellular antioxidant systems were activated as indicated by anincrease in total cellular glutathione content. Additional studies are needed to determine if the cellular reduction is caused by a direct effect of the X-rays targeting the DNA or an indirect effect of the X-ray targeting the cellular membrane, which then generates radicals that target cell cycle checkpoints or DNA damage. In conclusion, fibroblast cells can be used to determine early and late events of cellular function following exposure to harmful levels of radiation exposure and results of exposure can be seen within twenty four hours.

  19. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    International Nuclear Information System (INIS)

    Nakajima, Hideaki; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-01-01

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix

  20. Changing the Properties of Multipotent Mesenchymal Stromal Cells by IFNγ Administration.

    Science.gov (United States)

    Petinati, N A; Kapranov, N M; Bigil'deev, A E; Popova, M D; Davydova, Yu O; Gal'tseva, I V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

    2017-06-01

    We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

  1. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    International Nuclear Information System (INIS)

    Perkins, S.; Fleischman, R.A.

    1988-01-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells

  2. Aging up-regulates ARA55 in stromal cells, inducing androgen-mediated prostate cancer cell proliferation and migration.

    Science.gov (United States)

    Zou, Qingsong; Cui, Di; Liang, Shengjie; Xia, Shujie; Jing, Yifeng; Han, Bangmin

    2016-06-01

    Stromal cells in the peripheral zone (PZ) of the prostate from older males (PZ-old) could significantly promote Prostate cancer (PCa) growth compared with stromal cells from young males (PZ-young). But the mechanism is still unknown. In the co-culture system with PZ-old cells, Pc3/Du145 cells showed advanced proliferation and migration after Dihydrotestosterone (DHT) incubation, but DHT didn't show the similar effect in PZ-young co-culture system. Also, higher androgen/AR signal pathway activity and AR-related cytokines secretion (FGF-2, KGF, IGF-1) were found in PZ-old cells. As AR exprssison was equivalent in PZ-old and PZ-young cells, we focused on Androgen receptor associated protein-55(ARA55), a stromal-specific androgen receptor (AR) coactivator. ARA55 expression was higher in PZ-old cells compared with PZ-young cells in vitro. After knocking down ARA55 expression in PZ-old cells, the PCa growth- promoting effect from the PZ-old cells was diminished, which may be explained by the decreased the progressive cytokines secretion (FGF-2, KGF, IGF-1) from PZ-old stromal cells. In vivo, the consistent results were also found: PZ-old cells promoted prostate cancer cells growth, but this effect receded when knocking down ARA55 expression in PZ-old cells. From our study, we found PZ stromal cells presented age-related effects in proliferation and migration of prostate cancer cells in the androgen/AR dependent manner. As aging increased, more ARA55 were expressed in PZ stromal cells, leading to more sensitive androgen/androgen receptor (AR) signal pathway, then constituting a more feasible environment to cancer cells.

  3. Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

    Czech Academy of Sciences Publication Activity Database

    Petrenko, Yuriy; Petrenko, A.; Martin, I.; Wendt, D.

    2017-01-01

    Roč. 76, jun. (2017), s. 150-153 ISSN 0011-2240 Institutional support: RVO:68378041 Keywords : cryopreservation * tissue engineering * mesenchymal stromal cells Subject RIV: FP - Other Medical Disciplines OBOR OECD: Cell biology Impact factor: 1.996, year: 2016

  4. Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood

    DEFF Research Database (Denmark)

    Berg, Lise Charlotte; Koch, Thomas Gadegaard; Heerkens, T.

    2009-01-01

    Objective: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenthymal stromal cells (MSC) have been derived from multiple sources in the horse...

  5. Aging of marrow stromal (skeletal) stem cells and their contribution to age-related bone loss

    DEFF Research Database (Denmark)

    Bellantuono, Ilaria; Aldahmash, Abdullah; Kassem, Moustapha

    2009-01-01

    Marrow stromal cells (MSC) are thought to be stem cells with osteogenic potential and therefore responsible for the repair and maintenance of the skeleton. Age related bone loss is one of the most prevalent diseases in the elder population. It is controversial whether MSC undergo a process of agi...

  6. Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion.

    NARCIS (Netherlands)

    Torensma, R.; Brugge, P.J. ter; Jansen, J.A.; Figdor, C.G.

    2003-01-01

    The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion

  7. Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Hiroto Kinoshita

    Full Text Available Interleukin-6 (IL-6 is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial-stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6-positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6(-/- mice with wild-type (WT mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6(-/- mice, compared with WT mice. Impaired tumor development in IL-6(-/- mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3-related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell-conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1 receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.

  8. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas

    2011-01-01

    cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously...... or in a critical size calvarial defect in immune deficient mice for 10weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable...

  9. Mesenchymal Stromal Cells: What Is the Mechanism in Acute Graft-Versus-Host Disease?

    Directory of Open Access Journals (Sweden)

    Neil Dunavin

    2017-07-01

    Full Text Available After more than a decade of preclinical and clinical development, therapeutic infusion of mesenchymal stromal cells is now a leading investigational strategy for the treatment of acute graft-versus-host disease (GVHD. While their clinical use continues to expand, it is still unknown which of their immunomodulatory properties contributes most to their therapeutic activity. Herein we describe the proposed mechanisms, focusing on the inhibitory activity of mesenchymal stromal cells (MSCs at immunologic checkpoints. A deeper understanding of the mechanism of action will allow us to design more effective treatment strategies.

  10. Endometrial stromal cell attachment and matrix homeostasis in abdominal wall endometriomas.

    Science.gov (United States)

    Itoh, Hiroko; Mogami, Haruta; Bou Nemer, Laurice; Word, Larry; Rogers, David; Miller, Rodney; Word, R Ann

    2018-02-01

    How does progesterone alter matrix remodeling in abdominal wall endometriomas compared with normal endometrium? Progesterone may prevent attachment of endometrial cells to the abdominal wall, but does not ameliorate abnormal stromal cell responses of abdominal wall endometriomas. Menstruation is a tightly orchestrated physiologic event in which steroid hormones and inflammatory cells cooperatively initiate shedding of the endometrium. Abdominal wall endometriomas represent a unique form of endometriosis in which endometrial cells inoculate fascia or dermis at the time of obstetrical or gynecologic surgery. Invasion of endometrium into ectopic sites requires matrix metalloproteinases (MMPs) for tissue remodeling but endometrium is not shed externally. Observational study in 14 cases and 19 controls. Tissues and stromal cells isolated from 14 abdominal wall endometriomas were compared with 19 normal cycling endometrium using immunohistochemistry, quantitative PCR, gelatin zymography and cell attachment assays. P values cell preps to provide scientific rigor to the conclusions. The results indicate that MMP2 and MMP9 are not increased by TGFβ1 in endometrioma stromal cells. Although progesterone prevents attachment of endometrioma cells to matrix components of the abdominal wall, it does not ameliorate these abnormal stromal cell responses to TGFβ1. N/A. Endometriomas were collected from women identified pre-operatively. Not all endometriomas were collected. Stromal cells from normal endometrium were from different patients, not women undergoing endometrioma resection. This work provides insight into the mechanisms by which progesterone may prevent abdominal wall endometriomas but, once established, are refractory to progesterone treatment. Tissue acquisition was supported by NIH P01HD087150. Authors have no competing interests. © The Author(s) 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All

  11. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  12. Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

    Science.gov (United States)

    Poltavtsev, A M; Poltavtseva, R A; Yushina, M N; Pavlovich, S V; Svirshchevskaya, E V

    2017-08-01

    We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

  13. A rare ovarian tumor, leydig stromal cell tumor, presenting with virilization: a case report

    Directory of Open Access Journals (Sweden)

    Soheila Aminimoghaddam

    2012-11-01

    Full Text Available  Abstract Leydig stromal cell tumor is a rare ovarian tumor that belongs to the group of sex-cord stromal tumors. They produce testosterone leading to hyperandrogenism. We present a 41yr old woman with symptoms of virilization and a mass of right adenex via ultra Sonography, and a rise of total and free serum testosterone. An ovarian source of androgen was suspected and a surgery performed. A diagnosis of leydig-stromal cell tumor was confirmed. Our report is a reminder that although idiopathic hirsutism and other benign androgen excess disorder like Polycystic Ovarian Syndrome (PCOs are common, ovarian mass should be considered in differential diagnosis. 

  14. Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

    Directory of Open Access Journals (Sweden)

    Kristin Dawson

    Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

  15. Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

    Science.gov (United States)

    Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

    2011-01-01

    Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

  16. IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Roni M. Shtein

    2012-01-01

    Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

  17. Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

    Directory of Open Access Journals (Sweden)

    Chiquita Prahasanti

    2018-04-01

    Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

  18. Role of Stromal Paracrine Signals in Proliferative Diseases of the Aging Human Prostate

    Directory of Open Access Journals (Sweden)

    Kenichiro Ishii

    2018-04-01

    Full Text Available Androgens are essential for the development, differentiation, growth, and function of the prostate through epithelial–stromal interactions. However, androgen concentrations in the hypertrophic human prostate decrease significantly with age, suggesting an inverse correlation between androgen levels and proliferative diseases of the aging prostate. In elderly males, age- and/or androgen-related stromal remodeling is spontaneously induced, i.e., increased fibroblast and myofibroblast numbers, but decreased smooth muscle cell numbers in the prostatic stroma. These fibroblasts produce not only growth factors, cytokines, and extracellular matrix proteins, but also microRNAs as stromal paracrine signals that stimulate prostate epithelial cell proliferation. Surgical or chemical castration is the standard systemic therapy for patients with advanced prostate cancer. Androgen deprivation therapy induces temporary remission, but the majority of patients eventually progress to castration-resistant prostate cancer, which is associated with a high mortality rate. Androgen deprivation therapy-induced stromal remodeling may be involved in the development and progression of castration-resistant prostate cancer. In the tumor microenvironment, activated fibroblasts stimulating prostate cancer cell proliferation are called carcinoma-associated fibroblasts. In this review, we summarize the role of stromal paracrine signals in proliferative diseases of the aging human prostate and discuss the potential clinical applications of carcinoma-associated fibroblast-derived exosomal microRNAs as promising biomarkers.

  19. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC...... population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high...... and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone...

  20. Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

    International Nuclear Information System (INIS)

    Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

    2005-01-01

    The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

  1. Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy

    OpenAIRE

    Costa, M.; Cerqueira, Mariana Teixeira; Santos, T. C.; Marques, Belém Sampaio; Ludovico, Paula; Marques, A. P.; Pirraco, Rogério P.; Reis, R. L.

    2017-01-01

    Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditi...

  2. In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

    Science.gov (United States)

    Dave, Shruti D; Patel, Chetan N; Vanikar, Aruna V; Trivedi, Hargovind L

    2018-01-01

    Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 37 0 C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 37 0 C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 10 3/ μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease.

  3. Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro.

    Science.gov (United States)

    Rosu-Myles, Michael; Fair, Joel; Pearce, Nelson; Mehic, Jelica

    2010-10-01

    The ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood. C57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU. At a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105+). A 3-fold reduction in the percentage of CD105+ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105+ cells coincided with a 10-20% increase in the frequency of proliferating CD105(-) cells. Removal of CD105(-) stroma caused increased proliferation in CD105+ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105+ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105(-) cells. This work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.

  4. Reciprocal upregulation of Notch signaling molecules in hematopoietic progenitor and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Kikuchi Y

    2011-01-01

    Full Text Available Although mesenchymal stem cells (MSCs play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells, focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54 and myoblasts (M1601. The stromal cell derivatives were cocultured with murine HSCs (Lineage-Sca1+, and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with ninefold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Delta-like 3 were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was upregulated in the HSCs cocultured with A54 cells. A transwell assay revealed that the reciprocal upregulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal upregulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy.

  5. Effect of microemulsions on cell viability of human dermal fibroblasts

    Science.gov (United States)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  6. Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2010-09-01

    Full Text Available Abstract Background Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. Conclusion This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

  7. Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics

    DEFF Research Database (Denmark)

    Rumman, Mohammad; Majumder, Abhijit; Harkness, Linda

    2018-01-01

    Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell...... properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene...... expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared...

