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Sample records for cell differentiation

  1. Ghrelin and cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Geyang Xu; Yin Li; Wenjiao An; Weizhen Zhang

    2008-01-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is a gastric hormone that has been found to have a wide variety of biological functions. This review summarizes our current understanding of the effects of ghrelin on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells.

  2. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  3. Embryonic stem cell differentiation: A chromatin perspective

    OpenAIRE

    Rasmussen Theodore P

    2003-01-01

    Abstract Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to ...

  4. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells

    OpenAIRE

    Sally M Lansley; Searles, Richelle G.; Hoi, Aina; Thomas, Carla; Moneta, Helena; Herrick, Sarah E; Thompson, Philip J; Mark, Newman; Sterrett, Gregory F; Prêle, Cecilia M; Mutsaers, Steven E.

    2011-01-01

    Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm i...

  5. Differentiation into Endoderm Lineage: Pancreatic differentiation from Embryonic Stem Cells

    OpenAIRE

    2011-01-01

    The endoderm gives rise to digestive and respiratory tracts, thyroid, liver, and pancreas. Representative disease of endoderm lineages is type 1 diabetes resulting from destruction of the insulin-producing β cells. Generation of functional β cells from human embryonic stem (ES) cells in vitro can be practical, renewable cell source for replacement cell therapy for type 1 diabetes. It has been achieved by progressive instructive differentiation through each of the developmental stages. In this...

  6. Regulating cell differentiation at different layers

    Institute of Scientific and Technical Information of China (English)

    Jiarui Wu

    2011-01-01

    Cell differentiation is a basic behavior in the developmental process of multi-cellular organisms,through which various cell types are generated from one embryonic cell for further building different tissues and organs of animals or plants.It is estimated that there are more than two hundred cell types in a human body.To understand the molecular mechanisms of cell differentiation,researchers usually focus on a question how particular genes are selectively expressed during the differentiation process.However,more and more evidence indicates that the regulation of cell differentiation is far beyond simply controlling the expression of genetic program,which is supported by the collection of four research articles in this issue that the regulation of cell differentiation involves various factors at different layers,including epigenetics,metabolism and cell-cell interaction.

  7. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  8. Isologous diversification a theory of cell differentiation

    CERN Document Server

    Kaneko, K; Kaneko, Kunihiko; Yomo, Tetsuya

    1996-01-01

    Isologous diversification theory for cell differentiation is proposed, based on simulations of interacting cells with biochemical networks and cell division process following consumption of some chemicals. According to the simulations of the interaction-based dynamical systems model, the following scenario of the cell differentiation is proposed. (1) Up to some threshold number, divisions bring about almost identical cells with synchronized biochemical oscillations. (2)As the number is increased the oscillations lose the synchrony, leading to groups of cells with different phases of oscillations. (3)Amplitudes of oscillation and averaged chemical compositions start to differ by groups of cells. The differentiated behavior of states is transmitted to daughter cells. (4)Recursivity is formed so that the daughter cells keep the identical chemical character. This ``memory" is made possible through the transfer of initial conditions. (5) Successive differentiation proceeds. Mechanism of tumor cell formation, origi...

  9. Embryonic stem cell differentiation: a chromatin perspective.

    Science.gov (United States)

    Rasmussen, Theodore P

    2003-11-13

    Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  10. Embryonic stem cell differentiation: A chromatin perspective

    Directory of Open Access Journals (Sweden)

    Rasmussen Theodore P

    2003-11-01

    Full Text Available Abstract Embryonic stem (ES cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  11. Neurogenic differentiation of amniotic fluid stem cells.

    Science.gov (United States)

    Rosner, M; Mikula, M; Preitschopf, A; Feichtinger, M; Schipany, K; Hengstschläger, M

    2012-05-01

    In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

  12. Cell Division, Differentiation and Dynamic Clustering

    CERN Document Server

    Kaneko, K; Kaneko, Kunihiko; Yomo, Tetsuya

    1993-01-01

    A novel mechanism for cell differentiation is proposed, based on the dynamic clustering in a globally coupled chaotic system. A simple model with metabolic reaction, active transport of chemicals from media, and cell division is found to show three successive stages with the growth of the number of cells; coherent growth, dynamic clustering, and fixed cell differentiation. At the last stage, disparity in activities, germ line segregation, somatic cell differentiation, and homeochaotic stability against external perturbation are found. Our results, in consistency with the experiments of the preceding paper, imply that cell differentiation can occur without a spatial pattern. From dynamical systems viewpoint, the new concept of ``open chaos" is proposed, as a novel and general scenario for systems with growing numbers of elements, also seen in economics and sociology.A

  13. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen Lillelund;

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...... in the transcriptome was observed during the differentiation of the Caco-2 cells. 8762 of the 18149 genes analysed were expressed above background level in the undifferentiated Caco-2 cells, whereas only 5767 genes were expressed above background in differentiated Caco-2 cells. This pattern of expression was caused...... by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial...

  14. Stem cell differentiation and human liver disease

    Institute of Scientific and Technical Information of China (English)

    Wen-Li Zhou; Claire N Medine; Liang Zhu; David C Hay

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology.

  15. A Structured Population Model of Cell Differentiation

    CERN Document Server

    Doumic, Marie; Perthame, Benoit; Zubelli, Jorge P

    2010-01-01

    We introduce and analyze several aspects of a new model for cell differentiation. It assumes that differentiation of progenitor cells is a continuous process. From the mathematical point of view, it is based on partial differential equations of transport type. Specifically, it consists of a structured population equation with a nonlinear feedback loop. This models the signaling process due to cytokines, which regulate the differentiation and proliferation process. We compare the continuous model to its discrete counterpart, a multi-compartmental model of a discrete collection of cell subpopulations recently proposed by Marciniak-Czochra et al. in 2009 to investigate the dynamics of the hematopoietic system. We obtain uniform bounds for the solutions, characterize steady state solutions, and analyze their linearized stability. We show how persistence or extinction might occur according to values of parameters that characterize the stem cells self-renewal. We also perform numerical simulations and discuss the q...

  16. Differential geometry meets the cell.

    Science.gov (United States)

    Marshall, Wallace F

    2013-07-18

    A new study by Terasaki et al. highlights the role of physical forces in biological form by showing that connections between stacked endoplasmic reticulum cisternae have a shape well known in classical differential geometry, the helicoid, and that this shape is a predictable consequence of membrane physics.

  17. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

    2007-01-03

    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  18. Osteogenic Differentiation of Dental Follicle Stem Cells

    Directory of Open Access Journals (Sweden)

    Giorgio Mori, Andrea Ballini, Claudia Carbone, Angela Oranger, Giacomina Brunetti, Adriana Di Benedetto, Biagio Rapone, Stefania Cantore, Mariasevera Di Comite, Silvia Colucci, Maria Grano, Felice R. Grassi

    2012-01-01

    Full Text Available Background: Stem cells are defined as clonogenic cells capable of self-renewal and multi-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF.Dental Follicle Stem Cells (DFSCs were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mesenchymal tissues.Aim: The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers.Methods: We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR.Results: We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, producing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP and Collagen I (Coll I.Conclusion: This study suggests that DFSCs may provide a cell source for tissue engineering of bone.

  19. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells.

    Science.gov (United States)

    Lansley, Sally M; Searles, Richelle G; Hoi, Aina; Thomas, Carla; Moneta, Helena; Herrick, Sarah E; Thompson, Philip J; Newman, Mark; Sterrett, Gregory F; Prêle, Cecilia M; Mutsaers, Steven E

    2011-10-01

    Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0-26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.

  20. Biophysical regulation of stem cell differentiation.

    Science.gov (United States)

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  1. Nanotopographical Control of Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Laura E. McNamara

    2010-01-01

    Full Text Available Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated and direct (force-mediated mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

  2. Differential effects of lenalidomide during plasma cell differentiation.

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-05-10

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide.

  3. Differential effects of lenalidomide during plasma cell differentiation

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-01-01

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide. PMID:27057635

  4. Directed hepatic differentiation from embryonic stem cells

    OpenAIRE

    Chen, Xuesong; Zeng, Fanyi

    2011-01-01

    The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell trans...

  5. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  6. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen;

    2002-01-01

    in the transcriptome was observed during the differentiation of the Caco-2 cells. 8762 of the 18149 genes analysed were expressed above background level in the undifferentiated Caco-2 cells, whereas only 5767 genes were expressed above background in differentiated Caco-2 cells. This pattern of expression was caused...... by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial...... cells by performing reverse transcriptase-polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one - SART3 - was identified as a marker for human colonic crypts....

  7. Bioprinting and Differentiation of Stem Cells.

    Science.gov (United States)

    Irvine, Scott A; Venkatraman, Subbu S

    2016-09-08

    The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

  8. Bioprinting and Differentiation of Stem Cells

    Directory of Open Access Journals (Sweden)

    Scott A. Irvine

    2016-09-01

    Full Text Available The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

  9. Dazl Promotes Germ Cell Differentiation from Embryonic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Zhuo Yu; Ping Ji; Jinping Cao; Shu Zhu; Yao Li; Lin Zheng; Xuejin Chen; Lixin Feng

    2009-01-01

    It has been demonstrated that through the formation of embryoid bodies (Ebs) germ cells can be derived from embryonic stem (ES) cells. Here, we describe a transgene expression approach to derive germ cells directly from ES cells in vitro without EB formation. Through the ectopic expression of Deleted in Azoospermia-Like (Dazl), a germ cell-specific RNA-binding protein,both motile tailed-sperm and oocytes were induced from mouse ES (mES) cells in culture. Furthermore, transient overexpression of Dazl led to suppression of Nanog but induced germ cell nuclear antigen in mES cells. Dazl knockdown resulted in reduction in the expression of germ cell markers including Stella, MVH and Prdm1. Our study indicates that Dazl is a master gene controlling germ cell differentiation and that ectopic expression of Dazl promotes the dynamic differentiation of mouse ES cells into gametes in vitro.

  10. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    Science.gov (United States)

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  11. Signaling involved in stem cell reprogramming and differentiation

    Institute of Scientific and Technical Information of China (English)

    Shihori; Tanabe

    2015-01-01

    Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have reve-aled that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell pro-gramming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review,the molecular interactions and signaling pathways related to stem cell differentiation are discussed.

  12. Bioprinting and Differentiation of Stem Cells

    OpenAIRE

    2016-01-01

    The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink poly...

  13. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  14. Incidentally detected clear cell renal cell carcinoma with rhabdoid differentiation.

    Science.gov (United States)

    Krishnamoorthy, Venkatesh; Gowda, Kiran Krishne; Rao, Raman Narayana

    2016-01-01

    Renal cell carcinoma with rhabdoid differentiation (RCC-R) has an aggressive biologic behavior and poor prognosis. A recent consensus statement of the International Society of Urological Pathology (ISUP) proposed a nucleolar grading system (ISUP grade) for RCC to replace Fuhrman system and recommended reporting the presence of rhabdoid differentiation and considering tumors with rhabdoid differentiation to be ISUP Grade 4. We report a case of incidentally detected clear cell RCC-R in a 52-year-old man. This is one of the earliest cases of RCC-R (pT1b) detected and first such case from Indian subcontinent.

  15. Incidentally detected clear cell renal cell carcinoma with rhabdoid differentiation

    Directory of Open Access Journals (Sweden)

    Venkatesh Krishnamoorthy

    2016-01-01

    Full Text Available Renal cell carcinoma with rhabdoid differentiation (RCC-R has an aggressive biologic behavior and poor prognosis. A recent consensus statement of the International Society of Urological Pathology (ISUP proposed a nucleolar grading system (ISUP grade for RCC to replace Fuhrman system and recommended reporting the presence of rhabdoid differentiation and considering tumors with rhabdoid differentiation to be ISUP Grade 4. We report a case of incidentally detected clear cell RCC-R in a 52-year-old man. This is one of the earliest cases of RCC-R (pT1b detected and first such case from Indian subcontinent.

  16. Replication of prions in differentiated muscle cells.

    Science.gov (United States)

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication. (1) We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP(Sc), differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.

  17. Molecular mechanisms of male germ cell differentiation.

    Science.gov (United States)

    Hecht, N B

    1998-07-01

    During spermatogenesis, diploid stem cells differentiate, undergo meiosis, and transform into haploid spermatozoa. As this precisely timed series of events proceeds, chromosomal ploidy is reduced and the nucleosomes of the chromatin are replaced by a transcriptionally quiescent protamine-containing nucleus. The premature termination of transcription during the haploid phase of spermatogenesis necessitates an especially prominent role for posttranscriptional regulation in the temporal and spatial expression of many testis-specific proteins and isozymes. In this review article, discussion will focus on novel mechanisms regulating gene expression in mammalian male germ cells from genome to protein.

  18. [Human pluripotent stem cell and neural differentiation].