  8. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    EBs using BMP2 (bone morphogenic protein 2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold......Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...

  9. Paracrine action of mesenchymal stromal cells delivered by microspheres contributes to cutaneous wound healing and prevents scar formation in mice.

    Science.gov (United States)

    Huang, Sha; Wu, Yan; Gao, Dongyun; Fu, Xiaobing

    2015-07-01

    Accumulating evidence suggests that mesenchymal stromal cells (MSCs) participate in wound healing to favor tissue regeneration and inhibit fibrotic tissue formation. However, the evidence of MSCs to suppress cutaneous scar is extremely rare, and the mechanism remains unidentified. This study aimed to demonstrate whether MSCs-as the result of their paracrine actions on damaged tissues-would accelerate wound healing and prevent cutaneous fibrosis. For efficient delivery of MSCs to skin wounds, microspheres were used to maintain MSC potency. Whether MSCs can accelerate wound healing and alleviate cutaneous fibrosis through paracrine action was investigated with the use of a Transwell co-culture system in vitro and a murine model in vivo. MSCs cultured on gelatin microspheres fully retained their cell surface marker expression profile, proliferation, differentiation and paracrine potential. Co-cultures of MSCs and fibroblasts indicated that the benefits of MSCs on suppressing fibroblast proliferation and its fibrotic behavior induced by inflammatory cytokines probably were caused by paracrine actions. Importantly, microspheres successfully delivered MSCs into wound margins and significantly accelerated wound healing and concomitantly reduced the fibrotic activities of cells within the wounds and excessive accumulation of extracellular matrix as well as the transforming growth factor-β1/transforming growth factor-β3 ratio. This study provides insight into what we believe to be a previously undescribed, multifaceted role of MSC-released protein in reducing cutaneous fibrotic formation. Paracrine action of MSCs delivered by microspheres may thus qualify as a promising strategy to enhance tissue repair and to prevent excessive fibrosis during cutaneous wound healing. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

    Directory of Open Access Journals (Sweden)

    Richard J Cuthbert

    Full Text Available Current clinical trials utilize mesenchymal stromal cells (MSCs expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.MSCs were isolated from bone marrow (BM aspirates or surgical waste materials; enzymatically digested femoral heads (FHs and reamer irrigator aspirator (RIA waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH. MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH, with the highest up-regulation detected for CXCL12 in BM (>1300-fold. Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product

  11. Engineering Cartilage Tissue by Pellet Coculture of Chondrocytes and Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Wu, Ling; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes; Westendorf, Jennifer J.; van Wijnen, Andre J.

    2015-01-01

    Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made

  12. Low-frequency vibration treatment of bone marrow stromal cells induces bone repair in vivo.

    Science.gov (United States)

    He, Shengwei; Zhao, Wenzhi; Zhang, Lu; Mi, Lidong; Du, Guangyu; Sun, Chuanxiu; Sun, Xuegang

    2017-01-01

    To study the effect of low-frequency vibration on bone marrow stromal cell differentiation and potential bone repair in vivo . Forty New Zealand rabbits were randomly divided into five groups with eight rabbits in each group. For each group, bone defects were generated in the left humerus of four rabbits, and in the right humerus of the other four rabbits. To test differentiation, bones were isolated and demineralized, supplemented with bone marrow stromal cells, and implanted into humerus bone defects. Varying frequencies of vibration (0, 12.5, 25, 50, and 100 Hz) were applied to each group for 30 min each day for four weeks. When the bone defects integrated, they were then removed for histological examination. mRNA transcript levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor κ-B ligan, and pre-collagen type 1 α were measured. Humeri implanted with bone marrow stromal cells displayed elevated callus levels and wider, more prevalent, and denser trabeculae following treatment at 25 and 50 Hz. The mRNA levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor κ-B ligand, and pre-collagen type 1 α were also markedly higher following 25 and 50 Hz treatment. Low frequency (25-50 Hz) vibration in vivo can promote bone marrow stromal cell differentiation and repair bone injury.

  13. Low-frequency vibration treatment of bone marrow stromal cells induces bone repair in vivo

    Directory of Open Access Journals (Sweden)

    Shengwei He

    2017-01-01

    Full Text Available Objective(s:To study the effect of low-frequency vibration on bone marrow stromal cell differentiation and potential bone repair in vivo. Materials and Methods:Forty New Zealand rabbits were randomly divided into five groups with eight rabbits in each group. For each group, bone defects were generated in the left humerus of four rabbits, and in the right humerus of the other four rabbits. To test differentiation, bones were isolated and demineralized, supplemented with bone marrow stromal cells, and implanted into humerus bone defects. Varying frequencies of vibration (0, 12.5, 25, 50, and 100 Hz were applied to each group for 30 min each day for four weeks. When the bone defects integrated, they were then removed for histological examination. mRNA transcript levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor k-B ligan, and pre-collagen type 1 a were measured. Results:Humeri implanted with bone marrow stromal cells displayed elevated callus levels and wider, more prevalent, and denser trabeculae following treatment at 25 and 50 Hz. The mRNA levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor k-B ligand, and pre-collagen type 1 a were also markedly higher following 25 and 50 Hz treatment. Conclusion:Low frequency (25–50 Hz vibration in vivo can promote bone marrow stromal cell differentiation and repair bone injury.

  14. Human multipotent mesenchymal stromal cells in the treatment of postoperative temporal bone defect: an animal model

    Czech Academy of Sciences Publication Activity Database

    Školoudík, L.; Chrobok, V.; Kalfert, D.; Kočí, Zuzana; Syková, Eva; Chumak, Tetyana; Popelář, Jiří; Syka, Josef; Laco, J.; Dědková, J.; Dayanithi, Govindan; Filip, S.

    2016-01-01

    Roč. 25, č. 7 (2016), s. 1405-1414 ISSN 0963-6897 R&D Projects: GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : Human bone marrow * Human mesenchymal stromal cells (hMSCs) * Middle ear surgery * Temporal bone Subject RIV: FP - Other Medical Disciplines Impact factor: 3.006, year: 2016

  15. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells

    DEFF Research Database (Denmark)

    Dokkedahl, Karin Stenderup; Justesen, Jeannette; Clausen, Christian

    2003-01-01

    Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maxi...

  16. Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2007-01-01

    There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker g...

  17. Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs).

    Science.gov (United States)

    Bartosh, Thomas J; Ullah, Mujib; Zeitouni, Suzanne; Beaver, Joshua; Prockop, Darwin J

    2016-10-18

    Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.

  18. Honokiol, a constituent of Magnolia species, inhibits adrenergic contraction of human prostate strips and induces stromal cell death

    Directory of Open Access Journals (Sweden)

    Daniel Herrmann

    2014-09-01

    Conclusions: Honokiol inhibits smooth muscle contraction in the human prostate, and induces cell death in cultured stromal cells. Because prostate smooth muscle tone and prostate growth may cause LUTS, it appears possible that honokiol improves voiding symptoms.

  19. Mesenchymal stromal cells for cardiovascular repair: current status and future challenges

    DEFF Research Database (Denmark)

    Mathiasen, Anders Bruun; Haack-Sørensen, Mandana; Kastrup, Jens

    2009-01-01

    of treatments in patients with heart failure, the 1-year mortality is still approximately 20% after the diagnosis has been established. Treatment with stem cells with the potential to regenerate the damaged myocardium is a relatively new approach. Mesenchymal stromal cells are a promising source of stem cells...... studies are promising, but there are still many unanswered questions. In this review, we explore present preclinical and clinical knowledge regarding the use of stem cells in cardiovascular regenerative medicine, with special focus on mesenchymal stromal cells. We take a closer look at sources of stem...... for regenerative therapy. Clinical studies on stem cell therapy for cardiac regeneration have shown significant improvements in ventricular pump function, ventricular remodeling, myocardial perfusion, exercise potential and clinical symptoms compared with conventionally treated control groups. The results of most...

  20. Kinetic analysis of thymocyte attachment to thymus stromal cells in culture by using phase-contrast and scanning electron microscopy

    International Nuclear Information System (INIS)

    LaRochelle, G.G.; Jones, K.H.

    1989-01-01

    Direct cellular contact between thymocytes and thymus stromal cells within the thymus appears to contribute to the maturation of thymocytes. Thymocyte-stromal cell complexes, formed in vivo, have been isolated by others and postulated to play a role in T-cell differentiation. These previous studies have been hampered, however, by a time-consuming isolation procedure from which only small numbers of these complexes are recovered. We have examined a model to study thymocyte-stromal cell complexes in vitro in which thymocytes are added to primary cultures of thymus stromal cells. In the present study, we found that thymocytes were histotypically selective in their attachment to thymus stromal cells. We also investigated the kinetics of thymocyte attachment to these thymus stromal cells. Cultures were examined at selected time intervals from 5 min through 3 days of incubation. Thymocyte attachment to stromal cells was a biphasic interaction, with maximum surface attachment at 15 min of cocultivation, followed by migration of thymocytes into the cultures. Morphological studies were confirmed by using 3 H-leucine-labeled thymocytes and liquid scintigraphy. With increased time in culture, thymocytes became amoeboid and migrated between the layers of stromal cells where thymocyte mitotic figures were seen at 4 and 8 hr. In some cases it appeared that stromal cells, which often grew two to three cell layers deep, played an active role in enclosing thymocytes within the cultures. Large numbers of viable thymocytes were observed in the cultures at 24 hr. The number of thymocytes then decreased progressively on days 2 and 3, when relatively few were found within the layers of the culture

  1. A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

    Science.gov (United States)

    Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

    2013-01-01

    We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

  2. Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

    International Nuclear Information System (INIS)

    Kreja, L.; Selig, C.; Plappert, U.; Nothdurft, W.

    1996-01-01

    Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose-effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D 0 value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D 0 values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile site was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as open-quotes cometsclose quotes by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. 37 refs., 2 figs., 1 tab

  3. The dependence of fibroblast radiosensitivity on cell pH

    International Nuclear Information System (INIS)

    Veksler, A.M.; Kublik, L.N.; Degtyareva, O.V.; Ehjdus, L.Kh.

    1983-01-01

    The problem of the change of radiosensitivity of Chinese hamster fibroblasts, irradiated under aerobic and hypoxic conditions in the course of intracellular pH (pHsub(intr.)) change by means of a phosphate buffer has been studied. It has been found that pHsub(intr.) reduction considerably increases the radiosensitivity, the effect being more pronounced on hypoxic cells which is essential for radiotherapy of tumors. The survival rate of cell irradiated under hypoxia conditions does not depend on season while cell resistance in case of irradiation in open air in spring and autumn is different. The effect discovery in case of pHsub(intr.) reduction upon irradiation shows up the influence of the studied factor on repair processes

  4. In Vivo Functional Selection Identifies Cardiotrophin-1 as a Cardiac Engraftment Factor for Mesenchymal Stromal Cells.

    Science.gov (United States)

    Bortolotti, Francesca; Ruozi, Giulia; Falcione, Antonella; Doimo, Sara; Dal Ferro, Matteo; Lesizza, Pierluigi; Zentilin, Lorena; Banks, Lawrence; Zacchigna, Serena; Giacca, Mauro

    2017-10-17

    Transplantation of cells into the infarcted heart has significant potential to improve myocardial recovery; however, low efficacy of cell engraftment still limits therapeutic benefit. Here, we describe a method for the unbiased, in vivo selection of cytokines that improve mesenchymal stromal cell engraftment into the heart both in normal conditions and after myocardial infarction. An arrayed library of 80 secreted factors, including most of the currently known interleukins and chemokines, were individually cloned into adeno-associated viral vectors. Pools from this library were then used for the batch transduction of bone marrow-derived mesenchymal stromal cells ex vivo, followed by intramyocardial cell administration in normal and infarcted mice. Three weeks after injection, vector genomes were recovered from the few persisting cells and identified by sequencing DNA barcodes uniquely labeling each of the tested cytokines. The most effective molecule identified by this competitive engraftment screening was cardiotrophin-1, a member of the interleukin-6 family. Intracardiac injection of mesenchymal stromal cells transiently preconditioned with cardiotrophin-1 preserved cardiac function and reduced infarct size, parallel to the persistence of the transplanted cells in the healing hearts for at least 2 months after injection. Engraftment of cardiotrophin-1-treated mesenchymal stromal cells was consequent to signal transducer and activator of transcription 3-mediated activation of the focal adhesion kinase and its associated focal adhesion complex and the consequent acquisition of adhesive properties by the cells. These results support the feasibility of selecting molecules in vivo for their functional properties with adeno-associated viral vector libraries and identify cardiotrophin-1 as a powerful cytokine promoting cell engraftment and thus improving cell therapy of the infarcted myocardium. © 2017 American Heart Association, Inc.

  5. Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0251 TITLE: “Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA...Heterogeneity in Metabolic Disease Using Single- Cell RNA-Seq 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Linus Tzu-Yen...ABSTRACT We have developed a robust protocol to generate single cell transcriptional profiles from subcutaneous adipose tissue samples of both human

  6. Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0512 TITLE: Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer PRINCIPAL INVESTIGATOR: Andrew...SUBTITLE 5a. CONTRACT NUMBER Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer 5b. GRANT NUMBER W81XWH-15-1-0512 5c. PROGRAM...blocked by the addition of Pim inhibitors. These results suggest that the Pim protein kinase can regulate stromal cell biology to modulate epithelial

  7. Pancreatic Fibroblasts Stimulate the Motility of Pancreatic Cancer Cells through IGF1/IGF1R Signaling under Hypoxia.

    Directory of Open Access Journals (Sweden)

    Toshiki Hirakawa

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is characterized by its hypovascularity, with an extremely poor prognosis because of its highly invasive nature. PDAC proliferates with abundant stromal cells, suggesting that its invasive activity might be controlled by intercellular interactions between cancer cells and fibroblasts. Using four PDAC cell lines and two pancreas cancer-associated fibroblasts (CAFs, the expression of insulin-like growth factor-1 (IGF1 and IGF1 receptor (IGF1R was evaluated by RT-PCR, FACScan, western blot, or ELISA. Correlation between IGF1R and the hypoxia marker carbonic anhydrase 9 (CA9 was examined by immunohistochemical staining of 120 pancreatic specimens. The effects of CAFs, IGF1, and IGF1R inhibitors on the motility of cancer cells were examined by wound-healing assay or invasion assay under normoxia (20% O2 and hypoxia (1% O2. IGF1R expression was significantly higher in RWP-1, MiaPaCa-2, and OCUP-AT cells than in Panc-1 cells. Hypoxia increased the expression level of IGF1R in RWP-1, MiaPaCa-2, and OCUP-AT cells. CA9 expression was correlated with IGF1R expression in pancreatic specimens. CAFs produced IGF1 under hypoxia, but PDAC cells did not. A conditioned medium from CAFs, which expressed αSMA, stimulated the migration and invasion ability of MiaPaCa-2, RWP-1, and OCUP-AT cells. The motility of all PDAC cells was greater under hypoxia than under normoxia. The motility-stimulating ability of CAFs was decreased by IGF1R inhibitors. These findings might suggest that pancreas CAFs stimulate the invasion activity of PDAC cells through paracrine IGF1/IGF1R signaling, especially under hypoxia. Therefore the targeting of IGF1R signaling might represent a promising therapeutic approach in IGF1R-dependent PDAC.

  8. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

    Directory of Open Access Journals (Sweden)

    Mohammad Rasoul Khazaei

    2016-09-01

    Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P

  9. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  10. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  11. Decidualization and syndecan-1 knock down sensitize endometrial stromal cells to apoptosis induced by embryonic stimuli.

    Directory of Open Access Journals (Sweden)

    Sarah Jean Boeddeker

    Full Text Available Human embryo invasion and implantation into the inner wall of the maternal uterus, the endometrium, is the pivotal process for a successful pregnancy. Whereas disruption of the endometrial epithelial layer was already correlated with the programmed cell death, the role of apoptosis of the subjacent endometrial stromal cells during implantation is indistinct. The aim was to clarify whether apoptosis plays a role in the stromal invasion and to characterize if the apoptotic susceptibility of endometrial stromal cells to embryonic stimuli is influenced by decidualization and Syndecan-1. Therefore, the immortalized human endometrial stromal cell line St-T1 was used to first generate a new cell line with a stable Syndecan-1 knock down (KdS1, and second to further decidualize the cells with progesterone. As a replacement for the ethically inapplicable embryo all cells were treated with the embryonic factors and secretion products interleukin-1β, interferon-γ, tumor necrosis factor-α, transforming growth factor-β1 and anti-Fas antibody to mimic the embryo contact. Detection of apoptosis was verified via Caspase ELISAs, PARP cleavage and Annexin V staining. Apoptosis-related proteins were investigated via antibody arrays and underlying signaling pathways were analyzed by Western blot. Non-decidualized endometrial stromal cells showed a resistance towards apoptosis which was rescinded by decidualization and Syndecan-1 knock down independent of decidualization. This was correlated with an altered expression of several pro- and anti-apoptotic proteins and connected to a higher activation of pro-survival Akt in non-differentiated St-T1 as an upstream mediator of apoptotis-related proteins. This study provides insight into the largely elusive process of implantation, proposing an important role for stromal cell apoptosis to successfully establish a pregnancy. The impact of Syndecan-1 in attenuating the apoptotic signal is particularly interesting in the

  12. Collagen matrix as a tool in studying fibroblastic cell behavior.

    Science.gov (United States)

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.

  13. Analysis of bone marrow stromal cell transferred bacterial {beta}-galactosidase gene by PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Kumakawa, Toshiro [Tokyo Metropolitan Geriatric Hospital, Tokyo (Japan). Dept. of Blood Transfusion and Hematology; Hibino, Hitoshi; Tani, Kenzaburo; Asano, Shigetaka; Futatugawa, Shouji; Sera, Kouichiro

    1997-12-31

    PIXE, Particle Induced X-ray Emission, is a powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial {beta}-galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell. (author)

  14. Stromal cell contributions to the homeostasis and functionality of the immune system.

    Science.gov (United States)

    Mueller, Scott N; Germain, Ronald N

    2009-09-01

    A defining characteristic of the immune system is the constant movement of many of its constituent cells through the secondary lymphoid tissues, mainly the spleen and lymph nodes, where crucial interactions that underlie homeostatic regulation, peripheral tolerance and the effective development of adaptive immune responses take place. What has only recently been recognized is the role that non-haematopoietic stromal elements have in many aspects of immune cell migration, activation and survival. In this Review, we summarize our current understanding of lymphoid compartment stromal cells, examine their possible heterogeneity, discuss how these cells contribute to immune homeostasis and the efficient initiation of adaptive immune responses, and highlight how targeting of these elements by some pathogens can influence the host immune response.

  15. Cell survival of human tumor cells compared with normal fibroblasts following 60Co gamma irradiation

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Henning, C.B.; Reynolds, S.D.; Holmblad, G.L.; Trier, J.E.

    1982-01-01

    Three tumor cell lines, two of which were shown to be HeLa cells, were irradiated with 60 Co gamma irradiation, together with two cell cultures of normal human diploid fibroblasts. Cell survival was studied in three different experiments over a dose range of 2 to 14 gray. All the tumor cell lines showed a very wide shoulder in the dose response curves in contrast to the extremely narrow shoulder of the normal fibroblasts. In addition, the D/sub o/ values for the tumor cell lines were somewhat greater. These two characteristics of the dose response curves resulted in up to 2 orders of magnitude less sensitivity for cell inactivation of HeLa cells when compared with normal cells at high doses (10 gray). Because of these large differences, the extrapolation of results from the irradiation of HeLa cells concerning the mechanisms of normal cell killing should be interpreted with great caution

  16. Patterns of proliferation and differentiation of irradiated haemopoietic stem cells cultured on normal 'stromal' cell colonies in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.

    1981-01-01

    Experiments were designed to elucidate whether or not the irradiated bone marrow cells receive any stimulation for the self-replication and differentiation from normal 'stromal' cell colonies in the bone marrow cell culture in vitro. When irradiated or unirradiated bone marrow cells were overlaid on the normal adherent cell colonies, the proliferation of haemopoietic stem cells was supported, the degree of the stimulation depending on the starting cellular concentration. There was, however, no significant changes in the concentration of either CFUs or CFUc regardless of the dose of irradiation on the bone marrow cells overlaid. This was a great contrast to the dose-dependent decrease of CFUs or CFUc within the culture in which both the stem cells and stromal cells were simultaneously irradiated. These results suggest that the balance of self-replication and differentiation of the haemopoietic stem cells is affected only when haemopoietic microenvironment is perturbed. (author)

  17. Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Luan, Anna; Aitzetmüller, Matthias M; Brett, Elizabeth A; Atashroo, David; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Houschyar, Khosrow S; Schilling, Arndt F; Machens, Hans-Guenther; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

    2017-12-01

    Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34 + CD31 - CD45 - ). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

    International Nuclear Information System (INIS)

    Borek, C.

    1982-01-01

    In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

  19. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    OpenAIRE

    Selleri, Silvia; Bifsha, Panojot; Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Ren?e; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-...

  20. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise

    2013-01-01

    BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit...... differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which...... persists independent of cell density can be used for clinical therapies....

  1. Derivation of Insulin Producing Cells From Human Endometrial Stromal Stem Cells and Use in the Treatment of Murine Diabetes

    OpenAIRE

    Santamaria, Xavier; Massasa, Efi E; Feng, Yuzhe; Wolff, Erin; Taylor, Hugh S

    2011-01-01

    Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using ...

  2. Characteristics and function of bone marrow stromal adherent cells in normal and irradiated mice and guinea pigs

    Energy Technology Data Exchange (ETDEWEB)

    Changyu, Zheng; Ji, Liu; Xiaoying, Bi

    1986-04-01

    It has been shown from cytochemical and other characteristic studies of bone marrow stromal cells in CFU-F that there are seven types of stromal cells in the stromal adherent cell layer of normal and irradiated C/sub 57/ mice whereas there are only six types in guinea pigs. On the other hand, a radioresistant cell subtype appears in adherent layer after irradiation of both C/sub 57/ mice and guinea pig since the supernatant of cultured CFU-F of the normal and irradiated C/sub 57/ mice can stimulate production of CFU-Gm. It is justifiable that the bone marrow stromal adherent cells of the C/sub 57/ mice could produce CSF.

  3. Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.

    Science.gov (United States)

    Ezzelarab, Mohamed; Ezzelarab, Corin; Wilhite, Tyler; Kumar, Goutham; Hara, Hidetaka; Ayares, David; Cooper, David K C

    2011-01-01

    Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied. Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T-cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL-2, IFN-γ, TNF-α, and sCD40L correlated with the downregulation of T-cell proliferation by all types of MSC. Genetically modified pMSC is significantly less

  4. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

    1979-01-01

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  5. DNA repair in a Fanconi's anemia fibroblast cell strain

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

    1979-01-26

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

  6. Endometrial Stromal Cells and Immune Cell Populations Within Lymph Nodes in a Nonhuman Primate Model of Endometriosis

    Science.gov (United States)

    Fazleabas, A. T.; Braundmeier, A. G.; Markham, R.; Fraser, I. S.; Berbic, M.