    Science.gov (United States)

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  19. Proteomic cornerstones of hematopoietic stem cell differentiation

    DEFF Research Database (Denmark)

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

  20. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  1. Differentiation of Bone Marrow Mesenchymal Cells to Neural Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the possibility and condition of differentiation of bone marrow mesenchymal cells (BMSCs) to neural cells in vitro, BMSCs from whole bone marrow of rats were cultured. The BMSCs of passage 3 were identified with immunocytochemical staining of CD44 ( + ), CD71 ( + )and CD45(-). There were type Ⅰ and type Ⅱ cells in BMSCs. Type Ⅰ BMSCs were spindleshaped and strong positive in immunocytochemical staining of CD44 and CD71, whereas flat and big type Ⅱ BMSCs were lightly stained. The BMSCs of same passage were induced to differentiate into neural cells by β-mercaptoethanol (BME). After induction by BME, the type Ⅰ BMSCs withdrew to form neuron-like round soma and axon-like and dendrite-like processes, and were stained positively for neurofilament (NF). The type Ⅱ BMSCs did not change in the BME medium and were negatively or slightly stained of NF.

  2. Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Zhao HE; Jing-jing LI; Chang-hong ZHEN; Lin-ying FENG; Xiao-yan DING

    2006-01-01

    Aim: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. Methods: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-gem layers was compared. Results: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. Conclusion:These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

  3. Probing stem cell differentiation using atomic force microscopy

    Science.gov (United States)

    Liang, Xiaobin; Shi, Xuetao; Ostrovidov, Serge; Wu, Hongkai; Nakajima, Ken

    2016-03-01

    A real-time method using atomic force microscopy (AFM) was developed to probe stem cell differentiation by measuring the mechanical properties of cells and the extracellular matrix (ECM). The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. It is clear that AFM is a facile and useful tool for monitoring the differentiation of stem cells in a non-invasive manner.

  4. Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

    Science.gov (United States)

    Martínez-Zárate, Alma Delia; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Cisneros, Bulmaro; Winder, Steve J; Cerecedo, Doris

    2014-06-01

    Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

  5. Oligodendrocyte differentiation and implantation : new insights for remyelinating cell therapy

    NARCIS (Netherlands)

    Sher, Falak; Balasubramaniyan, Veerakumar; Boddeke, Erik; Copray, Sjef

    2008-01-01

    Purpose of review Recent research on oligodendrocyte development has yielded new insights on the involvement of morphogens and differentiation factors in oligodendrogenesis. This knowledge has improved strategies to control neural stem cell-derived oligodendrocyte differentiation and functional matu

  6. Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse.

    Directory of Open Access Journals (Sweden)

    Rene Cortese

    Full Text Available Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs in undifferentiated embryonic stem cells (ESCs, in in-vitro induced neural stem cells (NSCs and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p1.96 enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

  7. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    Science.gov (United States)

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  8. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

  9. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    Science.gov (United States)

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  10. Differentiation of pluripotent stem cells for regenerative medicine.

    Science.gov (United States)

    Li, Ke; Kong, Yan; Zhang, Mingliang; Xie, Fei; Liu, Peng; Xu, Shaohua

    2016-02-26

    A long-standing goal in regenerative medicine is to obtain scalable functional cells on demand to replenish cells lost in various conditions, including relevant diseases, injuries, and aging. As an unlimited cell source, pluripotent stem cells (PSCs) are invaluable for regenerative medicine, because they have the potential to give rise to any cell type in an organism. For therapeutic purposes, it is important to develop specific approach to directing PSC differentiation towards desired cell types efficiently. Through directed differentiation, PSCs could give rise to scalable, clinically relevant cells for in vivo transplantation, as well as for studying diseases in vitro and discovering drugs to treat them. Over the past few years, significant progress has been made in directing differentiation of PSCs into a variety of cell types. In this review, we discuss recent progress in directed differentiation of PSCs, clinical translation of PSC-based cell replacement therapies, and remaining challenges.

  11. Non-genetic heterogeneity, criticality and cell differentiation

    Science.gov (United States)

    Pal, Mainak; Ghosh, Sayantari; Bose, Indrani

    2015-02-01

    The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

  12. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive wh

  13. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency.

    Science.gov (United States)

    Ohno, Yohei; Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Tohyama, Shugo; Saito, Yuki; Kunitomi, Akira; Shimoji, Kenichiro; Onizuka, Takeshi; Kageyama, Toshimi; Yae, Kojiro; Tanaka, Tomofumi; Kaneda, Ruri; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi

    2013-01-01

    Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  14. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

    Directory of Open Access Journals (Sweden)

    Yohei Ohno

    2013-01-01

    Full Text Available Patient-specific induced pluripotent stem (iPS cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1 the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2 the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  15. The transcriptional landscape of alpha beta T cell differentiation

    NARCIS (Netherlands)

    Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe; Best, Adam J.; Knell, Jamie; Goldrath, Ananda; Jojic, Vladimir; Koller, Daphne; Shay, Tal; Regev, Aviv; Cohen, Nadia; Brennan, Patrick; Brenner, Michael; Kim, Francis; Rao, Tata Nageswara; Wagers, Amy; Heng, Tracy; Ericson, Jeffrey; Rothamel, Katherine; Ortiz-Lopez, Adriana; Mathis, Diane; Bezman, Natalie A.; Sun, Joseph C.; Min-Oo, Gundula; Kim, Charlie C.; Lanier, Lewis L.; Miller, Jennifer; Brown, Brian; Merad, Miriam; Gautier, Emmanuel L.; Jakubzick, Claudia; Randolph, Gwendalyn J.; Monach, Paul; Blair, David A.; Dustin, Michael L.; Shinton, Susan A.; Hardy, Richard R.; Laidlaw, David; Collins, Jim; Gazit, Roi; Rossi, Derrick J.; Malhotra, Nidhi; Sylvia, Katelyn; Kang, Joonsoo; Kreslavsky, Taras; Fletcher, Anne; Elpek, Kutlu; Bellemare-Pelletier, Angelique; Malhotra, Deepali; Turley, Shannon

    2013-01-01

    The differentiation of abT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes

  16. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhe-Hao Zhang

    Full Text Available Nicotinic acid adenine dinucleotide phosphate (NAADP is an endogenous Ca(2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca(2+ from acidic organelles through two pore channel 2 (TPC2 in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

  17. Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells.

    Science.gov (United States)

    Abdelalim, Essam M; Emara, Mohamed M

    2015-01-26

    Pluripotent stem cells (PSCs) are able to differentiate into several cell types, including pancreatic β cells. Differentiation of pancreatic β cells depends on certain transcription factors, which function in a coordinated way during pancreas development. The existing protocols for in vitro differentiation produce pancreatic β cells, which are not highly responsive to glucose stimulation except after their transplantation into immune-compromised mice and allowing several weeks for further differentiation to ensure the maturation of these cells in vivo. Thus, although the substantial improvement that has been made for the differentiation of induced PSCs and embryonic stem cells toward pancreatic β cells, several challenges still hindering their full generation. Here, we summarize recent advances in the differentiation of PSCs into pancreatic β cells and discuss the challenges facing their differentiation as well as the different applications of these potential PSC-derived β cells.

  18. Early specification of dopaminergic phenotype during ES cell differentiation

    Directory of Open Access Journals (Sweden)

    Li Meng

    2007-07-01

    Full Text Available Abstract Background Understanding how lineage choices are made during embryonic stem (ES cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA. However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. Results Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. Conclusion Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.

  19. NOV/CCN3 impairs muscle cell commitment and differentiation.

    Science.gov (United States)

    Calhabeu, Frederico; Lafont, Jérome; Le Dreau, Gwenvael; Laurent, Maryvonne; Kazazian, Chantal; Schaeffer, Laurent; Martinerie, Cécile; Dubois, Catherine

    2006-06-10

    NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

  20. Derivation, propagation and differentiation of human embryonic stem cells.

    Science.gov (United States)

    Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

    2004-04-01

    Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

  1. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    Science.gov (United States)

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

  2. Differentiation of neuroepithelia from human embryonic stem cells

    OpenAIRE

    2009-01-01

    We describe the method for in vitro differentiation of neuroepithelial cells from human embryonic stem cells under a chemically defined condition. The protocol is established following the fundamental principle of in vivo neuroectodermal specification. The primitive neuroepithelial cells generated by this protocol can be further induced into neuronal and glia cells with forebrain, mid/hind brain, and spinal cord identities.

  3. Differentiation of embryonic stem cells into corneal epithelium

    Institute of Scientific and Technical Information of China (English)

    WANG Zhichong; LIU Jingbo; GE Jian; HUANG Bing; GAO Qianying; LIU Bingqian; WANG Linghua; YU Ling; FAN Zhigang; LU Xiaoming

    2005-01-01

    Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

  4. Differentiation of monkey embryonic stem cells into neural lineages.

    Science.gov (United States)

    Kuo, Hung-Chih; Pau, K-Y Francis; Yeoman, Richard R; Mitalipov, Shoukhrat M; Okano, Hideyuki; Wolf, Don P

    2003-05-01

    Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

  5. Parallel differentiation of embryonic stem cells into different cell types by a single gene-based differentiation system.

    Science.gov (United States)

    Thoma, Eva C; Maurus, Katja; Wagner, Toni U; Schartl, Manfred

    2012-04-01

    The generation of defined somatic cell types from pluripotent stem cells represents a promising system for many applications for regenerative therapy or developmental studies. Certain key developmental genes have been shown to be able to influence the fate determination of differentiating stem cells suggesting an alternative differentiation strategy to conventional medium-based methods. Here, we present a system allowing controlled, directed differentiation of embryonic stem cells (ESCs) solely by ectopic expression of single genes. We demonstrate that the myogenic master regulator myoD1 is sufficient to induce formation of skeletal muscle. In contrast to previous studies, our data suggest that myoD1-induced differentiation is independent of additional differentiation-inducing or lineage-promoting signals and occurs even under pluripotency-promoting conditions. Moreover, we demonstrate that single gene-induced differentiation enables the controlled formation of two distinct cell types in parallel. By mixing ES cell lines expressing myoD1 or the neural transcription factor ngn2, respectively, we generated a mixed culture of myocytes and neurons. Our findings provide new insights in the role of key developmental genes during cell fate decisions. Furthermore, this study represents an interesting strategy to obtain mixed cultures of different cells from stem cells, suggesting a valuable tool for cellular development and cell-cell interaction studies.

  6. The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation

    Science.gov (United States)

    Doty, Stephen B.; Stiner, Dalina; Telford, William G.

    2000-01-01

    In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

  7. Differentiation of stem cells upon deprivation of exogenous FGF2

    DEFF Research Database (Denmark)

    Kjartansdóttir, Kristín Rós; Gabrielsen, Anette; Reda, Ahmed

    2012-01-01

    Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous...... fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive...... impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added...

  8. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  9. Efficient differentiation of mouse embryonic stem cells into motor neurons.

    Science.gov (United States)

    Wu, Chia-Yen; Whye, Dosh; Mason, Robert W; Wang, Wenlan

    2012-06-09

    Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

  10. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Chung, Chong-Pyoung [Department of Periodontology, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  11. Interplay of Matrix Stiffness and Cell-Cell Contact in Regulating Differentiation of Stem Cells.

    Science.gov (United States)

    Ye, Kai; Cao, Luping; Li, Shiyu; Yu, Lin; Ding, Jiandong

    2016-08-31

    Stem cells are capable of sensing and responding to the mechanical properties of extracellular matrixes (ECMs). It is well-known that, while osteogenesis is promoted on the stiff matrixes, adipogenesis is enhanced on the soft ones. Herein, we report an "abnormal" tendency of matrix-stiffness-directed stem cell differentiation. Well-defined nanoarrays of cell-adhesive arginine-glycine-aspartate (RGD) peptides were modified onto the surfaces of persistently nonfouling poly(ethylene glycol) (PEG) hydrogels to achieve controlled specific cell adhesion and simultaneously eliminate nonspecific protein adsorption. Mesenchymal stem cells were cultivated on the RGD-nanopatterned PEG hydrogels with the same RGD nanospacing but different hydrogel stiffnesses and incubated in the induction medium to examine the effect of matrix stiffness on osteogenic and adipogenic differentiation extents. When stem cells were kept at a low density during the induction period, the differentiation tendency was consistent with the previous reports in the literature; however, both lineage commitments were favored on the stiff matrices at a high cell density. We interpreted such a complicated stiffness effect at a high cell density in two-dimensional culture as the interplay of matrix stiffness and cell-cell contact. As a result, this study strengthens the essence of the stiffness effect and highlights the combinatory effects of ECM cues and cell cues on stem cell differentiation.

  12. Granulosa cell proliferation differentiation and its role in follicular development

    Institute of Scientific and Technical Information of China (English)

    LU Cuiling; YANG Wei; HU Zhaoyuan; LIU Yixun

    2005-01-01

    Granuiosa cells (GCs) are the most important cells in the ovary that undergo serious changes morphologically and physiologically during the processes of follicular proliferation, differentiation, ovulation, lutenization and atresia. Oocyte (OC) directs GC proliferation and differentiation, while GCs influence OC maturation. Many ovarian factors are involved in the regulation of these processes via different molecular mechanisms and signal pathways. P38MAPK can selectively regulate steroidogenesis in GCs controlled by FSH; Transcript factors LRH-1 and DAX-1 play an important role in this process; FSH induces GC prolfferation and differentiation by stimulating PCNA and StAR expression and steroidogenesis. Activated ERK1/2 signal pathway may be involved in the FSH-regulated GC proliferation and differentiation. Therefore, GC is an ideal model for studying cell proliferation, differentiation and interaction,as well as signal transduction. This review briefly summarizes the latest data in the literature, including the results achieved in our laboratory.

  13. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  14. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immuno

  15. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew indefin

  16. Division of Labor in Biofilms : the Ecology of Cell Differentiation

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-01-01

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental

  17. Functional Implications of Neuroendocrine Differentiated Cells in Prostate Cancer

    NARCIS (Netherlands)

    J. Jongsma (Johan)

    2000-01-01

    textabstractThis thesis focuses on NE differentiation in prostate cancer, especially in prostate cancer models. We studied the effects of androgen depletion on the NE differentiated status of in vivo and in vitro prostatic tumor models. Knowledge concerning the function of NE cells in the normal hum

  18. Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh.

    Science.gov (United States)

    Kishimoto, Naotaka; Momota, Yoshihiro; Hashimoto, Yoshiya; Ando, Kayoko; Omasa, Takeshi; Kotani, Junichiro

    2013-01-01

    Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, L-ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering.

  19. NEW DESIGNED HMBA AGENTS AS INDUCERS OF ERYTHROLEUKEMIA CELL DIFFERENTIATION

    Institute of Scientific and Technical Information of China (English)

    王华力; 张世馥; 周建平; 章静波

    2002-01-01

    Objective.Searching for more potent and less toxic HMBA related agents. Methods.Human erythroleukemia cell K562,murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA,then analyzed the activity of inducing differentiation of two new designed HMBA derivatives:HMBPA [hexamethylenebi (3 pyridin) amide] and Co HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology,cytochemical and molecular biology techniques. Results.We found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive,B+ ~76% ).Co HDTA can inhibit the growth of MEL DS19,but induces differentiation just in a small population (B+ 2% ~4.5% ).Between 0.02~5μ mol/L,HMBPA induces 3% ~8% cells committed to differentiation with little inhibition of cell proliferation.1μ mol/L HMBPA and 2mmol/L HMBA together,can obviously increase the percentage of differentiated cell (B+ ~72% ),inhibit DNA synthesis and accelerate β globin transcription. Conclusion.The new HMBA derivatives may provide potential cancer differentiation inducers.

  20. Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

    Science.gov (United States)

    Saxena, Monika; Prashar, Paritosh; Yadav, Prem Swaroop; Sen, Jonaki

    2016-01-01

    Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

  1. The Therapeutic Potential of Differentiated Lung Cells from Embryonic Stem Cells in Lung Diseases.

    Science.gov (United States)

    Mokhber Dezfouli, Mohammad Reza; Chaleshtori, Sirous Sadeghian; Dehghan, Mohammad Mehdi; Tavanaeimanesh, Hamid; Baharvand, Hossein; Tahamtani, Yaser

    2017-01-01

    Lung diseases cause great morbidity and mortality. The choice of effective medical treatment is limited and the number of lung diseases are difficult to treat with current treatments. The embryonic stem cells (ESCs) have the potential to differentiate into cell types of all three germinal layers, including lung epithelial cells. So they can be a potential source for new cell therapies for hereditary or acquired diseases of the airways and lungs. One method for treatment of lung diseases is cell therapy and the use of ESCs that can replace the damaged epithelial and endothelial cells. Progress using ESCs has developed slowly for lung regeneration because differentiation of lung cells from ESCs is more difficult as compared to differentiation of other cells. The review studies the therapeutic effects of differentiated lung cells from embryonic stem cells in lung diseases. There are few studies of differentiation of ESCs into a lineage of respiratory and then investigation of this cell in experimental model of lung diseases.

  2. COMPUTATION MODELING OF TCDD DISRUPTION OF B CELL TERMINAL DIFFERENTIATION

    Science.gov (United States)

    In this study, we established a computational model describing the molecular circuit underlying B cell terminal differentiation and how TCDD may affect this process by impinging upon various molecular targets.

  3. Bone marrow cells differentiation into organ cells using stem cell therapy.

    Science.gov (United States)

    Yang, Y-J; Li, X-L; Xue, Y; Zhang, C-X; Wang, Y; Hu, X; Dai, Q

    2016-07-01

    Bone marrow cells (BMC) are progenitors of bone, cartilage, skeletal tissue, the hematopoiesis-supporting stroma and adipocyte cells. BMCs have the potential to differentiate into neural cells, cardiac myocytes, liver hepatocytes, chondrocytes, renal, corneal, blood, and myogenic cells. The bone marrow cell cultures from stromal and mesenchymal cells are called multipotent adult progenitor cells (MAPCs). MAPCs can differentiate into mesenchymal cells, visceral mesoderm, neuroectoderm and endoderm in vitro. It has been shown that the stem cells derived from bone marrow cells (BMCs) can regenerate cardiac myocytes after myocardial infarction (MI). Adult bone marrow mesenchymal stem cells have the ability to regenerate neural cells. Neural stem/progenitor cells (NS/PC) are ideal for treating central nervous system (CNS) diseases, such as Alzheimer's, Parkinson's and Huntington disease. However, there are important ethical issues about the therapeutic use of stem cells. Neurons, cardiac myocytes, hepatocytes, renal cells, blood cells, chondrocytes and adipocytes regeneration from BMCs are very important in disease control. It is known that limbal epithelial stem cells in the cornea can repair the eye sight and remove symptoms of blindness. Stem cell therapy (SCT) is progressing well in animal models, but the use of SCT in human remains to be explored further.

  4. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

  5. Extracellular vesicles derived from preosteoblasts influence embryonic stem cell differentiation.

    Science.gov (United States)

    Nair, Rekha; Santos, Lívia; Awasthi, Siddhant; von Erlach, Thomas; Chow, Lesley W; Bertazzo, Sergio; Stevens, Molly M

    2014-07-15

    Embryonic stem cells (ESCs) can differentiate into all cell types of the body and, therefore, hold tremendous promise for cell-based regenerative medicine therapies. One significant challenge that should be addressed before using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of this heterogeneous cell population. The work presented here examines the potential of harnessing naturally derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be ∼170 nm in diameter and to express the CD40 surface marker. Multiple interactions were visualized between vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted. Incubation with vesicles caused significant changes in ESC gene expression, including persistence of pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well as the cells from which they were derived were examined using a small microRNA (miRNA) gene array. Interestingly, ∼20% of the examined miRNAs were increased more than twofold in the vesicles compared with preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may improve the clinical potential of these physiological liposomes for therapeutic applications.

  6. Multiplicity of Sites for Extrathymic T-cell Differentiation

    OpenAIRE

    Abo, Toru; Watanabe, Hisami; Sekikawa, Hiroho

    1993-01-01

    In addition to an intrathymic pathway of T-cell differentiation, extrathymic pathways of T-cell differentiation have been found to exist at multiple sites in the living bodies of both mice and humans. Such sites include the sinusoids in the liver, intraepithelial sites in the intestine, the splenic red pulp, the thymic medulla, the decidua in the uterus, and the omentum in the peritoneal cavity. Although extrathymic pathways are minimal in youth, they become predominant with aging and under c...

  7. Differentiation of mammary stem cells in vivo and in vitro.

    Science.gov (United States)

    Barraclough, R; Rudland, P S

    1989-03-01

    The fully differentiated cells of the rat mammary parenchyma, the ductal epithelial, alveolar, and myoepithelial cells, are distinguished by their ultrastructure and by their accumulation of immunocytochemically detectable marker proteins. The different cell types probably develop from primative ductal structures called terminal end buds, which are present in the developing rat mammary glands, and these structures contain relatively undifferentiated cells. Clonal epithelial stem cell lines, obtained from normal rat mammary glands or benign mammary tumors, differentiate under appropriate conditions along a pathway to droplet-cell/doming cultures of primative alveolarlike cells. Under different culture conditions, the epithelial stem cells differentiate along a separate pathway to myoepitheliallike cells. They accumulate some of the specific marker proteins of myoepithelial cells in vivo, including type IV collagen, laminin, and Thy-1 antigen. In addition, these myoepitheliallike cells in culture contain an abundance of a potential calcium-binding protein, p9Ka, which also occurs in myoepithelial cells of histological sections from mammary glands. The accumulation of type IV collagen, laminin, Thy-1, and p9Ka occurs asynchronously along the pathway to the myoepitheliallike cells in vitro. Furthermore, the steady-state levels of these different marker proteins arise by alterations in the controls at the transcriptional, the posttranscriptional processing, and the translational stages of their production. These results suggest a stepwise control of synthesis of myoepithelial cell marker proteins, and in the case of p9Ka and Thy-1 antigen, this altered control may arise through their possession of novel transcriptional promoters.

  8. Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

    Science.gov (United States)

    Willems, Christophe; Vankelecom, Hugo

    2014-01-01

    The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

  9. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    Science.gov (United States)

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  10. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

    Science.gov (United States)

    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  11. Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiao-lei SHI; Yu-dong QIU; Qiang LI; Ting XIE; Zhang-hua ZHU; Lei-lei CHEN; Lei LI; Yi-tao DING

    2005-01-01

    Aim: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. Methods: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentia tion of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. Results:Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3β (HNF-3β), albumin (ALB), cytokeratin 18 (CK18), transthyretin (TFR), glucose-6-phosphate (G6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3β, ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. Conclusion: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

  12. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cell compartments, as consumption of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. On-going research in our lab aims at describing strain-dependent effects of lactic acid...... determined by ELISA. Co-incubation of NK cells and a Lactobacillus acidophilus strain caused increased proliferation of the NK cells and induced IFN-gamma production. The proliferative response was further enhanced in the presence of autologous monocytes, probably because cytokines, secreted by monocytes...... having engulfed bacteria, stimulated the growth of the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various lactobacilli have the capacity to activate NK cells in vitro, in a monocyte dependent...

  13. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

    Science.gov (United States)

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao

    2013-01-01

    Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

  14. Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Hans-Lothar Fuchsbauer

    2011-08-01

    Full Text Available For back disorders, cell therapy is one approach for a real regeneration of a degenerated nucleus pulposus. Human mesenchymal stem cells (hMSC could be differentiated into nucleus pulposus (NP-like cells and used for cell therapy. Therefore it is necessary to find a suitable biocompatible matrix, which supports differentiation. It could be shown that a differentiation of hMSC in a microbial transglutaminase cross-linked gelatin matrix is possible, but resulted in a more chondrocyte-like cell type. The addition of porcine NP extract to the gelatin matrix caused a differentiation closer to the desired NP cell phenotype. This concludes that a hydrogel containing NP extract without any other supplements could be suitable for differentiation of hMSCs into NP cells. The NP extract itself can be cross-linked by transglutaminase to build a hydrogel free of NP atypical substrates. As shown by side-specific biotinylation, the NP extract contains molecules with free glutamine and lysine residues available for the transglutaminase.

  15. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells

    Institute of Scientific and Technical Information of China (English)

    Li-Bo Chen; Xiao-Bing Jiang; Lian Yang

    2004-01-01

    AIM: To explore the possibility of marrow mesenchymal stem cells (MSC)in vitro differentiating into functional isletlike cells and to test the diabetes therapeutic potency of Islet-like cells.METHODS: Rat MSCs were isolated from Wistar rats and cultured. Passaged MSCs were induced to differentiate into islet-like cells under following conditions: pre-induction with L-DMEM including 10 mmol/L nicotinamide+1 mmol/L β-mercaptoethanol+200 mL/L fetal calf serum (FSC) for 24 h,followed by induction with serum free H-DMEM solution including 10 mmol/L nicotinamide+ 1 mmol/L,β-mercaptoethanol for 10 h. Differentiated cells were observed under inverse microscopy, insulin and nestin expressed in differentiated cells were detected with immunocytochemistry. Insulin excreted from differentiated cells was tested with radioimmunoassay. Rat diabetic models were made to test in vivo function of differentiated MSCs.RESULTS: Typical islet -like clustered cells were observed.Insulin mRNA and protein expressions were positive in differentiated cells, and nestin could be detected in predifferentiated cells. Insulin excreted from differentiated MSCs (446.93±102.28 IU/L) was much higher than that from pre-differentiated MSCs (2.45±0.81 IU/L (P<0.01).Injected differentiated MSCs cells could down-regulate glucose level in diabetic rats.CONCLUSION: Islet-like functional cells can be differentiated from marrow mesenchymal stem cells, which may be a new procedure for clinical diabetes stem -cell therapy, these cells can control blood glucose level in diabetic rats. MSCs may play an important role in diabetes therapy by islet differentiation and transplantation.

  16. Molecular ties between the cell cycle and differentiation in embryonic stem cells.

    Science.gov (United States)

    Li, Victor C; Kirschner, Marc W

    2014-07-01

    Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells.

  17. Cell Shape and Cardiosphere Differentiation: A Revelation by Proteomic Profiling

    Directory of Open Access Journals (Sweden)

    Nanako Kawaguchi

    2013-01-01

    Full Text Available Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

  18. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito;

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...

  19. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

    Directory of Open Access Journals (Sweden)

    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

  20. Cartilage stem cells: regulation of differentiation.

    Science.gov (United States)

    Solursh, M

    1989-01-01

    The developing limb bud is a useful source of cartilage stem cells for studies on the regulation of chondrogenesis. In high density cultures these cells can progress through all stages of chondrogenesis to produce mineralized hypertrophic cartilage. If the cells are maintained in a spherical shape, single stem cells can progress through a similar sequence. The actin cytoskeleton is implicated in the regulation of chondrogenesis since conditions that favor its disruption promote chondrogenesis and conditions that favor actin assembly inhibit chondrogenesis. Since a number of extracellular matrix receptors mediate effects of the extracellular matrix on cytoskeletal organization and some of these receptors are developmentally regulated, it is proposed that matrix receptor expression plays a central role in the divergence of connective tissue cells during development.