    2011-01-01

    Mounting evidence suggests that immunological responses may be altered in endometriosis. The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis. The objective of the current study was to investigate for the first time immunological changes within uterine and peritoneal draining lymph nodes in a nonhuman primate baboon model of endometriosis. Paraffin-embedded femoral lymph nodes were obtained from 22 normally cycling female baboons (induced endometriosis n = 11; control n = 11). Immunohistochemical staining was performed with antibodies for endometrial stromal cells, T cells, immature and mature dendritic cells, and B cells. Lymph nodes were evaluated using an automated cellular imaging system. Endometrial stromal cells were significantly increased in lymph nodes from animals with induced endometriosis, compared to control animals (P = .033). In animals with induced endometriosis, some lymph node immune cell populations including T cells, dendritic cells and B cells were increased, suggesting an efficient early response or peritoneal drainage. PMID:21617251

  7. Enhanced Hematopoietic Stem Cell Self-Renewal-Promoting Ability of Clonal Primary Mesenchymal Stromal/Stem cells Versus Their Osteogenic Progeny.

    Science.gov (United States)

    He, Qiling; Scott Swindle, Claude; Wan, Chao; Flynn, Robert J; Oster, Robert A; Chen, Dongquan; Zhang, Fengjie; Shu, Yinglan; Klug, Christopher A

    2017-02-01

    Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin + Lepr + Sca-1 + CD146 + and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo. Stem Cells 2017;35:473-484. © 2016 AlphaMed Press.

  8. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  9. Generation of hiPSTZ16 (ISMMSi003-A cell line from normal human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    Marion Dejosez

    2018-01-01

    Full Text Available Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A. These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

  10. Osteogenic potential of bone marrow stromal cells on smooth, roughened, and tricalcium phosphate-modified titanium alloy surfaces.

    LENUS (Irish Health Repository)

    Colombo, John S

    2012-09-01

    This study investigated the influence of smooth, roughened, and tricalcium phosphate (TCP)-coated roughened titanium-aluminum-vanadium (Ti-6Al-4V) surfaces on the osteogenic potential of rat bone marrow stromal cells (BMSCs).

  11. Morphological evaluation during in vitro chondrogenesis of dental pulp stromal cells

    Directory of Open Access Journals (Sweden)

    Choo-Ryung Chung

    2012-02-01

    Full Text Available Objectives The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis. Materials and Methods Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets. Results Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction. Conclusions Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day of differentiation.

  12. Differentiation of Equine Mesenchymal Stromal Cells into Cells of Neural Lineage: Potential for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Claudia Cruz Villagrán

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSCs are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from young and adult horses. Following demonstration of stemness, MSCs were neurally induced and microscopically assessed at different time points. Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation. Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype. Expression of neural progenitor proteins, was assessed via western blot or immunofluorescence. In our study, MSCs generated from young and middle-aged horses did not show differences in their ability to undergo differentiation. The effect of cell passage number, however, is inconsistent and further experiments are needed. Ongoing work is aimed at transdifferentiating these cells into Schwann cells for transplantation into a peripheral nerve injury model in horses.

  13. In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

    Directory of Open Access Journals (Sweden)

    T.I. Wodewotzky

    2012-12-01

    Full Text Available Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

  14. In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

    International Nuclear Information System (INIS)

    Wodewotzky, T.I.; Lima-Neto, J.F.; Pereira-Júnior, O.C.M.; Sudano, M.J.; Lima, S.A.F.; Bersano, P.R.O.; Yoshioka, S.A.; Landim-Alvarenga, F.C.

    2012-01-01

    Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium

  15. In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Wodewotzky, T.I.; Lima-Neto, J.F. [Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil); Pereira-Júnior, O.C.M. [Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil); Departamento de Cirurgia e Anestesiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil); Sudano, M.J.; Lima, S.A.F. [Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil); Bersano, P.R.O. [Departamento de Patologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil); Yoshioka, S.A. [Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP (Brazil); Landim-Alvarenga, F.C. [Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual de São Paulo, Botucatu, SP (Brazil)

    2012-09-21

    Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

  16. Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

    Science.gov (United States)

    Santos, Jorge M; Camões, Sérgio P; Filipe, Elysse; Cipriano, Madalena; Barcia, Rita N; Filipe, Mariana; Teixeira, Mariana; Simões, Sandra; Gaspar, Manuela; Mosqueira, Diogo; Nascimento, Diana S; Pinto-do-Ó, Perpétua; Cruz, Pedro; Cruz, Helder; Castro, Matilde; Miranda, Joana P

    2015-05-09

    The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM

  17. Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells.

    Science.gov (United States)

    Jin, Cheng-Yu; Zhu, Bang-Shang; Wang, Xue-Feng; Lu, Qing-Hua

    2008-09-01

    Nanotitanium dioxide (TiO2) is an important industrial material that is widely used as an additive in cosmetics, pharmaceuticals, and food colorants. Although the small size of the TiO2 nanoparticle is useful in various applications, the biosafety of this material needs to be evaluated. In this study, mouse fibroblast (L929) cells were used to evaluate the cytotoxicity of different concentrations (3-600 microg/mL) of homogeneous and weakly aggregated TiO2 nanoparticles in aqueous solution. The L929 cells became round and even shrank as the concentration of TiO2 nanoparticles increased. Moreover, TiO2 nanoparticle-treated cells had condensed fragmented chromatin or were directly necrosed, as observed by acridine orange (AO) staining. The transmission electron microscopy (TEM) analysis showed that in cells cultured in a medium containing 300 microg/mL TiO2, the number of lysosomes increased, and some cytoplasmic organelles were damaged. In addition, there was a significant increase in oxidative stress at higher TiO2 nanoparticle concentrations (>60 microg/mL). As the concentration of TiO2 nanoparticles increased in the culture medium, the levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) increased, while those of methyl tetrazolium cytotoxicity (MTT), glutathione (GSH), and superoxide dismutase (SOD) decreased. A possible mechanism for the cytotoxicity of TiO2 nanoparticles is also discussed.

  18. Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

    Science.gov (United States)

    Kerkelä, Erja; Laitinen, Anita; Räbinä, Jarkko; Valkonen, Sami; Takatalo, Maarit; Larjo, Antti; Veijola, Johanna; Lampinen, Milla; Siljander, Pia; Lehenkari, Petri; Alfthan, Kaija; Laitinen, Saara

    2016-03-01

    Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. © 2016 AlphaMed Press.

  19. Effect of pirfenidone on the proliferation of rat corneal stromal cells

    Directory of Open Access Journals (Sweden)

    Jun-Jie Chen

    2015-02-01

    Full Text Available AIM: To investigate the effects of pirfenidone(PFDon the proliferation and transfomring growth factor-β1(TGF-β1expression in vitro culture rat corneal stromal cells. METHODS: Corneal stromal cells from 8 to 10wk SD rats were isolated, cultured and treated with different concentrations of PFD 0mg/mL(control group, 0.15mg/mL(experimental group Ⅰ, 0.3mg/mL(experimental group Ⅱ, 1mg/mL(experimental group Ⅲfor 48h. CCK-8 assay was performed to assess cell proliferation, while immunocytochemistry and Western Blot were used to detect the expression of ki-67 and TGF-β1 expression, respectively. RESULTS: Compared with control group, PFD significantly inhibited the proliferation in a dose-dependent manner(all P1 in a dose-dependent manner(PCONCLUSION: Pirfenidone can significantly inhibit the proliferation of rat corneal stromal cell by down regulating TGF-β1 expression, therefore, it has potential prospect in lightening the corneal wound healing reaction.

  20. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

    Full Text Available Haematopoietic stem cell (HSC transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK haematopoietic stem/progenitor cells (HPC demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

  1. Bone marrow stromal cell therapy for ischemic stroke: A meta-analysis of randomized control animal trials.

    Science.gov (United States)

    Wu, Qing; Wang, Yuexiang; Demaerschalk, Bart M; Ghimire, Saruna; Wellik, Kay E; Qu, Wenchun

    2017-04-01

    Background Results of animal studies assessing efficacy of bone marrow stromal cell therapy for ischemic stroke remain inconsistent. Aims The aims are to assess efficacy of bone marrow stromal cell therapy for ischemic stroke in animal studies. Methods Randomized controlled animal trials assessing efficacy of bone marrow stromal cell therapy were eligible. Stroke therapy academic industry round table was used to assess methodologic quality of included studies. Primary outcomes were total infarction volume and modified Neurological Severity Score. Multiple prespecified sensitivity analyses and subgroup analyses were conducted. Random effects models were used for meta-analysis. Results Thirty-three randomized animal trials were included with a total of 796 animals. The median quality score was 6 (interquartile range, 5-7). Bone marrow stromal cell therapy decreased total infarction volume (standardized mean difference, 0.897; 95% confidence interval, 0.553-1.241; P animals treated with bone marrow stromal cell and controls was 2.47 (95% confidence interval, 1.84-3.11; P animal studies. Conclusions Bone marrow stromal cell therapy significantly decreased total infarction volume and increased neural functional recovery in randomized controlled animal models of ischemic stroke.

  2. Neoplastic and stromal cells contribute to an extracellular matrix gene expression profile defining a breast cancer subtype likely to progress.

    Directory of Open Access Journals (Sweden)

    Tiziana Triulzi

    Full Text Available We recently showed that differential expression of extracellular matrix (ECM genes delineates four subgroups of breast carcinomas (ECM1, -2, -3- and -4 with different clinical outcome. To further investigate the characteristics of ECM signature and its impact on tumor progression, we conducted unsupervised clustering analyses in 6 additional independent datasets of invasive breast tumors from different platforms for a total of 643 samples. Use of four different clustering algorithms identified ECM3 tumors as an independent group in all datasets tested. ECM3 showed a homogeneous gene pattern, consisting of 58 genes encoding 43 structural ECM proteins. From 26 to 41% of the cases were ECM3-enriched, and analysis of datasets relevant to gene expression in neoplastic or corresponding stromal cells showed that both stromal and breast carcinoma cells can coordinately express ECM3 genes. In in vitro experiments, β-estradiol induced ECM3 gene production in ER-positive breast carcinoma cell lines, whereas TGFβ induced upregulation of the genes leading to ECM3 gene classification, especially in ER-negative breast carcinoma cells and in fibroblasts. Multivariate analysis of distant metastasis-free survival in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade (p = 0.001. Cox models, estimated separately in grade I-II and grade III tumors, indicated a highly significant association between ECM3 and worse survival probability only in grade III tumors (HR = 3.0, 95% CI = 1.3-7.0, p = 0.0098. Gene Set Enrichment analysis of ECM3 compared to non-ECM3 tumors revealed significant enrichment of epithelial-mesenchymal transition (EMT genes in both grade I-II and grade III subsets of ECM3 tumors. Thus, ECM3 is a robust cluster that identifies breast carcinomas with EMT features but with accelerated metastatic potential only in the undifferentiated (grade III phenotype. These findings support the

  3. Epimorphin Regulates the Intestinal Stem Cell Niche via Effects on the Stromal Microenvironment.

    Science.gov (United States)

    Vishy, Courtney E; Swietlicki, Elzbieta A; Gazit, Vered; Amara, Suneetha; Heslop, Gabriela; Lu, Jianyun; Levin, Marc S; Rubin, Deborah C

    2018-04-06

    Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin (Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission and small bowel length in vivo. Stem cell derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim -/- and WT mice. Enteroids co-cultured with ISEMFS had increased growth and crypt-like budding compared to enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared to co-cultures with WT ISEMFs. In primary crypt cultures, Epim -/- enteroids had significantly increased surface area and budding compared WTs. However stem cell assays comparing the number of Epim -/- vs WT colony forming units after first passage showed no differences in the absence of ISEMF support. Epim -/- vs. WT ISEMFs had increased Wnt4 expression and addition of Wnt4 to WT co-cultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment.

  4. Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces

    DEFF Research Database (Denmark)

    Dolatshahi-Pirouz, A; Jensen, Thomas Hartvig Lindkjær; Kolind, Kristian

    2011-01-01

    In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin...

  5. Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells

    Czech Academy of Sciences Publication Activity Database

    Matusková, M.; Baranovicová, L.; Kozovská, Z.; Duriniková, E.; Pastoráková, A.; Hunaková, L.; Waczulíková, I.; Nencka, Radim; Kučerová, L.