  1. Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

    Directory of Open Access Journals (Sweden)

    Cornelia Brendel

    Full Text Available RAS mutations are frequently found among acute myeloid leukemia patients (AML, generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1 in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC driven differentiation. Taken together, our findings show that AML with inv(16 and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

  2. Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells.

    Directory of Open Access Journals (Sweden)

    Opas Traitanon

    Full Text Available The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC and mTOR inhibitor (Sirolimus, SRL on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml profoundly inhibited CD19(+ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27(+ memory B cells were affected more by SRL than naïve CD27- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19(+CD138(+ and Blimp1(+/Pax5(low cells even at low dose (2 ng/ml, and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25(+/CD69(+ and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4(+CD25(- T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.

  3. Heterogeneous differentiation patterns of individual CD8+ T cells.

    Science.gov (United States)

    Gerlach, Carmen; Rohr, Jan C; Perié, Leïla; van Rooij, Nienke; van Heijst, Jeroen W J; Velds, Arno; Urbanus, Jos; Naik, Shalin H; Jacobs, Heinz; Beltman, Joost B; de Boer, Rob J; Schumacher, Ton N M

    2013-05-03

    Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

  4. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  5. Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Xingrong Yan; Liwen Li; Fulin Chen; Yanhong Yang; Wei Liu; Wenxin Geng; Huichong Du; Jihong Cui; Xin Xie; Jinlian Hua; Shumin Yu

    2013-01-01

    Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stem cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, βIII-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neurogenesis related genes (Sox-1, Nestin, GABA, Pax6, Zic5 and Pitx1) in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

  6. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    Science.gov (United States)

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  7. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  8. 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 main

  9. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during different

  10. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  11. Differential Protein Network Analysis of the Immune Cell Lineage

    Directory of Open Access Journals (Sweden)

    Trevor Clancy

    2014-01-01

    Full Text Available Recently, the Immunological Genome Project (ImmGen completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks.

  12. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  13. PPARγ agonists promote oligodendrocyte differentiation of neural stem cells by modulating stemness and differentiation genes.

    Directory of Open Access Journals (Sweden)

    Saravanan Kanakasabai

    Full Text Available Neural stem cells (NSCs are a small population of resident cells that can grow, migrate and differentiate into neuro-glial cells in the central nervous system (CNS. Peroxisome proliferator-activated receptor gamma (PPARγ is a nuclear receptor transcription factor that regulates cell growth and differentiation. In this study we analyzed the influence of PPARγ agonists on neural stem cell growth and differentiation in culture. We found that in vitro culture of mouse NSCs in neurobasal medium with B27 in the presence of epidermal growth factor (EGF and basic fibroblast growth factor (bFGF induced their growth and expansion as neurospheres. Addition of all-trans retinoic acid (ATRA and PPARγ agonist ciglitazone or 15-Deoxy-Δ(12,14-Prostaglandin J(2 (15d-PGJ2 resulted in a dose-dependent inhibition of cell viability and proliferation of NSCs in culture. Interestingly, NSCs cultured with PPARγ agonists, but not ATRA, showed significant increase in oligodendrocyte precursor-specific O4 and NG2 reactivity with a reduction in NSC marker nestin, in 3-7 days. In vitro treatment with PPARγ agonists and ATRA also induced modest increase in the expression of neuronal β-III tubulin and astrocyte-specific GFAP in NSCs in 3-7 days. Further analyses showed that PPARγ agonists and ATRA induced significant alterations in the expression of many stemness and differentiation genes associated with neuro-glial differentiation in NSCs. These findings highlight the influence of PPARγ agonists in promoting neuro-glial differentiation of NSCs and its significance in the treatment of neurodegenerative diseases.

  14. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Giada Graziana Genchi

    2015-01-01

    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  15. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    Science.gov (United States)

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  16. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  17. Role of Hox genes in stem cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Anne Seifert; David F Werheid; Silvana M Knapp; Edda Tobiasch

    2015-01-01

    Hox genes are an evolutionary highly conserved genefamily. They determine the anterior-posterior body axisin bilateral organisms and influence the developmentalfate of cells. Embryonic stem cells are usually devoidof any Hox gene expression, but these transcriptionfactors are activated in varying spatial and temporalpatterns defining the development of various bodyregions. In the adult body, Hox genes are among othersresponsible for driving the differentiation of tissuestem cells towards their respective lineages in order torepair and maintain the correct function of tissues andorgans. Due to their involvement in the embryonic andadult body, they have been suggested to be useable forimproving stem cell differentiations in vitro and in vivo .In many studies Hox genes have been found as drivingfactors in stem cell differentiation towards adipogenesis,in lineages involved in bone and joint formation, mainlychondrogenesis and osteogenesis, in cardiovascularlineages including endothelial and smooth muscle celldifferentiations, and in neurogenesis. As life expectancyis rising, the demand for tissue reconstruction continuesto increase. Stem cells have become an increasinglypopular choice for creating therapies in regenerativemedicine due to their self-renewal and differentiationpotential. Especially mesenchymal stem cells are usedmore and more frequently due to their easy handlingand accessibility, combined with a low tumorgenicityand little ethical concerns. This review therefore intendsto summarize to date known correlations betweennatural Hox gene expression patterns in body tissuesand during the differentiation of various stem cellstowards their respective lineages with a major focus onmesenchymal stem cell differentiations. This overviewshall help to understand the complex interactions of Hoxgenes and differentiation processes all over the bodyas well as in vitro for further improvement of stem celltreatments in future regenerative medicine approaches.

  18. Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Qiaoli Gu

    2012-01-01

    Full Text Available Background: Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO-1 expression. Curcumin can potently induce HO-1 expression. Objective: The present study describes the effects of curcumin on rat MSC (rMSCs differentiation into osteoblasts and adipocytes. Materials and Methods: Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2 and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2 and CCAAT/enhancer-binding protein (C/EBP α. Results: Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs. Conclusions: These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

  19. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  20. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  1. Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

    Science.gov (United States)

    2015-10-01

    supplemented with 10% fetal bovine serum. Detection and quantification of neurite outgrowth Cells were plated and treated in 96-well plates. For measuring...inhibits the stemness of glioma stem cells by target- ing RTVP-1. Oncotarget 2013; 4(5):665-76; PMID:23714687 14. Trang P, Wiggins JF, Daige CL, Cho C...Award Number: W81XWH-13-1-0241 TITLE: Identifying that Regulate Neuroblastoma Cell Differentiation PRINCIPAL INVESTIGATOR: Dr. Liqin Du

  2. The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

    Science.gov (United States)

    Cha, Min-Ji; Choi, Eunhyun; Lee, Seahyoung; Song, Byeong-Wook; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-02-15

    In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3β (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

  3. Interleukin-1 regulates proliferation and differentiation of oligodendrocyte progenitor cells.

    Science.gov (United States)

    Vela, José M; Molina-Holgado, Eduardo; Arévalo-Martín, Angel; Almazán, Guillermina; Guaza, Carmen

    2002-07-01

    Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1beta in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1beta were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1beta and express IL-1 receptors. Unlike other proinflammatory cytokines (TNFalpha and IFNgamma), IL-1beta was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1beta blocked proliferation at the late progenitor/prooligodendrocyte (O4+) stage but did not affect proliferation of early progenitors (A2B5+). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab+ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1beta promoted maturation to the MBP+ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1beta activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1beta also induced a time-dependent inhibition of PFGF-Ralpha gene expression. These results support a role for IL-1beta in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating

  4. Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells

    Institute of Scientific and Technical Information of China (English)

    Essam M Abdelalim; Mohamed M Emara

    2015-01-01

    Pluripotent stem cells (PSCs) are able to differentiate intoseveral cell types, including pancreatic β cells. Differentiationof pancreatic β cells depends on certain transcriptionfactors, which function in a coordinated way duringpancreas development. The existing protocols for in vitrodifferentiation produce pancreatic β cells, which are nothighly responsive to glucose stimulation except after theirtransplantation into immune-compromised mice and allowingseveral weeks for further differentiation to ensurethe maturation of these cells in vivo . Thus, although thesubstantial improvement that has been made for the differentiationof induced PSCs and embryonic stem cellstoward pancreatic β cells, several challenges still hinderingtheir full generation. Here, we summarize recent advancesin the differentiation of PSCs into pancreatic β cells anddiscuss the challenges facing their differentiation as wellas the different applications of these potential PSC-derivedβ cells.

  5. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

    Science.gov (United States)

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed

    2016-01-25

    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

  6. Energy Metabolism in Mesenchymal Stem Cells During Osteogenic Differentiation

    Science.gov (United States)

    Shum, Laura C.; White, Noelle S.; Mills, Bradley N.; de Mesy Bentley, Karen L.

    2016-01-01

    There is emerging interest in stem cell energy metabolism and its effect on differentiation. Bioenergetic changes in differentiating bone marrow mesenchymal stem cells (MSCs) are poorly understood and were the focus of our study. Using bioenergetic profiling and transcriptomics, we have established that MSCs activate the mitochondrial process of oxidative phosphorylation (OxPhos) during osteogenic differentiation, but they maintain levels of glycolysis similar to undifferentiated cells. Consistent with their glycolytic phenotype, undifferentiated MSCs have high levels of hypoxia-inducible factor 1 (HIF-1). Osteogenically induced MSCs downregulate HIF-1 and this downregulation is required for activation of OxPhos. In summary, our work provides important insights on MSC bioenergetics and proposes a HIF-based mechanism of regulation of mitochondrial OxPhos in MSCs. PMID:26487485

  7. Geometric cues for directing the differentiation of mesenchymal stem cells

    Science.gov (United States)

    Kilian, Kristopher A.; Bugarija, Branimir; Lahn, Bruce T.; Mrksich, Milan

    2010-01-01

    Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates. PMID:20194780

  8. Derivation and differentiation of haploid human embryonic stem cells.

    Science.gov (United States)

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-07

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  9. Differential expression of genes involved in the epigenetic regulation of cell identity in normal human mammary cell commitment and differentiation

    Institute of Scientific and Technical Information of China (English)

    Danila Coradini; Patrizia Boracchi; Saro Oriana; Elia Biganzoli; Federico Ambrogi

    2014-01-01

    The establishment and maintenance of mammary epithelial cell identity depends on the activity of a group of proteins, collectively called maintenance proteins, that act as epigenetic regulators of gene transcription through DNA methylation, histone modification, and chromatin remodeling. Increasing evidence indicates that dysregulation of these crucial proteins may disrupt epithelial cellintegrity and trigger breast tumor initiation. Therefore, we exploredin silico the expression pattern of a panel of 369 genes known to be involved in the establishment and maintenance of epithelial cellidentity and mammary gland remodeling in cell subpopulations isolated from normal human mammary tissue and selectively enriched in their content of bipotent progenitors, committed luminal progenitors, and differentiated myoepithelial or differentiated luminal cells. The results indicated that, compared to bipotent cells, differentiated myoepithelial and luminal subpopulations were both characterized by the differential expression of 4 genes involved in cell identity maintenance:CBX6 andPCGF2, encoding proteins belonging to the Polycomb group, andSMARCD3 andSMARCE1, encoding proteins belonging to the Trithorax group. In addition to these common genes, the myoepithelial phenotype was associated with the differential expression of HDAC1, which encodes histone deacetylase 1, whereas the luminal phenotype was associated with the differential expression ofSMARCA4 andHAT1, which encode a Trithorax protein and histone acetylase 1, respectively. The luminal compartment was further characterized by the overexpression ofALDH1A3 and GATA3, and the down-regulation ofNOTCH4and CCNB1, with the latter suggesting a block in cell cycle progression at the G2 phase. In contrast, myoepithelial differentiation was associated with the overexpression ofMYC and the down-regulation ofCCNE1, with the latter suggesting a block in cellcycle progression at the G1 phase.

  10. Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

    Science.gov (United States)

    Czeisler, Catherine; Short, Aaron; Nelson, Tyler; Gygli, Patrick; Ortiz, Cristina; Catacutan, Fay Patsy; Stocker, Ben; Cronin, James; Lannutti, John; Winter, Jessica; Otero, José Javier

    2016-12-01

    We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.

  11. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  12. Structural properties of scaffolds: Crucial parameterstowards stem cells differentiation

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Tissue engineering is a multidisciplinary field thatapplies the principles of engineering and life-sciencesfor regeneration of damaged tissues. Stem cells haveattracted much interest in tissue engineering as a cellsource due to their ability to proliferate in an undifferentiatedstate for prolonged time and capability ofdifferentiating to different cell types after induction.Scaffolds play an important role in tissue engineeringas a substrate that can mimic the native extracellularmatrix and the properties of scaffolds have been shownto affect the cell behavior such as the cell attachment,proliferation and differentiation. Here, we focus on therecent reports that investigated the various aspectsof scaffolds including the materials used for scaffoldfabrication, surface modification of scaffolds, topographyand mechanical properties of scaffolds towards stemcells differentiation effect. We will present a moredetailed overview on the effect of mechanical propertiesof scaffolds on stem cells fate.

  13. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  14. Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas

    DEFF Research Database (Denmark)

    Kim, Yung Hae; Larsen, Hjalte List; Rué, Paul;

    2015-01-01

    Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine...

  15. DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLEATION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    韩代书; 赵青; 葛晔华; 周建平; 马静; 陈克铨; 薛社普

    2002-01-01

    Objective. To investigate the roles of mouse erythroid differentiation and denueleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation.Methods. Mouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcD-NA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate,mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and β-globin genes were analysed by semi-quantitative RT-PCR.Results. MEL ceils transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index,and colony-forming rate in semi-solid medium ( P<0.01 ). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of β-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3. 1 ), and the expression of c-myc decreased by 66.3%.Conclusions. MEDDF can induce differentiation of MEL cell and suppress its malignancy.

  16. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    Science.gov (United States)

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  17. MicroRNA in cell differentiation and development

    Institute of Scientific and Technical Information of China (English)

    SHI Yi; JIN YouXin

    2009-01-01

    The regulation of gene expression by microRNAs (miRNAs) Is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal develop-ment and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are po-tential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  18. MicroRNA in cell differentiation and development

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The regulation of gene expression by microRNAs(miRNAs) is a recently discovered pattern of gene regulation in animals and plants.MiRNAs have been implicated in various aspects of animal development and cell differentiation,such as early embryonic development,neuronal development,muscle development,and lymphocyte development,by the analysis of genetic deletions of individual miRNAs in mammals.These studies show that miRNAs are key regulators in animal development and are potential causes of human diseases.Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  19. Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

    Science.gov (United States)

    Roberts, R Michael; Loh, Kyle M; Amita, Mitsuyoshi; Bernardo, Andreia S; Adachi, Katsuyuki; Alexenko, Andrei P; Schust, Danny J; Schulz, Laura C; Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Pedersen, Roger A

    2014-05-01

    It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  20. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi;

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  1. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  2. Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

    Science.gov (United States)

    Tsurumachi, Niina; Akita, Daisuke; Kano, Koichiro; Matsumoto, Taro; Toriumi, Taku; Kazama, Tomohiko; Oki, Yoshinao; Tamura, Yoko; Tonogi, Morio; Isokawa, Keitaro; Shimizu, Noriyoshi; Honda, Masaki

    2016-03-01

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 μm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-μm nylon mesh filters: cell diameters less than 40 μm (small adipocytes: S-adipocytes) and cell diameters of 40-100 μm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells.

  3. Differentiation of human mesenchymal stem cell spheroids under microgravity conditions

    Directory of Open Access Journals (Sweden)

    Cerwinka Wolfgang H

    2012-06-01

    Full Text Available Abstract To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 106 cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

  4. Transcription factor interplay in T helper cell differentiation.

    Science.gov (United States)

    Evans, Catherine M; Jenner, Richard G

    2013-11-01

    The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

  5. Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

    Science.gov (United States)

    Maarof, Ghyath; Bouchet-Delbos, Laurence; Gary-Gouy, Hélène; Durand-Gasselin, Ingrid; Krzysiek, Roman; Dalloul, Ali

    2010-03-04

    Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

  6. The production and directed differentiation of human embryonic stem cells.

    Science.gov (United States)

    Trounson, Alan

    2006-04-01

    Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of

  7. NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation.

    Science.gov (United States)

    Wang, Linghao; Chen, Ke; Wan, Xinxing; Wang, Fang; Guo, Zi; Mo, Zhaohui

    2017-03-18

    Osteoporosis is one of the most common skeletal disease featured by osteopenia and adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis.

  8. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  9. Delivery of differentiation factors by mesoporous silica particles assists advanced differentiation of transplanted murine embryonic stem cells

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; Kozhevnikova, Mariya; König, Niclas;

    2013-01-01

    Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long-term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application...... neurotrophic factor and glial cell line-derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long-term survival in vivo. We propose that the delivery of growth factors...... by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation....

  10. Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

    Institute of Scientific and Technical Information of China (English)

    Takashi Nakamura; Yuta Chiba; Masahiro Naruse; Kan Saito; Hidemitsu Harada; Satoshi Fukumoto

    2016-01-01

    Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

  11. Correlation between membrane fluidity cellular development and stem cell differentiation

    KAUST Repository

    Noutsi, Pakiza

    2016-12-01

    Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  12. Optical imaging for stem cell differentiation to neuronal lineage.

    Science.gov (United States)

    Hwang, Do Won; Lee, Dong Soo

    2012-03-01

    In regenerative medicine, the prospect of stem cell therapy holds great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell- or tissue-specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating into a neuronal lineage. The detection limit of weak promoters or reporter genes can be greatly enhanced by adopting a yeast GAL4 amplification system or an engineering-enhanced luciferase reporter gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  13. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  14. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  15. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  16. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds

    DEFF Research Database (Denmark)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette

    2007-01-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We...... polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer h......MSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small...

  17. Arsenic inhibits hedgehog signaling during P19 cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jui Tung [Environmental Toxicology Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2014-12-15

    Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mouse embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH

  18. Plant phosphoglycerolipids: the gatekeepers of vascular cell differentiation

    Directory of Open Access Journals (Sweden)

    Bojan eGujas

    2016-02-01

    Full Text Available In higher plants, the plant vascular system has evolved as an inter-organ communication network essential to deliver a wide range of signaling factors among distantly separated organs. To become conductive elements, phloem and xylem cells undergo a drastic differentiation program that involves the degradation of the majority of their organelles. While the molecular mechanisms regulating such complex process remain poorly understood, it is nowadays clear that phosphoglycerolipids display a pivotal role in the regulation of vascular formation. In animal cells, this class of lipids is known to mediate acute responses as signal transducers and also act as constitutive signals that help defining organelle identity. Their rapid turnover, asymmetrical distribution across subcellular compartments as well as their ability to rearrange cytoskeleton fibers make phosphoglycerolipids excellent candidates to regulate complex morphogenetic processes such as vascular differentiation. Therefore, in this review we aim to summarize, emphasize and connect our current understanding about the involvement of phosphoglycerolipids in phloem and xylem differentiation.

  19. Inorganic arsenic impairs differentiation and functions of human dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Macoch, Mélinda; Morzadec, Claudie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes (France); Vernhet, Laurent, E-mail: laurent.vernhet@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France)

    2013-01-15

    Experimental studies have demonstrated that the antileukemic trivalent inorganic arsenic prevents the development of severe pro-inflammatory diseases mediated by excessive Th1 and Th17 cell responses. Differentiation of Th1 and Th17 subsets is mainly regulated by interleukins (ILs) secreted from dendritic cells (DCs) and the ability of inorganic arsenic to impair interferon-γ and IL-17 secretion by interfering with the physiology of DCs is unknown. In the present study, we demonstrate that high concentrations of sodium arsenite (As(III), 1–2 μM) clinically achievable in plasma of arsenic-treated patients, block differentiation of human peripheral blood monocytes into immature DCs (iDCs) by inducing their necrosis. Differentiation of monocytes in the presence of non-cytotoxic concentrations of As(III) (0.1 to 0.5 μM) only slightly impacts endocytotic activity of iDCs or expression of co-stimulatory molecules in cells activated with lipopolysaccharide. However, this differentiation in the presence of As(III) strongly represses secretion of IL-12p70 and IL-23, two major regulators of Th1 and Th17 activities, from iDCs stimulated with different toll-like receptor (TLR) agonists in metalloid-free medium. Such As(III)-exposed DCs also exhibit reduced mRNA levels of IL12A and/or IL12B genes when activated with TLR agonists. Finally, differentiation of monocytes with non-cytotoxic concentrations of As(III) subsequently reduces the ability of activated DCs to stimulate the release of interferon-γ and IL-17 from Th cells. In conclusion, our results demonstrate that clinically relevant concentrations of inorganic arsenic markedly impair in vitro differentiation and functions of DCs, which may contribute to the putative beneficial effects of the metalloid towards inflammatory autoimmune diseases. Highlights: ► Inorganic arsenic impairs differentiation and functions of human dendritic cells (DCs) ► Arsenite (> 1 μM) blocks differentiation of dendritic cells by

  20. Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

    Science.gov (United States)

    Werner, Maike; Blanquer, Sébastien B. G.; Haimi, Suvi P.; Korus, Gabriela; Dunlop, John W. C.; Duda, Georg N.; Grijpma, Dirk. W.

    2016-01-01

    Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well‐defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward‐lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin‐A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell‐instructive material parameter in the field of biomaterial‐guided tissue regeneration.

  1. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  2. Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation.

    Directory of Open Access Journals (Sweden)

    Zhilong Li

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

  3. Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Jin YANG; Guo-qiang LIU; Rui WEI; Wen-fang HOU; Mei-juan GAO; Ming-xia ZHU; Hai-ning WANG; Gui-an CHEN; Tian-pei HONG

    2011-01-01

    Aim:Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells.The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and,if so,whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α).Methods:Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol.The cumulative percentage of beating EBs was calculated.The expression of cardiac-specific markers including cardiac troponin Ⅰ (cTnl) and α-myosin heavy chain (α-MHC) was detected using RT-PCR,real-time PCR and Western blot.The dispersed beating EBs were examined using immunofluorescent staining.Results:The percentage of beating EBs and the expression of cTnl were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium.From 6 to 18 d of differentiation,the increased expression of cTnl and α-MHC by ghrelin (1 nmol/L)was time-dependent,and in line with the alteration of the percentages of beating EBs.Furthermore,the dispersed beating EBs were double-positively immunostained with antibodies against cTnl and α-actinin.However,blockage of GHS-R1α with its specific antagonist D-[lys3]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation.Conclusion:Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells,which is not mediated via GHS-R1α.

  4. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Changqing Ye; Xiaodong Yuan; Hui Liu; Yanan Cai; Ya Ou

    2010-01-01

    β-mercaptoethanol induces in vitro adult adipose-derived stromal cells (ADSCs) to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptcethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker (nestin), a neuronal marker (neuron-specific enolase), and a glial marker (glial fibrillary acidic protein). In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

  5. Human pluripotent stem cell differentiation into authentic striatal projection neurons.

    Science.gov (United States)

    Delli Carri, Alessia; Onorati, Marco; Castiglioni, Valentina; Faedo, Andrea; Camnasio, Stefano; Toselli, Mauro; Biella, Gerardo; Cattaneo, Elena

    2013-08-01

    Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington's Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1(+)/GSX2(+) progenitor cells of the lateral ganglionic eminence (LGE) at day 15-25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2(+)/GABA(+) neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs.The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

  6. Silk scaffolds with tunable mechanical capability for cell differentiation.

    Science.gov (United States)

    Bai, Shumeng; Han, Hongyan; Huang, Xiaowei; Xu, Weian; Kaplan, David L; Zhu, Hesun; Lu, Qiang

    2015-07-01

    Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an "inert" material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high β-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall β-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells toward myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features.

  7. Selenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells.

    Science.gov (United States)

    Yan, Jidong; Fei, Yao; Han, Yan; Lu, Shemin

    2016-10-01

    Selenoprotein O (Sel O) is a selenium-containing protein, but its function is still unclear. In the present study, we observed that the mRNA and protein expression levels of Sel O increased during chondrogenic induction of ATDC5 cells. The effects of Sel O on chondrocyte differentiation were then examined through shRNA-mediated gene silencing technique. The expression of Sel O was significantly suppressed at both mRNA and protein levels in a stable cell line transfected with a Sel O-specific target shRNA construct. Thereafter, we demonstrated that Sel O deficiencies suppress chondrogenic differentiation of ATDC5 cells. Sel O deficiencies inhibited expression of chondrogenic gene Sox9, Col II, and aggrecan. Sel O-deficient cells also accumulated a few cartilage glycosaminoglycans (GAGs) and decreased the activity of alkaline phosphatase (ALP). In addition, Sel O deficiencies inhibited chondrocyte proliferation through delayed cell cycle progression by suppression of cyclin D1 expression. Moreover, Sel O deficiencies induced chondrocyte death through cell apoptosis. In summary, we describe the expression patterns and the essential roles of Sel O in chondrocyte viability, proliferation, and chondrogenic differentiation. Additionally, Sel O deficiency-mediated impaired chondrogenesis may illustrate the mechanisms of Se deficiency in the pathophysiological process of the endemic osteoarthropathy.

  8. Differentiation: A Central Topic in Developmental and Cell Biology

    Science.gov (United States)

    Müller, W. A.

    The concept of "differentiation" encompasses all processes leading to differently specialized cell types, beginning with the progressive divergence of developmental pathways and ending with the successive programming and final elaboration of each particular cell type. Guidance and positional information are provided by external cues, by differentially allotted cytoplasmic determinants such as mRNA for transcription factors, and by cascades of intercellular signals. Eventually cell type specific selector genes, such as the muscle cell determining MyoD/myogenin genes and neural key genes (e.g., achaete scute-C, neurogenin), are switched on which control entire sets of subordinate effector genes. In multiplying cells "cell heredity" based on an epigenetic cellular memory enables transmission of the cell type determining program from parental to daughter cells. This memory can be based on autocatalytic self-activation of cell type specific selector genes and on the enduring action of gene groups such as the Polycomb and thrithorax complexes that code for proteins which bind to DNA sequences called cellular memory modules. These modules confer permanent accessibility (potentiation) or inaccessibility (silencing) upon many different gene loci on the chromosomes.

  9. Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Javad Verdi; Seyed Abdolreza Mortazavi-Tabatabaei; Shiva Sharif; Hadi Verdi; Alireza Shoae-Hassani

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

  10. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells.