    2012-01-01

    Roč. 14, č. 12 (2012), s. 776-787 ISSN 1099-498X Institutional research plan: CEZ:AV0Z40550506 Keywords : bystander effect * cancer gene therapy * mesenchymal stromal cells Subject RIV: CC - Organic Chemistry Impact factor: 2.163, year: 2012

  6. Single-Stage Cell-Based Cartilage Regeneration Using a Combination of Chondrons and Mesenchymal Stromal Cells: Comparison With Microfracture

    NARCIS (Netherlands)

    Bekkers, J.E.J.; Tsuchida, A.I.; van Rijen, M.H.P.; Vonk, L.A.; Dhert, W.J.A.; Saris, Daniël B.F.

    2013-01-01

    Background: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed

  7. Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

    DEFF Research Database (Denmark)

    Haack-Sorensen, M.; Friis, T.; Bindslev, L.

    2008-01-01

    OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under...... conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly...... analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation...

  8. Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

    Science.gov (United States)

    Philippeos, Christina; Telerman, Stephanie B; Oulès, Bénédicte; Pisco, Angela O; Shaw, Tanya J; Elgueta, Raul; Lombardi, Giovanna; Driskell, Ryan R; Soldin, Mark; Lynch, Magnus D; Watt, Fiona M

    2018-04-01

    Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Good Preservation of Stromal Cells and No Apoptosis in Human Ovarian Tissue after Vitrification

    Directory of Open Access Journals (Sweden)

    Raffaella Fabbri

    2014-01-01

    Full Text Available The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

  10. Columnar cell lesions and pseudoangiomatous hyperplasia like stroma: is there an epithelial-stromal interaction?

    Science.gov (United States)

    Recavarren, Rosemary A; Chivukula, Mamatha; Carter, Gloria; Dabbs, David J

    2009-10-10

    The significance of association between cancer and its microenvironment has been increasingly recognized. It has been shown in animal models that interaction between neoplastic epithelial cells and adjacent stroma can modulate tumor behavior. Carcinoma associated stromal cells can transform normal epithelial cells into neoplastic cells. In breast, columnar cell lesions are non-obligate precursors of low grade ductal carcinoma in situ. Columnar cell lesions can be seen intimately associated with PASH-like-stroma, a lesion we termed as CCPLS. Our aim is to investigate epithelial-stromal interactions in CCPLS and compare them to PASH without columnar cell lesions in breast core needle biopsies. Normal terminal duct lobular unit (TDLU) epithelium was seen in association with columnar cell lesions as well as PASH. Eight (8) cases of each category were examined by a panel of immunostains: CD117 (C-kit), CD34, CD105, bFGF, AR, ER-beta, MIB-1. We observed a markedly decreased expression of c-kit in columnar cell lesions compared to TDLU-epithelium. CD105 showed a quantitative increase in activated vessels in CCPLS compared to PASH. A subset of CCPLS and PASH were androgen receptor positive. A strong nuclear positivity for ER-beta is observed in the epithelium and stroma of all CCPLS cases. We conclude that (1) activated blood vessels predominate in CCPLS; (2) A molecular alteration is signified by c-kit loss in columnar cell lesions; (3) ER-beta and androgen receptor positivity indicate CCPLS are hormonally responsive lesions. Our study suggests an intimate vascular and hormone dependent epithelial-stromal interaction exists in CCPLS lesions.

  11. Aging- and Senescence-associated Changes of Mesenchymal Stromal Cells in Myelodysplastic Syndromes.

    Science.gov (United States)

    Mattiucci, Domenico; Maurizi, Giulia; Leoni, Pietro; Poloni, Antonella

    2018-01-01

    Hematopoietic stem and progenitor cells reside within the bone marrow (BM) microenvironment. By a well-balanced interplay between self-renewal and differentiation, they ensure a lifelong supply of mature blood cells. Physiologically, multiple different cell types contribute to the regulation of stem and progenitor cells in the BM microenvironment by cell-extrinsic and cell-intrinsic mechanisms. During the last decades, mesenchymal stromal cells (MSCs) have been identified as one of the main cellular components of the BM microenvironment holding an indispensable role for normal hematopoiesis. During aging, MSCs diminish their functional and regenerative capacities and in some cases encounter replicative senescence, promoting inflammation and cancer progression. It is now evident that alterations in specific stromal cells that comprise the BM microenvironment can contribute to hematologic malignancies, and there is growing interest regarding the contribution of MSCs to the pathogenesis of myelodysplastic syndromes (MDSs), a clonal hematological disorder, occurring mostly in the elderly, characterized by ineffective hematopoiesis and increased tendency to acute myeloid leukemia evolution. The pathogenesis of MDS has been associated with specific genetic and epigenetic events occurring both in hematopoietic stem cells (HSCs) and in the whole BM microenvironment with an aberrant cross talk between hematopoietic elements and stromal compartment. This review highlights the role of MSCs in MDS showing functional and molecular alterations such as altered cell-cycle regulation with impaired proliferative potential, dysregulated cytokine secretion, and an abnormal gene expression profile. Here, the current knowledge of impaired functional properties of both aged MSCs and MSCs in MDS have been described with a special focus on inflammation and senescence induced changes in the BM microenvironment. Furthermore, a better understanding of aberrant BM microenvironment could

  12. A Color-coded Imageable Syngeneic Mouse Model of Stromal-cell Recruitment by Metastatic Lymphoma.

    Science.gov (United States)

    Matsumoto, Takuro; Suetsugu, Atsushi; Shibata, Yuhei; Nakamura, Nobuhiko; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

    2015-09-01

    A syngeneic color-coded imageable lymphoma model has been developed to visualize recruitment of host stromal cells by malignant lymphoma during metastasis. The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were initially established. EL4-RFP cells were subsequently injected into the tail vein of C57/BL6-GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the upper mediastinum and in the liver 28 days after cell injection. Large EL4-RFP liver metastases in C57/BL6-GFP mice contained GFP-expressing stromal cells derived from the host. In addition, EL4-RFP lymphoma metastasis was formed in peri-gastric lymph nodes, which were also enriched in host GFP-expressing cells. Furthermore, EL4-RFP lymphoma cells were also observed in the peripheral blood and bone marrow of C57/BL6-GFP transgenic mice, where they were associated with GFP-expressing host cells. Lymph node, liver and bone marrow metastases were found approximately 4 weeks after transplantation and all RFP-expressing metastases were highly enriched in GFP-expressing host stromal cells. This model of malignant lymphoma can be used to study early tumor development, metastasis, and the role of the stroma, as well as for discovery and evaluation of novel therapeutics for this treatment-resistant disease. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)

    Science.gov (United States)

    Lai, Kuo-Pao; Huang, Chiung-Kuei; Fang, Lei-Ya; Izumi, Kouji; Lo, Chi-Wen; Wood, Ronald; Kindblom, Jon; Yeh, Shuyuan

    2013-01-01

    Stromal-epithelial interaction plays a pivotal role to mediate the normal prostate growth, the pathogenesis of benign prostatic hyperplasia (BPH), and prostate cancer development. Until now, the stromal androgen receptor (AR) functions in the BPH development, and the underlying mechanisms remain largely unknown. Here we used a genetic knockout approach to ablate stromal fibromuscular (fibroblasts and smooth muscle cells) AR in a probasin promoter-driven prolactin transgenic mouse model (Pb-PRL tg mice) that could spontaneously develop prostate hyperplasia to partially mimic human BPH development. We found Pb-PRL tg mice lacking stromal fibromuscular AR developed smaller prostates, with more marked changes in the dorsolateral prostate lobes with less proliferation index. Mechanistically, prolactin mediated hyperplastic prostate growth involved epithelial-stromal interaction through epithelial prolactin/prolactin receptor signals to regulate granulocyte macrophage-colony stimulating factor expression to facilitate stromal cell growth via sustaining signal transducer and activator of transcription-3 activity. Importantly, the stromal fibromuscular AR could modulate such epithelial-stromal interacting signals. Targeting stromal fibromuscular AR with the AR degradation enhancer, ASC-J9®, led to the reduction of prostate size, which could be used in future therapy. PMID:23893956

  14. Stromal cell derived factor-1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer

    International Nuclear Information System (INIS)

    Kang, Hua; Watkins, Gareth; Parr, Christian; Douglas-Jones, Anthony; Mansel, Robert E; Jiang, Wen G

    2005-01-01

    Stromal cell-derived factor (SDF)-1 (CXC chemokine ligand-12) is a member of the CXC subfamily of chemokines, which, through its cognate receptor (CXC chemokine receptor [CXCR]4), plays an important role in chemotaxis of cancer cells and in tumour metastasis. We conducted the present study to evaluate the effect of SDF-1 on the invasiveness and migration of breast cancer cells, and we analyzed the expression of SDF-1 and its relation to clinicopathological features and clinical outcomes in human breast cancer. Expression of SDF-1 mRNA in breast cancer, endothelial (HECV) and fibroblast (MRC5) cell lines and in human breast tissues were studied using RT-PCR. MDA-MB-231 cells were transfected with a SDF-1 expression vector, and their invasiveness and migration was tested in vitro. In addition, the expression of SDF-1 was investigated using immunohistochemistry and quantitative RT-PCR in samples of normal human mammary tissue (n = 32) and mammary tumour (n = 120). SDF-1 expression was identified in MRC5, MDA-MB-435s and MDA-MB-436 cell lines, but CXCR4 expression was detected in all cell lines and breast tissues. An autocrine loop was created following transfection of MDA-MB-231 (which was CXCR4 positive and SDF-1 negative) with a mammalian expression cassette encoding SDF-1 (MDA-MB-231SDF1 +/+ ) or with control plasmid pcDNA4/GFP (MDA-MB-231 +/- ). MDA-MB-231SDF1 +/+ cells exhibited significantly greater invasion and migration potential (in transfected cells versus in wild type and empty MDA-MB-231 +/- ; P < 0.01). In mammary tissues SDF-1 staining was primarily seen in stromal cells and weakly in mammary epithelial cells. Significantly higher levels of SDF-1 were seen in node-positive than in node-negative tumours (P = 0.05), in tumours that metastasized (P = 0.05), and tumours from patients who died (P = 0.03) than in tumours from patients who were disease free. It was most notable that levels of SDF-1 correlated significantly with overall survival (P = 0.001) and

  15. Production of Reactive Oxygen Species by Multipotent Stromal Cells/Mesenchymal Stem Cells Upon Exposure to Fas Ligand

    OpenAIRE

    Rodrigues, Melanie; Turner, Omari; Stolz, Donna; Griffith, Linda G.; Wells, Alan

    2011-01-01

    Multipotent stromal cells (MSCs) can be differentiated into osteoblasts and chondrocytes, making these cells candidates to regenerate cranio-facial injuries and lesions in long bones. A major problem with cell replacement therapy, however, is the loss of transplanted MSCs at the site of graft. Reactive oxygen species (ROS) and nonspecific inflammation generated at the ischemic site have been hypothesized to lead to MSCs loss; studies in vitro show MSCs dying both in the presence of ROS or cyt...

  16. Proliferation of Prostate Stromal Cell Induced by Benign Prostatic Hyperplasia Epithelial Cell Stimulated With Trichomonas vaginalis via Crosstalk With Mast Cell.