    Science.gov (United States)

    Sunitha, Manne Mudhu; Srikanth, Lokanathan; Santhosh Kumar, Pasupuleti; Chandrasekhar, Chodimella; Sarma, Potukuchi Venkata Gurunadha Krishna

    2016-10-01

    Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.

  11. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  12. Increased differentiation of Th22 cells in Hashimoto's thyroiditis.

    Science.gov (United States)

    Bai, Xue; Sun, Jie; Wang, Weiwei; Shan, Zhongyan; Zheng, Hongzhi; Li, Yushu; Zhao, Yuhang; Gong, Ming; Teng, Weiping

    2014-01-01

    As Th22 subsets are identified, their involvement in the pathogenesis of numerous autoimmune diseases has become apparent. In this study, we investigated differentiation of Th22 cells in the autoimmune thyroid diseases including Hashimoto's thyroiditis (HT) and Graves' disease (GD). Besides, we also explored the involvement of Th22 cells in an iodine-induced autoimmune thyroiditis (AIT) model (i.e., NOD.H-2(h4) mice). In HT patients, we showed the level of circulating Th22 cells correlated with the level of serum IL-22, and was significantly higher than in GD patients and healthy control subjects. Levels of serum IL-6, a major Th22 cell differentiation effector, were also higher in HT, and correlated with Th22 cells concentration. Peripheral blood mononuclear cells isolated from HT patients produced larger amounts of IL-6 in vitro than did those isolated from other groups. Furthermore, unlike those from GD patients, T lymphocytes from HT patients showed an enhanced differentiation in vitro into Th22 cells in the presence of recombinant IL-6 and TNF-α. In addition, levels of circulating Th22 cells and titers of thyroid peroxidase antibody were positively correlated in HT patients. In NOD.H-2(h4) mice, higher numbers of Th22 cells were observed in the spleens of the AIT group, while splenocytes of this group also produced larger amounts of IL-6 and IL-22 in vitro compared with the control. Intra-thyroid infiltrating IL-22+ lymphocytes were significantly increased in mice of the AIT group compared with the control. Our results indicate that Th22 cells may contribute to the pathogenesis of HT.

  13. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

    Directory of Open Access Journals (Sweden)

    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  14. The Vast Universe of T Cell Diversity: Subsets of Memory Cells and Their Differentiation.

    Science.gov (United States)

    Jandus, Camilla; Usatorre, Amaia Martínez; Viganò, Selena; Zhang, Lianjun; Romero, Pedro

    2017-01-01

    The T cell receptor confers specificity for antigen recognition to T cells. By the first encounter with the cognate antigen, reactive T cells initiate a program of expansion and differentiation that will define not only the ultimate quantity of specific cells that will be generated, but more importantly their quality and functional heterogeneity. Recent achievements using mouse model infection systems have helped to shed light into the complex network of factors that dictate and sustain memory T cell differentiation, ranging from antigen load, TCR signal strength, metabolic fitness, transcriptional programs, and proliferative potential. The different models of memory T cell differentiation are discussed in this chapter, and key phenotypic and functional attributes of memory T cell subsets are presented, both for mouse and human cells. Therapeutic manipulation of memory T cell generation is expected to provide novel unique ways to optimize current immunotherapies, both in infection and cancer.

  15. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    Science.gov (United States)

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  16. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E; Rehman, Jalees; Malik, Asrar B; Wary, Kishore K

    2016-01-01

    The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

  17. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E.; Rehman, Jalees; Malik, Asrar B.; Wary, Kishore K.

    2015-01-01

    Summary The studies of stem cell behavior and differentiation in a developmental context is complex, time-consuming and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation the embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder-layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture. PMID:25687301

  18. Differentiation of Human Cord Blood and Stromal Derived Stem Cells into Neuron Cells

    Directory of Open Access Journals (Sweden)

    Özlem Pamukçu Baran

    2007-01-01

    Full Text Available The most basic properties of stem cells are the capacities to self-renew indefinitely and to differentiate into multiple cell or tissue types. Umbilical cord blood has been utilized for human hematopoietic stem cell transplantation as an alternative source to bone marrow.The experiments show that Wharton’s jelly cells are easily attainable and can be expanded in vitro, maintained in culture, and induced to differentiate into neural cells. Almost recent studies it has been discovered that the cord blood-derived cells can differantiate not only to blood cells but also to various somatic cells like neuron or muscle cell with the signals taken from the envoirenment.Interestingly, neural cells obtained from umbilical cord blood show a relatively high spontaneous differentiation into oligodendrocytes, Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types.It has been shown that embryonic stem cells can be induced to differentiate into neurons and glia by treatment with retinoic acid or basic fibroblast growth factor. It has been studied that the diseases as Motor Neuron Disease, Parkinson, Alzheimer and degeneration of medulla spinalis and also paralysises could be treated with transplantation of cord blood-dericed stem cells.

  19. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  20. Chemo-mechanical control of neural stem cell differentiation

    Science.gov (United States)

    Geishecker, Emily R.

    Cellular processes such as adhesion, proliferation, and differentiation are controlled in part by cell interactions with the microenvironment. Cells can sense and respond to a variety of stimuli, including soluble and insoluble factors (such as proteins and small molecules) and externally applied mechanical stresses. Mechanical properties of the environment, such as substrate stiffness, have also been suggested to play an important role in cell processes. The roles of both biochemical and mechanical signaling in fate modification of stem cells have been explored independently. However, very few studies have been performed to study well-controlled chemo-mechanotransduction. The objective of this work is to design, synthesize, and characterize a chemo-mechanical substrate to encourage neuronal differentiation of C17.2 neural stem cells. In Chapter 2, Polyacrylamide (PA) gels of varying stiffnesses are functionalized with differing amounts of whole collagen to investigate the role of protein concentration in combination with substrate stiffness. As expected, neurons on the softest substrate were more in number and neuronal morphology than those on stiffer substrates. Neurons appeared locally aligned with an expansive network of neurites. Additional experiments would allow for statistical analysis to determine if and how collagen density impacts C17.2 differentiation in combination with substrate stiffness. Due to difficulties associated with whole protein approaches, a similar platform was developed using mixed adhesive peptides, derived from fibronectin and laminin, and is presented in Chapter 3. The matrix elasticity and peptide concentration can be individually modulated to systematically probe the effects of chemo-mechanical signaling on differentiation of C17.2 cells. Polyacrylamide gel stiffness was confirmed using rheological techniques and found to support values published by Yeung et al. [1]. Cellular growth and differentiation were assessed by cell counts

  1. Large-scale differential display analysis of T helper cell differentiation.

    Science.gov (United States)

    Ojala, Pekka; Virtanen, Eveliina; Chen, Zhi

    2007-03-01

    We have developed a novel large-scale multicapillary fluorescent differential display (FDD) platform amenable to further automation. The power of the method is demonstrated by the analysis of T helper cell differentiation. Eight RNA samples from wild type, Stat4 knockout and Stat6 knockout mice were analyzed with 16 anchoring primers and 24 arbitrary primers, resulting in 285 294 sample peaks. Visually selected patterns of differential expression suggest two major regulatory mechanisms: activation and Stat4 genotype. A subset of the findings is reproduced in the confirmatory differential display (DD) that included technical and biological replicates. In a small fragment identification pilot study, we identify Ifi27 and Cct8 to be up-regulated by T cell activation. We present a method for the analysis of electropherogram similarity across large datasets, based on correlation of low-resolution representations of electrophoretic data. We show how it can be applied to analyze experimental and technical variables. Using this method, we demonstrate the effect of activation and genotype. In addition, agreement of our real experimental data to the theoretical basis of DD, as well as issues in anchoring primer selectivity, are studied.

  2. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch.

    Science.gov (United States)

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-08-12

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

  3. Biological characteristics of breast carcinomas with neuroendocrine cell differentiation

    Institute of Scientific and Technical Information of China (English)

    姚根有; 周吉林; 赵仲生; 阮俊

    2004-01-01

    Background The aim of this study was to investigate DNA content and expression of c-erbB-2, PS2, and prostate-specific antigen (PSA) proteins in breast carcinomas with neuroendocrine (NE) cell differentiation.Methods Chromogranin, c-erbB-2, PS2, and PSA in 131 samples of breast cancer were detected immunohistochemically. Classic Feulgen staining image analysis techniques were used to quantify DNA content in 81 of the breast cancer samples.Results The c-erbB-2 positive rate in breast carcinoma samples containing neuroendocrine cells was 37.5% and the rate of high expression of c-erbB-2 (++ or +++) was 33.3%, both significantly lower than that in breast carcinomas without neuroendocrine cells (62.6% and 68.7%, respectively, P 5c aneuploidy cells, and rate of aneuploidy among cells were all lower than that in NE (-) breast carcinomas (P<0.01). In NE (+) grade I or II breast carcinomas, these indices were also all lower than that in the NE (-) breast carcinoma samples (P<0.01).Conclusion Breast carcinomas with neuroendocrine differentiation have a lower rate of malignancy. Neuroendocrine differentiation could serve as a prognostic marker in clinical practice.

  4. Photoresist Derived Carbon for Growth and Differentiation of Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Tie Zou

    2007-08-01

    Full Text Available Apoptosis or necrosis of neurons in the central nervous system (CNS is thehallmark of many neurodegenerative diseases and Traumatic Brain Injury (TBI. Theinability to regenerate in CNS offers little hope for naturally repairing the damagedneurons. However, with the rapid development of new technologies, regenerative medicineoffers great promises to patients with these disorders. Among many events for furtheradvancement of regenerative medicine, extracellular matrix (ECM plays a critical role forcellular migration and differentiation. To develop a biocompatible and electricallyconductive substrate that can be potentially used to promote growth and regeneration ofneurons and to record intracellular and multisite signals from brain as a probe, a polymericprecursor – SPR 220.7 was fabricated by pyrolysis at temperatures higher than 700 oC.Human Neuroblastoma cells - SK-N-MC, SY5Y, mouse teratocarcinoma cells P-19 and ratPC12 cells were found to attach and proliferate on photoresist derived carbon film.Significantly, neuronal differentiation of PC12 cells induced by NGF was demonstrated byobserving cell shape and size, and measuring the length of neurites under SEM. Our resultsindicated that fabricated carbon could potentially be explored in regenerative medicine forpromoting neuronal growth and differentiation in CNS with neurodegeneration.

  5. FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells

    Directory of Open Access Journals (Sweden)

    Gustavo Jesus Vazquez-Zapien

    2016-01-01

    Full Text Available Some of the greatest challenges in stem cells (SCs biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR, immunocytochemistry, and Fourier Transform Infrared (FTIR spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2. DPCs expressed endodermal genes (SOX17 and Pdx1 at day 11, an inductor gene of embryonic pancreas development (Pdx1 at day 17 and pancreas genes and proteins (Insulin and Glucagon at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells.

  6. FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells

    Science.gov (United States)

    Mata-Miranda, Monica Maribel; Sanchez-Monroy, Virginia; Delgado-Macuil, Raul Jacobo; Perez-Ishiwara, David Guillermo

    2016-01-01

    Some of the greatest challenges in stem cells (SCs) biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs) toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs) were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR), immunocytochemistry, and Fourier Transform Infrared (FTIR) spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2). DPCs expressed endodermal genes (SOX17 and Pdx1) at day 11, an inductor gene of embryonic pancreas development (Pdx1) at day 17 and pancreas genes and proteins (Insulin and Glucagon) at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days) were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells. PMID:27651798

  7. The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells.

    Science.gov (United States)

    Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F; Schett, Georg; Mielenz, Dirk; David, Jean-Pierre

    2014-10-20

    The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

  8. Poorly Differentiated Squamous Cell Carcinoma Arising in Tattooed Skin

    Directory of Open Access Journals (Sweden)

    Deba P. Sarma

    2010-01-01

    Full Text Available Introduction. Tattoos have increasingly become accepted by mainstream Western society. As a result, the incidence of tattoo-associated dermatoses is on the rise. The presence of a poorly differentiated squamous cell carcinoma in an old tattooed skin is of interest as it has not been previously documented. Case Presentation. A 79-year-old white homeless man of European descent presented to the dermatology clinic with a painless raised nodule on his left forearm arising in a tattooed area. A biopsy of the lesion revealed a poorly differentiated squamous cell carcinoma infiltrating into a tattoo. The lesion was completely excised and the patient remains disease-free one year later. Conclusion. All previous reports of squamous cell carcinomas arising in tattoos have been well-differentiated low-grade type or keratoacanthoma-type and are considered to be coincidental rather than related to any carcinogenic effect of the tattoo pigments. Tattoo-associated poorly differentiated invasive carcinoma appears to be extremely rare.

  9. Organ-derived dendritic cells have differential effects on alloreactive T cells

    OpenAIRE

    Kim, Theo D.; Terwey, Theis H.; Zakrzewski, Johannes L; Suh, David; Kochman, Adam A.; Chen, Megan E.; King, Chris G.; Borsotti, Chiara; Grubin, Jeremy; Smith, Odette M.; Heller, Glenn; Liu, Chen; Murphy, George F.; Alpdogan, Onder; Marcel R. M. van den Brink

    2008-01-01

    Dendritic cells (DCs) are considered critical for the induction of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been shown to induce organ-tropism through induction of specific homing molecules on T cells. Using adoptive transfer of CFSE-labeled cells, we first demonstrated that alloreactive T cells differentially up-regulate specific homing molecules in vivo. Host-type dendritic cells from the GVHD targe...

  10. Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

    Institute of Scientific and Technical Information of China (English)

    吴桂珠; 郑秀; 江忠清; 王金华; 宋岩峰

    2010-01-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells(ADSCs) into myoblasts,which may provide a new strategy for tissue engineering in patients with stress urinary incontinence(SUI).ADSCs,isolated and cultured ex vivo,were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine(5-aza),5% FBS,and 5% horse serum.Cellular morphology was observed under an i...

  11. Stem cells in light of a new concept for cell differentiation.

    Science.gov (United States)

    Kristeva, Marlene Anastassova

    2008-10-01

    My concept of cell differentiation involves genetic information from DNA being transcribed into mRNA proteins-morphogenes (mRNAs+ homeodomain proteins)-and stored in the ovoplasm as maternal inheritance, or cytoplasmic genetic memory. Feedback mechanism(s) allow these morphogenes to selectively unlock new genes, regulating the development of the embryo. The blastomeres and the embryonic pluripotent cells of the inner cell mass of early (5 day) blastocysts are loaded with morphogenes which hamper the production of cell lines and are responsible for the formation of embryoid bodies in vitro and teratomas in vivo. There are therefore legitimate concerns as to proposals to use embryonic pluripotent cells for cell therapy and regenerative medicine. An alternative cell therapy would involve the production of tailored growth-related genes-morphogenes-and hence selective in vitro differentiation of adult de-differentiated cells.

  12. Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Szu-Hsiu Liu

    2012-01-01

    Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

  13. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  14. Myoepithelial cell differentiation markers in ductal carcinoma in situ progression.

    Science.gov (United States)

    Russell, Tanya D; Jindal, Sonali; Agunbiade, Samiat; Gao, Dexiang; Troxell, Megan; Borges, Virginia F; Schedin, Pepper

    2015-11-01

    We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

  15. Interleukin 4: signalling mechanisms and control of T cell differentiation.

    Science.gov (United States)

    Paul, W E

    1997-01-01

    Interleukin 4 (IL-4) is a pleiotropic type I cytokine that controls both growth and differentiation among haemopoietic and non-haemopoietic cells. Its receptor is a heterodimer. One chain, the IL-4R alpha chain, binds IL-4 with high affinity and determines the nature of the biochemical signals that are induced. The second chain, gamma c, is required for the induction of such signals. IL-4-mediated growth depends upon activation events that involve phosphorylation of Y497 of IL-4R alpha, leading to the binding and phosphorylation of 4PS/IRS-2 in haemopoietic cells and of IRS-1 in non-haemopoietic cells. By contrast, IL-4-mediated differentiation events depend upon more distal regions of the IL-4R alpha chain that include a series of STAT-6 binding sites. The distinctive roles of these receptor domains was verified by receptor-reconstruction experiments. The 'growth' and 'differentiation' domains of the IL-4R alpha chain, independently expressed as chimeric structures with a truncated version of the IL-2R beta chain, were shown to convey their functions to the hybrid receptor. The critical role of STAT-6 in IL-4-mediated gene activation and differentiation was made clear by the finding that lymphocytes from STAT-6 knockout mice are strikingly deficient in these functions but have retained the capacity to grow, at least partially, in response to IL-4. IL-4 plays a central role in determining the phenotype of naive CD4+ T cells. In the presence of IL-4, newly primed naive T cells develop into IL-4 producers while in its absence they preferentially become gamma-interferon (IFN-gamma) producers. Recently, a specialized subpopulation of T cells, CD4+/NK1.1+ cells, has been shown to produce large amounts of IL-4 upon stimulation. Two examples of mice with deficiencies in these cells are described--beta 2-microglobulin knockout mice and SJL mice. Both show defects in the development of IL-4-producing cells and in the increase in serum IgE in response to stimulation with the

  16. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells.

    Science.gov (United States)

    Dyce, Paul W; Shen, Wei; Huynh, Evanna; Shao, Hua; Villagómez, Daniel A F; Kidder, Gerald M; King, W Allan; Li, Julang

    2011-05-01

    We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.

  17. Biophysical characteristics reveal neural stem cell differentiation potential.

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    Fatima H Labeed

    Full Text Available BACKGROUND: Distinguishing human neural stem/progenitor cell (huNSPC populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: We used dielectrophoresis (DEP to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. CONCLUSIONS/SIGNIFICANCE: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

  18. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  19. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    Science.gov (United States)

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  20. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Science.gov (United States)

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  1. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

    Science.gov (United States)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette; Li, Haisheng; Zou, Xuenong; Flyvbjerg, Allan; Kassem, Moustapha; Bünger, Cody

    2007-02-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

  2. Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation

    Science.gov (United States)

    Berríos, Verónica O.; Boukli, Nawal M.; Rodriguez, Jose W.; Negraes, Priscilla D.; Schwindt, Telma T.; Trujillo, Cleber A.; Oliveira, Sophia L. B.; Cubano, Luis A.; Ferchmin, P. A.; Eterovic, Vesna A.; Ulrich, Henning; Martins, Antonio H.

    2015-01-01

    Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyri-dostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 μM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 μM pyri-dostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected. PMID:25758980

  3. Enhancement of cardiomyocyte differentiation from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2’-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2’-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.

  4. The Hematopoietic Differentiation and Production of Mature Myeloid Cells from Human Pluripotent Stem Cells

    OpenAIRE

    Choi, Kyung-Dal; Vodyanik, Maxim; Slukvin, Igor I.

    2011-01-01

    Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and ...

  5. Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

    Science.gov (United States)

    Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

    2017-01-01

    Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

  6. Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells

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    Vaibhav Shinde

    2016-04-01

    Full Text Available Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs under simulated microgravity within a fast-rotating clinostat (clinorotation and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs. Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The

  7. [Differentiation of functional cells from iPS cells by efficient gene transfer].

    Science.gov (United States)

    Kawabata, Kenji; Tashiro, Katsuhisa; Mizuguchi, Hiroyuki

    2010-11-01

    Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

  8. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

    Science.gov (United States)

    Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

    2016-04-01

    Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms

  9. Equine induced pluripotent stem cells have a reduced tendon differentiation capacity compared to embryonic stem cells

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    Emma Patricia Bavin

    2015-11-01

    Full Text Available Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In 2-dimensional differentiation assays the iPSCs expressed tendon associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in 3-dimensional differentiation assays the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3-dimensional in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application.

  10. mTOR and the differentiation of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xinxin Xiang; Jing Zhao; Geyang Xu; Yin Li; Weizhen Zhang

    2011-01-01

    The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase,belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells, mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.

  11. Isolation and differentiation of medaka embryonic stem cells.

    Science.gov (United States)

    Hong, Yunhan; Schartl, Manfred

    2006-01-01

    Medaka is a small laboratory fish that daily produces eggs easily controllable by light cycles. This fish represents a unique lower vertebrate compared to mammals, in which embryonic stem (ES) cell lines can be derived from midblastula embryos (MBEs). Like mouse ES cells, medaka ES cells most resemble the totipotent embryonic cells at the blastula stage. Medaka ES cells retain a diploid karyotype, pluripotency in vitro, and chimera competence in vivo. They give rise to high efficiencies of transient and stable gene transfer and maintain their pluripotency after long-term drug selection for transgene integration. They can also be directed to differentiate into particular cell types. Medaka is the most distantly related vertebrate to mammals, and its ES cell lines provide an ideal reference to mammalian ES cells for the molecular analysis of stemness. More important, medaka ES cell lines on their own offer an excellent tool for studying stem cell biology in vitro and in vivo because production and observation of ES-derived chimeras as well as phenotypic analyses are very easy because of its external, transparent, and temperature-adjustable embryology.

  12. Snai1 represses Nanog to promote embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    F. Galvagni

    2015-06-01

    Full Text Available Embryonic stem cell (ESC self-renewal and pluripotency is maintained by an external signaling pathways and intrinsic regulatory networks involving ESC-specific transcriptional complexes (mainly formed by OCT3/4, Sox2 and Nanog proteins, the Polycomb repressive complex 2 (PRC2 and DNA methylation [1–8]. Among these, Nanog represents the more ESC specific factor and its repression correlates with the loss of pluripotency and ESC differentiation [9–11]. During ESC early differentiation, many development-associated genes become upregulated and although, in general, much is known about the pluripotency self-renewal circuitry, the molecular events that lead ESCs to exit from pluripotency and begin differentiation are largely unknown. Snai1 is one the most early induced genes during ESC differentiation in vitro and in vivo [12,13]. Here we show that Snai1 is able to directly repress several stemness-associated genes including Nanog. We use a ESC stable-line expressing a inducible Snai1 protein. We here show microarray analysis of embryonic stem cells (ESC expressing Snail-ER at various time points of induction with 4-OH. Data were deposited in Gene Expression Omnibus (GEO datasets under reference GSE57854 and here: http://epigenetics.hugef-research.org/data.php.

  13. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  14. Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Daniel J Hoover

    Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

  15. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  16. Clonal, self-renewing and differentiating human and porcine urothelial cells, a novel stem cell population.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available Although urothelial progenitor-like cells have been described in the human urinary tract, the existence of stem cells remains to be proven. Using a culture system that favors clonogenic epithelial cell growth, we evaluated and characterized clonal human urothelial cells. We isolated human urothelial cells that were clonogenic, capable of self-renewal and could develop into fully differentiated urothelium once re-implanted into the subcapsular space of nude mice. In addition to final urothelial cell differentiation, spontaneous formation of bladder-like microstructures was observed. By examining an epithelial stem cell signature marker, we found p63 to correlate with the self-renewal capacity of the isolated human urothelial clonal populations. Since a clinically relevant, long-term model for functional reconstitution of human cells does not exist, we sought to establish a culture method for porcine urothelial cells in a clinically relevant porcine model. We isolated cells from porcine ureter, urethra and bladder that were clonogenic and capable of self-renewal and differentiation into fully mature urothelium. In conclusion, we could isolate human and porcine cell populations, behaving as urothelial stem cells and showing clonogenicity, self-renewal and, once re-implanted, morphological differentiation.

  17. Differentially expressed genes in giant cell tumor of bone.

    Science.gov (United States)

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  18. Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation

    OpenAIRE

    Haremaki, Tomomi; Fraser, Stuart T.; Kuo, Yien-Ming; Baron, Margaret H.; Weinstein, Daniel C.

    2007-01-01

    Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these diff...

  19. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    NARCIS (Netherlands)

    Dekker, E. den; Grefte, S.; Huijs, T.; Dam, G.B. ten; Versteeg, E.M.M.; Berk, L.C.J. van den; Bladergroen, B.A.; Kuppevelt, A.H.M.S.M. van; Figdor, C.G.; Torensma, R.

    2008-01-01

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expr

  20. Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells

    Science.gov (United States)

    Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

    2016-01-01

    One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration. PMID:27733898

  1. High glucose suppresses embryonic stem cell differentiation into neural lineage cells.

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E Albert; Chen, Xi; Yang, Peixin

    2016-04-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

  2. Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

    Directory of Open Access Journals (Sweden)

    Deirdre A. Nelson

    2013-04-01

    Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells; cytokeratin 7 (ductal cells; and smooth muscle α-actin (myoepithelial cells and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.

  3. High glucose suppresses embryonic stem cell differentiation into neural lineage cells

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E. Albert; Chen, Xi; Yang, Peixin

    2017-01-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system. PMID:26940741

  4. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.

    Science.gov (United States)

    Mummery, Christine L; Zhang, Jianhua; Ng, Elizabeth S; Elliott, David A; Elefanty, Andrew G; Kamp, Timothy J

    2012-07-20

    Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

  5. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  6. Transcription factor ABF-1 suppresses plasma cell differentiation but facilitates memory B cell formation.

    Science.gov (United States)

    Chiu, Yi-Kai; Lin, I-Ying; Su, Shin-Tang; Wang, Kuan-Hsiung; Yang, Shii-Yi; Tsai, Dong-Yan; Hsieh, Yi-Ting; Lin, Kuo-I

    2014-09-01

    Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.

  7. Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.

    Science.gov (United States)

    Choi, Hyun Woo; Kim, Jong Soo; Choi, Sol; Hong, Yean Ju; Kim, Min Jung; Seo, Han Geuk; Do, Jeong Tae

    2014-10-01

    Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

  8. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  9. Differentiation mechanism and function of the cereal aleurone cells and hormone effects on them.

    Science.gov (United States)

    Zheng, Yankun; Wang, Zhong

    2014-11-01

    The cereal aleurone cells differentiate from the endosperm epidermis with the exception of endosperm transfer cells. Aleurone cells contain proteins, lipids, and minerals, and are important for digesting the endosperm storage products to nurse the embryo under effects of several hormones during the seed germination. The differentiation of aleurone cells is related to location effect and special gene expression. Moreover, the differentiation of aleurone cells is probably affected by the cues from maternal tissues. In the paper, differentiation mechanism and function of aleurone cells and hormone effects on them are reviewed. Some speculations about the differentiation mechanism of aleurone cells are given here.