    Science.gov (United States)

    Kim, Jung-Hyun; Kim, Sang-Su; Han, Ik-Hwan; Sim, Seobo; Ahn, Myoung-Hee; Ryu, Jae-Sook

    2016-11-01

    Chronic inflammation has a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. Mast cells have been detected in chronic inflammatory infiltrate of the prostate, and it is possible that the interaction between prostate epithelial cells and Trichomonas vaginalis influences the activity of mast cells in the prostate stroma. Activated mast cells might influence the biological functions of nearby tissues and cells. In this study, we investigated whether mast cells reacted with the culture supernatant of BPH epithelial cells infected with T. vaginalis may induce the proliferation of prostate stromal cells. To measure the proliferation of prostate stromal cells in response to chronic inflammation caused by the infection of BPH-1 cells with T. vaginalis, the CCK-8 assay and wound healing assay were used. ELISAs, quantitative real-time PCR, western blotting and immunofluorescence were used to measure the production and expression of inflammatory cytokine and cytokine receptor. BPH-1 cells incubated with live trichomonads produced increased levels of CCL2, IL-1β, IL-6, and CXCL8, and induced the migration of mast cells and monocytes. When the culture supernatant of BPH-1 cells stimulated with trichomonads (TCM) was added to mast cells, they became activated, as confirmed by release of β-hexosaminidase and CXCL8. Prostate stromal cells incubated with the culture supernatant of mast cells activated with TCM (M-TCM) proliferated and expressed increased levels of CXCL8, CCL2, and the cytokine receptors CXCR1 and CCR2. Blocking the chemokine receptors reduced the proliferation of stromal cells and also decreased the production of CXCL8 and CCL2. Moreover, the expression of FGF2, cyclin D1, and Bcl-2 was increased in the proliferated stromal cells stimulated with M-TCM. Additionally, the M-TCM-treated stromal cells were more invasive than control cells. The inflammatory mediators released by BPH epithelial cells in response to infection by

  17. Effect of Mesenchymal Stromal Cells on T Cells in a Septic Context: Immunosuppression or Immunostimulation?

    Science.gov (United States)

    Le Burel, Sébastien; Thepenier, Cédric; Boutin, Laetitia; Lataillade, Jean-Jacques; Peltzer, Juliette

    2017-10-15

    Sepsis is a complex process, including a first wave of damage partially due to the body's response to pathogens, followed by a phase of immune cell dysfunction. The efficacy of a pharmacological approach facing a rapidly evolving system implies a perfect timing of administration-this difficulty could explain the recent failure of clinical trials. Mesenchymal stromal cells (MSCs) are usually defined as immunosuppressive and their beneficial effects in preclinical models of acute sepsis have been shown to rely partly on such ability. If nonregulated, this phenotype could be harmful in the immunosuppressed context arising hours after sepsis onset. However, MSCs being environment sensitive, we hypothesized that they could reverse their immunosuppressive properties when confronted with suffering immune cells. Our objective was to evaluate the effect of human MSCs on activated human lymphocytes in an in vitro endotoxemia model. Peripheral blood mononuclear cells (PBMCs) underwent a 24-h lipopolysaccharide (LPS) intoxication and were stimulated with phytohemagglutinin (PHA) in contact with MSCs. MSCs induced a differential effect on lymphocytes depending on PBMC intoxication with LPS. Unintoxicated lymphocytes were highly proliferative with PHA and were inhibited by MSCs, whereas LPS-intoxicated lymphocytes showed a low proliferation rate, but were supported by MSCs, even when monocytes were depleted. These data, highlighting MSC plasticity in their immunomodulatory activity, pave the way for further studies investigating the mechanisms of mutual interactions between MSCs and immune cells in sepsis. Thus, MSCs might be able to fight against both early sepsis-induced hyperinflammatory response and later time points of immune dysfunction.

  18. Priming with ceramide-1 phosphate promotes the therapeutic effect of mesenchymal stem/stromal cells on pulmonary artery hypertension

    International Nuclear Information System (INIS)

    Lim, Jisun; Kim, YongHwan; Heo, Jinbeom; Kim, Kang-Hyun; Lee, Seungun; Lee, Sei Won; Kim, Kyunggon; Kim, In-Gyu; Shin, Dong-Myung

    2016-01-01

    Some molecules enriched in damaged organs can contribute to tissue repair by stimulating the mobilization of stem cells. These so-called “priming” factors include bioactive lipids, complement components, and cationic peptides. However, their therapeutic significance remains to be determined. Here, we show that priming of mesenchymal stromal/stem cells (MSCs) with ceramide-1 phosphate (C1P), a bioactive lipid, enhances their therapeutic efficacy in pulmonary artery hypertension (PAH). Human bone marrow (BM)-derived MSCs treated with 100 or 200 μM C1P showed improved migration activity in Transwell assays compared with non-primed MSCs and concomitantly activated MAPK p42/44 and AKT signaling cascades. Although C1P priming had little effect on cell surface marker phenotypes and the multipotency of MSCs, it potentiated their proliferative, colony-forming unit-fibroblast, and anti-inflammatory activities. In a monocrotaline-induced PAH animal model, a single administration of human MSCs primed with C1P significantly attenuated the PAH-related increase in right ventricular systolic pressure, right ventricular hypertrophy, and thickness of α-smooth muscle actin-positive cells around the vessel wall. Thus, this study shows that C1P priming increases the effects of MSC therapy by enhancing the migratory, self-renewal, and anti-inflammatory activity of MSCs and that MSC therapy optimized with priming protocols might be a promising option for the treatment of PAH patients. - Highlights: • Human BM-derived MSCs primed with C1P have enhanced migratory activity. • C1P primed MSCs increase proliferation, self-renewal, and anti-inflammatory capacity. • C1P priming enhances the therapeutic capacity of MSCs in a PAH animal model.

  19. Priming with ceramide-1 phosphate promotes the therapeutic effect of mesenchymal stem/stromal cells on pulmonary artery hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Jisun [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 88 Olympic-ro 43 gil, Songpa-gu, Seoul 05505 (Korea, Republic of); Kim, YongHwan; Heo, Jinbeom; Kim, Kang-Hyun; Lee, Seungun [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Sei Won [Department of Pulmonary and Critical Care Medicine and Clinical Research Center for Chronic Obstructive Airway Diseases, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Kyunggon [Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Clinical Proteomics Core Lab, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, In-Gyu, E-mail: igkim@plaza.snu.ac.kr [Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 88 Olympic-ro 43 gil, Songpa-gu, Seoul 05505 (Korea, Republic of); Shin, Dong-Myung, E-mail: d0shin03@amc.seoul.kr [Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Department of Physiology, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

    2016-04-22

    Some molecules enriched in damaged organs can contribute to tissue repair by stimulating the mobilization of stem cells. These so-called “priming” factors include bioactive lipids, complement components, and cationic peptides. However, their therapeutic significance remains to be determined. Here, we show that priming of mesenchymal stromal/stem cells (MSCs) with ceramide-1 phosphate (C1P), a bioactive lipid, enhances their therapeutic efficacy in pulmonary artery hypertension (PAH). Human bone marrow (BM)-derived MSCs treated with 100 or 200 μM C1P showed improved migration activity in Transwell assays compared with non-primed MSCs and concomitantly activated MAPK{sup p42/44} and AKT signaling cascades. Although C1P priming had little effect on cell surface marker phenotypes and the multipotency of MSCs, it potentiated their proliferative, colony-forming unit-fibroblast, and anti-inflammatory activities. In a monocrotaline-induced PAH animal model, a single administration of human MSCs primed with C1P significantly attenuated the PAH-related increase in right ventricular systolic pressure, right ventricular hypertrophy, and thickness of α-smooth muscle actin-positive cells around the vessel wall. Thus, this study shows that C1P priming increases the effects of MSC therapy by enhancing the migratory, self-renewal, and anti-inflammatory activity of MSCs and that MSC therapy optimized with priming protocols might be a promising option for the treatment of PAH patients. - Highlights: • Human BM-derived MSCs primed with C1P have enhanced migratory activity. • C1P primed MSCs increase proliferation, self-renewal, and anti-inflammatory capacity. • C1P priming enhances the therapeutic capacity of MSCs in a PAH animal model.

  20. Aging enhances the vulnerability of mesenchymal stromal cells to uniaxial tensile strain-induced apoptosis.

    Science.gov (United States)

    McKayed, Katey; Prendergast, Patrick J; Campbell, Veronica A

    2016-02-08

    Mechanical priming can be employed in tissue engineering strategies to control the fate and differentiation pattern of mesenchymal stromal cells. This is relevant to regenerative medicine whereby mechanical cues can promote the regeneration of a specific tissue type from mesenchymal precursors. The ability of cells to respond to mechanical forces is dependent upon mechanotransduction pathways that involve membrane-associated proteins, such as integrins. During the aging process changes in the mechanotransduction machinery may influence how cells from aged individuals respond to mechanical priming. In this study mesenchymal stromal cells were prepared from young adult and aged rats and exposed to uniaxial tensile strain at 5% and 10% for 3 days, or 2.5% for 7 days. Application of 5% tensile strain had no impact on cell viability. In contrast, application of 10% tensile strain evoked apoptosis and the strain-induced apoptosis was significantly higher in the mesenchymal stromal cells prepared from the aged rats. In parallel to the age-related difference in cellular responsiveness to strain, an age-related decrease in expression of α2 integrin and actin, and enhanced lipid peroxidation was observed. This study demonstrates that mesenchymal stem cells from aged animals have an altered membrane environment, are more vulnerable to the pro-apoptotic effects of 10% tensile strain and less responsive to the pro-osteogenic effects of 2.5% tensile strain. Thus, it is essential to consider how aged cells respond to mechanical stimuli in order to identify optimal mechanical priming strategies that minimise cell loss, particularly if this approach is to be applied to an aged population. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Automated Quantification of Hematopoietic CellStromal Cell Interactions in Histological Images of Undecalcified Bone

    Science.gov (United States)

    Zehentmeier, Sandra; Cseresnyes, Zoltan; Escribano Navarro, Juan; Niesner, Raluca A.; Hauser, Anja E.

    2015-01-01

    Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data. PMID:25938636

  2. Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct.

    Science.gov (United States)

    Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2017-02-01

    Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell-sheet technology non-invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature-responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post-transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 10 5 cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle-injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet-transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell-sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  3. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    Science.gov (United States)

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  4. Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

    Science.gov (United States)

    Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

    2014-12-01

    Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

  5. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Bolis, Sara; Lo Cicero, Viviana; Andriolo, Gabriella; Turchetto, Lucia

    2016-01-01

    The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed.

  6. A simple and efficient method for deriving neurospheres from bone marrow stromal cells

    International Nuclear Information System (INIS)

    Yang Qin; Mu Jun; Li Qi; Li Ao; Zeng Zhilei; Yang Jun; Zhang Xiaodong; Tang Jin; Xie Peng

    2008-01-01

    Bone marrow stromal cells (MSCs) can be differentiated into neuronal and glial-like cell types under appropriate experimental conditions. However, previously reported methods are complicated and involve the use of toxic reagents. Here, we present a simplified and nontoxic method for efficient conversion of rat MSCs into neurospheres that express the neuroectodermal marker nestin. These neurospheres can proliferate and differentiate into neuron, astrocyte, and oligodendrocyte phenotypes. We thus propose that MSCs are an emerging model cell for the treatment of a variety of neurological diseases

  7. Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

    Science.gov (United States)

    Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

    2018-01-06

    Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

  8. Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Jaroslaw Suchanski

    Full Text Available In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first

  9. Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

    Science.gov (United States)

    Suchanski, Jaroslaw; Tejchman, Anna; Zacharski, Maciej; Piotrowska, Aleksandra; Grzegrzolka, Jedrzej; Chodaczek, Grzegorz; Nowinska, Katarzyna; Rys, Janusz; Dziegiel, Piotr; Kieda, Claudine; Ugorski, Maciej

    2017-01-01

    In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such

  10. Macrophage inflammatory protein-3α influences growth of K562 leukemia cells in co-culture with anticancer drug-pretreated HS-5 stromal cells

    International Nuclear Information System (INIS)

    Lee, Y.C.; Chiou, T.-J.; Tzeng, W.-F.; Chu, S.T.

    2008-01-01

    Stromal cell monolayers have been an important means of studying the regulation of hematopoiesis, because they produce cytokines. Cytosine arabinoside, vincristine, daunorubicin, and doxorubicin are common drugs for hematological cancer therapy, and they may have some effects on bone marrow stroma during chemotherapy. The aim of this study was to elucidate interactions between the bone marrow stromal microenvironment and leukemic cells after drug treatment. We tested the hypothesis that human HS-5 stromal cells, pretreated with anticancer drugs, affected the growth of leukemic K562 cells by changing the cytokines in the culture microenvironment. Thereafter, proliferation of K562 cells increased nearly 2.5-fold compared the co-cultivation with drugs-pretreated HS-5 stromal cells and drugs-untreated HS-5 stromal cells. The results indicated that co-cultivation with HS-5 stromal cells pretreated with drugs caused significant K562 cell proliferation. Cytokines in the microenvironment were detected via the RayBio Human Cytokine Antibody Array Membrane. The levels of the cytokines CKβ, IL-12, IL-13, IGFBP-2, MCP-1, MCP-3, MCP-4, MDC, MIP-1β and MIP-1δ were decreased, with a particularly marked decrease in MIP-3α. In co-culture medium, there was a 20-fold decrease in MIP-3α in daunorubicin-pretreated HS-5 cells and at least a 3-fold decrease in Ara-C-pretreated cells. This indicated a significant effect of anticancer drugs on the stromal cell line. Using phosphorylated Erk and pRb proteins as cell proliferation markers, we found that phosphorylation of these markers in K562 cells was inhibited during co-cultivation with drug-pretreated stromal cells in MIP-3α-supplemented medium and restored by MIP-3α antibody supplement. In conclusion, anticancer drug pretreatment suppresses the negative control exerted by HS-5 cells on leukemic cell proliferation, via modulation of cytokines in the microenvironment, especially at the level of MIP-3α

  11. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  12. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    International Nuclear Information System (INIS)

    Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

    2011-01-01

    Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

  13. Quantification of stromal vascular cell mechanics with a linear cell monolayer rheometer

    Energy Technology Data Exchange (ETDEWEB)

    Elkins, Claire M., E-mail: cma9@stanford.edu; Fuller, Gerald G. [Department of Chemical Engineering, Stanford University, Stanford, California 94305 (United States); Shen, Wen-Jun; Khor, Victor K.; Kraemer, Fredric B. [Division of Endocrinology, Gerontology and Metabolism, Stanford University, Stanford, California 94305 and Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 (United States)

    2015-01-15

    Over the past few decades researchers have developed a variety of methods for measuring the mechanical properties of whole cells, including traction force microscopy, atomic force microscopy (AFM), and single-cell tensile testing. Though each of these techniques provides insight into cell mechanics, most also involve some nonideal conditions for acquiring live cell data, such as probing only one portion of a cell at a time, or placing the cell in a nonrepresentative geometry during testing. In the present work, we describe the development of a linear cell monolayer rheometer (LCMR) and its application to measure the mechanics of a live, confluent monolayer of stromal vascular cells. In the LCMR, a monolayer of cells is contacted on both top and bottom by two collagen-coated plates and allowed to adhere. The top plate then shears the monolayer by stepping forward to induce a predetermined step strain, while a force transducer attached to the top plate collects stress information. The stress and strain data are then used to determine the maximum relaxation modulus recorded after step-strain, G{sub r}{sup 0}, referred to as the zero-time relaxation modulus of the cell monolayer. The present study validates the ability of the LCMR to quantify cell mechanics by measuring the change in G{sub r}{sup 0} of a confluent cell monolayer upon the selective inhibition of three major cytoskeletal components (actin microfilaments, vimentin intermediate filaments, and microtubules). The LCMR results indicate that both actin- and vimentin-deficient cells had ∼50% lower G{sub r}{sup 0} values than wild-type, whereas tubulin deficiency resulted in ∼100% higher G{sub r}{sup 0} values. These findings constitute the first use of a cell monolayer rheometer to quantitatively distinguish the roles of different cytoskeletal elements in maintaining cell stiffness and structure. Significantly, they are consistent with results obtained using single-cell mechanical testing methods

  14. Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

    Science.gov (United States)

    Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

    2007-05-01

    Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

  15. Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.

    Science.gov (United States)

    de Soure, António M; Fernandes-Platzgummer, Ana; da Silva, Cláudia L; Cabral, Joaquim M S

    2016-10-20

    Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    International Nuclear Information System (INIS)

    Jewell, D.E.; Hausman, G.J.

    1986-01-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm 2 flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by [ 3 H]-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture

  17. Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

    International Nuclear Information System (INIS)

    Wang, J.-H.; Tuohimaa, Pentti

    2006-01-01

    Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

  18. Chemotaxis of primitive hematopoietic cells in response to stromal cell–derived factor-1

    Science.gov (United States)

    Jo, Deog-Yeon; Rafii, Shahin; Hamada, Tsuneyoshi; Moore, Malcolm A.S.

    2000-01-01

    Stromal cell–derived factor-1 (SDF-1) provides a potent chemotactic stimulus for CD34+ hematopoietic cells. We cultured mobilized peripheral blood (PB) and umbilical cord blood (CB) for up to 5 weeks and examined the migratory activity of cobblestone area–forming cells (CAFCs) and long-term culture–initiating cells (LTC-ICs) in a transwell assay. In this system, SDF-1 or MS-5 marrow stromal cells placed in the lower chamber induced transmembrane and transendothelial migration by 2- and 5-week-old CAFCs and LTC-ICs in 3 hours. Transmigration was blocked by preincubation of input CD34+ cells with antibody to CXCR4. Transendothelial migration of CB CAFCs and LTC-ICs was higher than that of PB. We expanded CD34+ cells from CB in serum-free medium with thrombopoietin, flk-2 ligand, and c-kit ligand, with or without IL-3 and found that CAFCs cultured in the absence of IL-3 had a chemotactic response equivalent to noncultured cells, even after 5 weeks. However, addition of IL-3 to the culture reduced this response by 20–50%. These data indicate that SDF-1 induces chemotaxis of primitive hematopoietic cells signaling through CXCR4 and that the chemoattraction could be downmodulated by culture ex vivo. PMID:10619866

  19. N-cadherin Expression in Testicular Germ Cell and Gonadal Stromal Tumors

    Directory of Open Access Journals (Sweden)

    Daniel J. Heidenberg, Joel H. Barton, Denise Young, Michael Grinkemeyer, Isabell A. Sesterhenn

    2012-01-01

    Full Text Available Neural-cadherin is a member of the cadherin gene family encoding the N-cadherin protein that mediates cell adhesion. N-cadherin is a marker of Sertoli cells and is also expressed in germ cells of varying stages of maturation. The purpose of this study was to determine the presence and distribution of this protein by immunohistochemistry in 105 germ cell tumors of both single and mixed histological types and 12 gonadal stromal tumors. Twenty-four germ cell tumors consisted of one cell type and the remaining were mixed. Of the 23 seminomas in either pure or mixed tumors, 74% were positive. Two spermatocytic seminomas were positive. Of the 83 cases with yolk sac tumor, 99% were positive for N-cadherin. The teratomas were positive in 73% in neuroectodermal and / or glandular components. In contrast, 87% of embryonal carcinomas did not express N-cadherin. Only 17% of the syncytiotrophoblastic cells were positive for N-cadherin. In conclusion, N-cadherin expression is very helpful in the identification of yolk sac tumors. In addition to glypican-3 and Sal-like protein 4, N-cadherin can be beneficial for the diagnosis and classification of this subtype of testicular germ cell tumor. Nine of the 12 gonadal stromal tumors were positive to a variable extent.

  20. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  1. Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice.

    Directory of Open Access Journals (Sweden)

    Subhrajit Saha

    Full Text Available Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS, resulting from direct cytocidal effects on intestinal stem cells (ISC and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome.Autologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy or abdominal irradiation (16-20 Gy in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p<0.0007 and p<0.0009 respectively beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated.Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated

  2. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-01-01

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  3. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  4. Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

    Science.gov (United States)

    Zhang, Jingzhu; Link, Daniel C

    2016-11-01

    The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

  5. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    Science.gov (United States)

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

  6. Transcriptional Profiling of Bone Marrow Stromal Cells in Response to Porphyromonas gingivalis Secreted Products

    Science.gov (United States)

    Reddi, Durga; Belibasakis, Georgios N.

    2012-01-01

    Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting (periodontal) tissues. Porphyromonas gingivalis is an oral pathogen highly implicated in the pathogenesis of this disease. It can exert its effects to a number of cells, including osteogenic bone marrow stromal cells which are important for homeostastic capacity of the tissues. By employing gene microarray technology, this study aimed to describe the overall transcriptional events (>2-fold regulation) elicited by P. gingivalis secreted products in bone marrow stromal cells, and to dissect further the categories of genes involved in bone metabolism, inflammatory and immune responses. After 6 h of challenge with P. gingivalis, 271 genes were up-regulated whereas 209 genes were down-regulated, whereas after 24 h, these numbers were 259 and 109, respectively. The early (6 h) response was characterised by regulation of genes associated with inhibition of cell cycle, induction of apoptosis and loss of structural integrity, whereas the late (24 h) response was characterised by induction of chemokines, cytokines and their associated intracellular pathways (such as NF-κB), mediators of connective tissue and bone destruction, and suppression of regulators of osteogenic differentiation. The most strongly up-regulated genes were lipocalin 2 (LCN2) and serum amyloid A3 (SAA3), both encoding for proteins of the acute phase inflammatory response. Collectively, these transcriptional changes elicited by P. gingivalis denote that the fundamental cellular functions are hindered, and that the cells acquire a phenotype commensurate with propagated innate immune response and inflammatory-mediated tissue destruction. In conclusion, the global transcriptional profile of bone marrow stromal cells in response to P. gingivalis is marked by deregulated homeostatic functions, with implications in the pathogenesis of periodontitis. PMID:22937121

  7. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

    Science.gov (United States)

    Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

    2018-02-07

    Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

  8. Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of Wagyu compared with those of Angus cattle.

    Science.gov (United States)

    Wei, S; Fu, X; Liang, X; Zhu, M J; Jiang, Z; Parish, S M; Dodson, M V; Zan, L; Du, M

    2015-03-01

    Japanese Wagyu cattle are well known for their extremely high marbling and lower subcutaneous adipose tissue compared with Angus cattle. However, mechanisms for differences in adipose deposition are unknown. The objective of this paper was to evaluate breed differences in the structure of subcutaneous adipose tissue, adipogenesis, and mitogenesis of stromal vascular (SV) cells between Wagyu and Angus cattle. Subcutaneous biopsy samples were obtained from 5 Wagyu (BW = 302 ± 9 kg) and 5 Angus (BW = 398 ± 12 kg) heifers at 12 mo of age, and samples were divided into 3 pieces for histological examination, biochemical analysis, and harvest of SV cells. Adipogenesis of SV cells was assessed by the expression of adipogenic markers and Oil Red-O staining, while mitogenesis was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium dromide) test, phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB; AKT). Based on histological analysis, Wagyu had larger adipocytes compared with Angus. At the tissue level, protein expression of peroxisome proliferator-activated receptor γ (PPARG) in Wagyu was much lower compared with that of Angus. Similarly, a lower mRNA expression of PPARG was found in Wagyu SV cells. No significant difference was observed for the zinc finger protein 423 (ZNF423) expression between Wagyu and Angus. As assessed by Oil Red-O staining, Wagyu SV cells possessed a notable trend of lower adipogenic capability. Interestingly, higher mitogenic ability was discovered in Wagyu SV cells, which was associated with an elevated phosphorylation of ERK1/2. There was no difference in AKT phosphorylation of SV cells between Wagyu and Angus. Moreover, exogenous fibroblast growth factor 2 (FGF2) enhanced mitogenesis and ERK1/2 phosphorylation of SV cells to a greater degree in Angus compared with that in Wagyu. Expression of transforming growth factor β 3 (TGFB3) and bone morphogenetic protein 2 (BMP2) in Wagyu SV

  9. Overexpression of matrix metalloproteinase-7 and -9 in NSCLC tumor and stromal cells: correlation with a favorable clinical outcome.

    Science.gov (United States)

    Stenvold, Helge; Donnem, Tom; Andersen, Sigve; Al-Saad, Samer; Al-Shibli, Khalid; Busund, Lill-Tove; Bremnes, Roy M

    2012-02-01

    Matrix metalloproteinases (MMPs) are considered important players in angiogenesis and cancer progression. Several drugs developed for targeting MMPs have until now been without clinical efficacy. As both malignant cells and cells of the surrounding stroma contribute to tumor growth, we have explored the impact of MMP-2, -7 and -9 expression in both the tumor and stromal compartment of non-small-cell lung cancers (NSCLC). From 335 unselected stage I to IIIA NSCLC carcinomas, duplicate tumor and tumor-associated stromal cores were collected in tissue microarrays (TMAs). Immunohistochemistry was used to detect the expression of MMP-2, -7 and -9 in tumor and stromal cells. In univariate analyses, high tumor cell MMP-7 expression (P=0.029) and high stromal MMP-9 expression (P=0.001) were positive prognostic factors. In the multivariate analysis, high tumor cell MMP-7 expression (HR 1.58, CI 1.08-2.32, P=0.020) and high stromal MMP-9 expression (HR 1.92, CI 1.25-2.96, P=0.003) were independent positive prognostic factors for disease-specific survival. High levels of MMP-7 in tumor cells and high levels of MMP-9 in tumor associated stroma were independent positive prognostic factors in NSCLC patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  10. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells.

    Science.gov (United States)

    Burns, Jorge S; Harkness, Linda; Aldahmash, Abdullah; Gautier, Laurent; Kassem, Moustapha

    2017-12-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures. Copyright © 2017. Published by Elsevier B.V.

  11. Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

    Science.gov (United States)

    Tsuzuki, Tomoko; Okada, Hidetaka; Cho, Hisayuu; Tsuji, Shoko; Nishigaki, Akemi; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2012-02-01

    Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P hypoxic conditions that could influence angiogenesis in the human endometrium.

  12. Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses.

    Directory of Open Access Journals (Sweden)

    Guido Moll

    Full Text Available Infusion of human third-party mesenchymal stromal cells (MSCs appears to be a promising therapy for acute graft-versus-host disease (aGvHD. To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46 and DAF (CD55, but were protected from complement lysis via expression of protectin (CD59. Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

  13. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

    Science.gov (United States)

    Sinha, Mithun; Sen, Chandan K; Singh, Kanhaiya; Das, Amitava; Ghatak, Subhadip; Rhea, Brian; Blackstone, Britani; Powell, Heather M; Khanna, Savita; Roy, Sashwati

    2018-03-05

    Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

  14. Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Koch, Thomas Gadegaard; Thomsen, Preben Dybdahl; Betts, Dean H.

    2009-01-01

      BACKGROUND AIMS: A robust methodology for the isolation of cord blood-derived multipotent mesenchymal stromal cells (CB-MSCs) from fresh umbilical cord blood has not been reported in any species. The objective of this study was to improve the isolation procedure for equine CB-MSCs. METHODS: Pre......-culture separation of red and white blood cells was done using either PrepaCyte?-EQ medium or Ficoll-Paque? PREMIUM density medium. Regular FBS and MSC-qualified FBS were compared for their ability to support the establishment of putative primary MSC colonies. RESULTS AND CONCLUSIONS: Our results indicate that Prepa...

  15. Aging of bone marrow mesenchymal stromal/stem cells: Implications on autologous regenerative medicine.

    Science.gov (United States)

    Charif, N; Li, Y Y; Targa, L; Zhang, L; Ye, J S; Li, Y P; Stoltz, J F; Han, H Z; de Isla, N

    2017-01-01

    With their proliferation, differentiation into specific cell types, and secretion properties, mesenchymal stromal/stem cells (MSC) are very interesting tools to be used in regenerative medicine. Bone marrow (BM) was the first MSC source characterized. In the frame of autologous MSC therapy, it is important to detect donor's parameters affecting MSC potency. Age of the donors appears as one parameter that could greatly affect MSC properties. Moreover, in vitro cell expansion is needed to obtain the number of cells necessary for clinical developments. It will lead to in vitro cell aging that could modify cell properties. This review recapitulates several studies evaluating the effect of in vitro and in vivo MSC aging on cell properties.

  16. COX-2 and Prostaglandin EP3/EP4 Signaling Regulate the Tumor Stromal Proangiogenic Microenvironment via CXCL12-CXCR4 Chemokine Systems

    Science.gov (United States)

    Katoh, Hiroshi; Hosono, Kanako; Ito, Yoshiya; Suzuki, Tatsunori; Ogawa, Yasufumi; Kubo, Hidefumi; Kamata, Hiroki; Mishima, Toshiaki; Tamaki, Hideaki; Sakagami, Hiroyuki; Sugimoto, Yukihiko; Narumiya, Shuh; Watanabe, Masahiko; Majima, Masataka

    2010-01-01

    Bone marrow (BM)–derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)−2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3−/− mice and EP4−/− mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins. PMID:20110411

  17. Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

    Science.gov (United States)

    Volak, Adrienn; LeRoy, Stanley G; Natasan, Jeya Shree; Park, David J; Cheah, Pike See; Maus, Andreas; Fitzpatrick, Zachary; Hudry, Eloise; Pinkham, Kelsey; Gandhi, Sheetal; Hyman, Bradley T; Mu, Dakai; GuhaSarkar, Dwijit; Stemmer-Rachamimov, Anat O; Sena-Esteves, Miguel; Badr, Christian E; Maguire, Casey A

    2018-05-16

    The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

  18. Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    DEFF Research Database (Denmark)

    Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

    2017-01-01

    Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

  19. Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

    International Nuclear Information System (INIS)

    Gery, Bernard; Little, John B.; Coppey, Jacques

    1996-01-01

    Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. Methods and Materials: The cell system consists of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. Results: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10 5 fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. Conclusions: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumors fragments

  20. An In Vivo Characterization of Trophic Factor Production Following Neural Precursor Cell or Bone Marrow Stromal Cell Transplantation for Spinal Cord Injury

    Science.gov (United States)

    Hawryluk, Gregory W.J.; Mothe, Andrea; Wang, Jian; Wang, Shelly; Tator, Charles

    2012-01-01

    Cellular transplantation strategies for repairing the injured spinal cord have shown consistent benefit in preclinical models, and human clinical trials have begun. Interactions between transplanted cells and host tissue remain poorly understood. Trophic factor secretion is postulated a primary or supplementary mechanism of action for many transplanted cells, however, there is little direct evidence to support trophin production by transplanted cells in situ. In the present study, trophic factor expression was characterized in uninjured, injured-untreated, injured-treated with transplanted cells, and corresponding control tissue from the adult rat spinal cord. Candidate trophic factors were identified in a literature search, and primers were designed for these genes. We examined in vivo trophin expression in 3 paradigms involving transplantation of either brain or spinal cord-derived neural precursor cells (NPCs) or bone marrow stromal cells (BMSCs). Injury without further treatment led to a significant elevation of nerve growth factor (NGF), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1), and lower expression of vascular endothelial growth factor isoform A (VEGF-A) and platelet-derived growth factor-A (PDGF-A). Transplantation of NPCs led to modest changes in trophin expression, and the co-administration of intrathecal trophins resulted in significant elevation of the neurotrophins, glial-derived neurotrophic factor (GDNF), LIF, and basic fibroblast growth factor (bFGF). BMSCs transplantation upregulated NGF, LIF, and IGF-1. NPCs isolated after transplantation into the injured spinal cord expressed the neurotrophins, ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), and bFGF at higher levels than host cord. These data show that trophin expression in the spinal cord is influenced by injury and cell transplantation, particularly when combined with intrathecal trophin infusion

  1. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  2. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Directory of Open Access Journals (Sweden)

    Djida Ghoubay-Benallaoua

    Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  3. Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.

    Science.gov (United States)

    Okada, Hidetaka; Okamoto, Rika; Tsuzuki, Tomoko; Tsuji, Shoko; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2011-09-01

    To investigate whether 17β-estradiol (E(2)) and progestins exert direct effects on vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1/CXCL12) in human endometrial stromal cells (ESCs) and thereby to clarify the regulatory function of these local angiogenic factors in the endometrium. In vitro experiment. Research laboratory at Kansai Medical University. Fourteen patients undergoing hysterectomy for benign reasons. ESCs were cultured with E(2) and/or various clinically relevant progestins (medroxyprogesterone acetate [MPA], norethisterone [NET], levonorgestrel [LNG], dienogest [DNG], and progesterone [P]). The mRNA levels and production of VEGF and SDF-1 were assessed by real-time reverse-transcription polymerase chain reaction and ELISA, respectively. E(2) significantly induced the mRNA levels and protein production of VEGF and SDF-1 in ESCs. MPA could antagonize the E(2)-stimulated effects in a time- and dose-dependent manner, and this effect could be reversed by RU-486 (P receptor antagonist). All of the progestins (MPA, NET, LNG, and DNG; 10(-9) to 10(-7) mol/L) attenuated E(2)-induced VEGF and SDF-1 production, whereas P showed these inhibitory effects only when present in a high concentration (10(-7) mol/L). Progestins have inhibitory effects on E(2)-induced VEGF and SDF-1 in ESCs. These results may indicate a potential mechanism for action of the female sex steroids in the human endometrium that can be helpful for various clinical applications. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  4. Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Kucerova, Lucia; Skolekova, Svetlana; Matuskova, Miroslava; Bohac, Martin; Kozovska, Zuzana

    2013-01-01

    Mesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3. The tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed. The AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil. Our work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses

  5. MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors

    NARCIS (Netherlands)

    Georgi, Nicole; Taipaleenmaeki, H.; Raiss, C.C.; Groen, N.; Portalska, K.K.; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine Nicole; van Wijnen, A.; Karperien, Hermanus Bernardus Johannes

    2015-01-01

    The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Because the differentiation potential of hMSCs differs between donors, it is necessary to establish

  6. Stromal cell-derived factor-1 alpha (SDF-1 alpha) improves neural recovery after spinal cord contusion in rats

    NARCIS (Netherlands)

    Zendedel, A.; Nobakht, M.; Bakhtiyari, M.; Beyer, C.; Kipp, M.; Baazm, M.; Joghataie, M.T.

    2012-01-01

    Stromal cell-derived factor-1 alpha (SDF-1α) is an important cytokine, implicated in the control of stem cell trafficking and bone marrow-derived stem cell mobilization. Generally, SDF-1α regulates multiple physiological processes such as embryonic development and organ homeostasis. There is also

  7. SERPINB2 is a novel TGFβ-responsive lineage fate determinant of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

    2017-01-01

    TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treat...

  8. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    NSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology......We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h......NSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation....

  9. Early postradition recovery of hematopoietic stromal precursor cells

    Energy Technology Data Exchange (ETDEWEB)

    Todriya, T.V.

    1985-04-01

    The aim of this investigation was an immunohistochemical study of alpha-endorphin-producing cells and also a study of rat mast cells (MC in the antral mucosa of the human stomach. Men aged 18 to 30 years undergoing in-patient treatment wre studied. According to the results of radioimmunoassay, antibodies against alpha-endorphin did not react with enkephalins, beta-endorphin, or the C-terminal fragment of beta-endorphin, but had cross reactivity of about 10% with gammaendorphin. Results were subjected to statistical analysis by Student's test at a 85% level of significance and they are shown. The facts presented here suggest that MC of human gastric mucosa include argyrophilic cells which contain alpha-endorphin.

  10. PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

    Science.gov (United States)

    Pincha, Neha; Hajam, Edries Yousaf; Badarinath, Krithika; Batta, Surya Prakash Rao; Masudi, Tafheem; Dey, Rakesh; Andreasen, Peter; Kawakami, Toshiaki; Samuel, Rekha; George, Renu; Danda, Debashish; Jacob, Paul Mazhuvanchary; Jamora, Colin

    2018-05-01

    Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

  11. Transcriptome analysis of bone marrow mesenchymal stromal cells from patients with primary myelofibrosis

    Directory of Open Access Journals (Sweden)

    Christophe Martinaud

    2015-09-01

    Full Text Available Primary myelofibrosis (PMF is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. W