  10. Derivation, characterization and retinal differentiation of induced pluripotent stem cells

    Indian Academy of Sciences (India)

    Subba Rao Mekala; Vasundhara Vauhini; Usha Nagarajan; Savitri Maddileti; Subhash Gaddipati; Indumathi Mariappan

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

  11. Regulation of germinal center B-cell differentiation.

    Science.gov (United States)

    Zhang, Yang; Garcia-Ibanez, Laura; Toellner, Kai-Michael

    2016-03-01

    Germinal centers (GC) are the main sites where antigen-activated B-cell clones expand and undergo immunoglobulin gene hypermutation and selection. Iterations of this process will lead to affinity maturation, replicating Darwinian evolution on the cellular level. GC B-cell selection can lead to four different outcomes: further expansion and evolution, apoptosis (non-selection), or output from the GC with differentiation into memory B cells or plasma cells. T-helper cells in GC have been shown to have a central role in regulating B-cell selection by sensing the density of major histocompatibility complex (MHC):peptide antigen complexes. Antigen is provided on follicular dendritic cells in the form of immune complex. Antibody on these immune complexes regulates antigen accessibility by shielding antigen from B-cell receptor access. Replacement of antibody on immune complexes by antibody generated from GC-derived plasma cell output will gradually reduce the availability of antigen. This antibody feedback can lead to a situation where a slow rise in selection stringency caused by a changing environment leads to directional evolution toward higher affinity antibody.

  12. Sulfated polysaccharides and cell differentiation in the sea urchin embryo.

    Science.gov (United States)

    Løvtrup-Rein, H; Løvtrup, S

    1984-01-01

    The synthesis of sulfated polysaccharides during the embryonic development of Paracentrotus lividus has been investigated by incorporation of radioactive sulfate, glucose, glucosamine and fucose. The following substances become labelled: fucan sulfate (approximately 60%), heparan sulfate (approximately 20%) and dermatan sulfate (approximately 20%), and possibly a very slight amount of chondroitin sulfate. In animalized and vegetalized embryos, the rate of incorporation is significantly reduced, and furthermore dermatan sulfate is almost absent in animalized embryos. It is concluded that this substance is associated with the differentiation of vegetative cells, possibly the mesenchyme cells.

  13. Large chromatin domains in pluripotent and differentiated cells

    Institute of Scientific and Technical Information of China (English)

    Shibin Hu; Lu Cheng; Bo Wen

    2012-01-01

    Pluripotent stem cells are able to proliferate unlimitedly and to generate all somatic cell types,thus holding a great promise in medical applications.Epigenetic modifications are believed to play crucial roles in regulating pluripotency and differentiation.Recent genome-wide studies on mammalian systems have revealed several types of large chromatin domains which are associated with higherorder organization of the genome.The elucidation of genomic distribution and dynamics of these domains have shed light on the mechanisms underling pluripotency and lineage commitment.

  14. Natural Killer Cell Differentiation From Hematopoietic Stem Cells: A Comparative Analysis of Heparin and Stromal Cell Supported Methods

    OpenAIRE

    2011-01-01

    Natural killer (NK) cells differentiated from hematopietic stem cells (HSCs)may have significant clinical benefits over those from adult donors, including the ability to choose allo-reactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study NK differentiation from HSCs. Stroma and cytokines support NK differentiation, but may have considerable regulatory hurdles. Recently, a clinical grade heparin-based method has been reported and could serve...

  15. Parameters Identification of Photovoltaic Cells Based on Differential Evolution Algorithm

    Directory of Open Access Journals (Sweden)

    Liao Hui

    2016-01-01

    Full Text Available For the complex nonlinear model of photovoltaic cells, traditional evolution strategy is easy to fall into the local optimal and its identification time is too long when taking parameters identification, then the difference algorithm is proposed in this study, which is to solve the problems of parameter identification in photovoltaic cell model, where it is very difficult to achieve with other identification algorithms. In this method, the random data is selected as the initial generation; the successful evolution to the next generation is done through a certain strategy of difference algorithm, which can achieve the effective identification of control parameters. It is proved that the method has a good global optimization and the fast convergence ability, and the simulation results are shown that the differential evolution has high identification ability and it is an effective method to identify the parameters of photovoltaic cells, where the photovoltaic cells can be widely used in other places with these parameters.

  16. In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells

    Directory of Open Access Journals (Sweden)

    Ruhma Mahmood

    2015-01-01

    Full Text Available Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs and umbilical cord mesenchymal stem cells (UC-MSCs, respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells.

  17. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  18. Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Puente, Pilar de la, E-mail: pilardelapuentegarcia@gmail.com [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain); Ludeña, Dolores [Pathology Service, University Hospital of Salamanca, P/San Vicente 58-182, 37007 Salamanca (Spain); López, Marta; Ramos, Jennifer; Iglesias, Javier [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain)

    2013-02-01

    Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.

  19. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

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    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  20. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

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    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  1. Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

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    Frech Moritz J

    2010-12-01

    Full Text Available Abstract Background Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process. Results In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis. Conclusion These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

  2. Differentiation of Equine Mesenchymal Stromal Cells into Cells of Neural Lineage: Potential for Clinical Applications

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    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.

  3. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

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    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  4. Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

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    Stolzing Alexandra

    2011-08-01

    Full Text Available Abstract Background Microglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L, astrocyte-conditioned medium (ACM and GM-CSF on the differentiation to microglia-like cells. Methods We assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80, but also for the first time for functional performance (phagocytosis, oxidative burst and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices. Results The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation. Conclusion We conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.

  5. Differentiated cell behavior: a multiscale approach using measure theory.

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    Colombi, Annachiara; Scianna, Marco; Tosin, Andrea

    2015-11-01

    This paper deals with the derivation of a collective model of cell populations out of an individual-based description of the underlying physical particle system. By looking at the spatial distribution of cells in terms of time-evolving measures, rather than at individual cell paths, we obtain an ensemble representation stemming from the phenomenological behavior of the single component cells. In particular, as a key advantage of our approach, the scale of representation of the system, i.e., microscopic/discrete vs. macroscopic/continuous, can be chosen a posteriori according only to the spatial structure given to the aforesaid measures. The paper focuses in particular on the use of different scales based on the specific functions performed by cells. A two-population hybrid system is considered, where cells with a specialized/differentiated phenotype are treated as a discrete population of point masses while unspecialized/undifferentiated cell aggregates are represented by a continuous approximation. Numerical simulations and analytical investigations emphasize the role of some biologically relevant parameters in determining the specific evolution of such a hybrid cell system.

  6. [Progress in early pancreas development and reprogramming of terminally differentiated cells into β cells].

    Science.gov (United States)

    Mingjun, Cao; Huansheng, Dong; Qingjie, Pan; Hongjun, Wang; Xiao, Dong

    2014-06-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which the immune system attacks insulin-secreting β cells, thus leading to an absolute deficiency of insulin. Patients must rely on exogenous insulin, which cannot effectively prevent diabetes complications. Generation of insulin-secreting cells by reprogramming of pluripotent stem cells or somatic cells is a potential approach for the treatment of T1DM. These cells can be used for cell therapy and drug screening, and may eventually provide a cure for the disease. Significant progress has been made in generating insulin-secreting cells through the expression of β cell specific transcription factors in stem cells or somatic cells. In this review, we summarize recent research progress in early pancreas development, β cell specific transcription factors and reprogramming of terminally differentiated cells into β cells.

  7. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  8. Inhibition of FGF signaling accelerates neural crest cell differentiation of human pluripotent stem cells.

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    Jaroonwitchawan, Thiranut; Muangchan, Pattamon; Noisa, Parinya

    2016-12-02

    Neural crest (NC) is a transient population, arising during embryonic development and capable of differentiating into various somatic cells. The defects of neural crest development leads to neurocristopathy. Several signaling pathways were revealed their significance in NC cell specification. Fibroblast growth factor (FGF) is recognized as an important signaling during NC development, for instance Xenopus and avian; however, its contributions in human species are remained elusive. Here we used human pluripotent stem cells (hPSCs) to investigate the consequences of FGF inhibition during NC cell differentiation. The specific-FGF receptor inhibitor, SU5402, was used in this investigation. The inhibition of FGF did not found to affect the proliferation or death of hPSC-derived NC cells, but promoted hPSCs to commit NC cell fate. NC-specific genes, including PAX3, SLUG, and TWIST1, were highly upregulated, while hPSC genes, such as OCT4, and E-CAD, rapidly reduced upon FGF signaling blockage. Noteworthy, TFAP-2α, a marker of migratory NC cells, abundantly presented in SU5402-induced cells. This accelerated NC cell differentiation could be due to the activation of Notch signaling upon the blockage of ERK1/2 phosphorylation, since NICD was increased by SU5402. Altogether, this study proposed the contributions of FGF signaling in controlling human NC cell differentiation from hPSCs, the crosstalk between FGF and Notch, and might imply to the influences of FGF signaling in neurocristophatic diseases.

  9. Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Li-ye; ZHENG Jia-kun; WANG Chao-yang; LI Wen-yu

    2005-01-01

    Objective: To investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells. Methods: Bone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry. Conclusions: Bone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

  10. Generation of patient-specific pluripotent stem cells and directed differentiation of embryonic stem cells for regenerative medicine

    Institute of Scientific and Technical Information of China (English)

    Minyue Ma; Jiahao Sha; Zuomin Zhou; Qi Zhou; Qingzhang Li

    2008-01-01

    Embryonic stem(ES) cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells: nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cell scurrently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.

  11. Mesenchymal stem cells from rat olfactory bulbs can differentiate into cells with cardiomyocyte characteristics.

    Science.gov (United States)

    Huang, Yuahn-Sieh; Li, I-Hsun; Chueh, Sheau-Huei; Hueng, Dueng-Yuan; Tai, Ming-Cheng; Liang, Chang-Min; Lien, Shiu-Bii; Sytwu, Huey-Kang; Ma, Kuo-Hsing

    2015-12-01

    Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC-like cells were also found to exist in rat olfactory bulb and are capable of inducing differentiation into mesenchymal lineages - osteocytes, chondrocytes and adipocytes. However, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb-derived MSCs could differentiate into myocardial cells in vitro. Fibroblast-like cells isolated from the olfactory bulb of neonatal rats were grown under four conditions: no treatment; in the presence of growth factors (neuregulin-1, bFGF and forskolin); co-cultured with cardiomyocytes; and co-cultured with cardiomyocytes plus neuregulin-1, bFGF and forskolin. Cell differentiation into myocardial cells was monitored by RT-PCR, light microscopy immunofluorescence, western blot analysis and contractile response to pharmacological treatments. The isolated olfactory bulb-derived fibroblast-like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of co-culture of MSCs with rat cardiomyocytes plus neuregulin-1, bFGF and forskolin. RT-PCR and western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α-actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacological treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb-derived fibroblast-like cells with MSC characteristics can differentiate into myocardial-like cells.

  12. Differentiation of Adipose-derived Stem Cells into Schwann Cell Phenotype in Comparison with Bone Marrow Stem Cells

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    Zolikha Golipoor

    2010-06-01

    Full Text Available Objective(sBone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs, but adipose tissue appears to be an alternative and more readily available source. In this study, rat adipose-derived stem cells (ADSCs were induced to differentiate into Schwann-like cells and compared with rat bone marrow stem cells (BMSCs for their Schwann-like cells differentiation potential. Materials and MethodsBMSCs and ADSCs were characterized for expression of MSCs-specific markers, osteogenic and adipogenic differentiation. They were induced to differentiate into Schwann-like cells and analyzed for expression of the Schwann specific markers. The immunocytochemical differentiation markers were S-100 and real time quantitative Real-time polymerase chain reaction (RT-PCR markers were S100, P75 and glial fibrillary acidic protein (GFAP. 3-(4, 5-Dimethylthiazol- 2-yl-2, 5-diphenyltetrazolium bromide (MTT assay and Annexin V-Fluorescein isothiocyanate (FITC/ Propidium iodide (PI double labeling method were employed to detect early stage cell apoptosis.ResultsBMSCs and ADSCs showed similarities in expression of the MSC-specific markers, osteogenic and adipogenic differentiation. Both quantitative RT-PCR and immunocytochemical analysis demonstrated that BMSCs and ADSCs had equal expression of the Schwann-specific markers following Schwann-like cells differentiation. However, gene expression of P75 was higher in BMSCs compared with ADSCs. MTT assay and flow cytometry found that of the total BMSCs and ADSCs in the culture medium, 20% to 30% of the cells died, but the remaining cell population remained strongly attached to the substrate and differentiated.ConclusionComparative analysis showed that Schwann-like cell differentiation potential of ADSCs was slightly decreased in comparison with BMSCs. Therefore, BMSCs are more favorable choice than ADSCs for tissue engineering.

  13. Capacity of Human Dental Follicle Cells to Differentiate into Neural Cells In Vitro

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    Shingo Kanao

    2017-01-01

    Full Text Available The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells (hDFCs have the capacity to commit to differentiation into multiple cell types. Here we investigated the capacity of hDFCs to differentiate into neural cells and the efficiency of a two-step strategy involving floating neurosphere-like bodies for neural differentiation. Undifferentiated hDFCs showed a spindle-like morphology and were positive for neural markers such as nestin, β-III-tubulin, and S100β. The cellular morphology of several cells was neuronal-like including branched dendrite-like processes and neurites. Next, hDFCs were used for neurosphere formation in serum-free medium containing basic fibroblast growth factor, epidermal growth factor, and B27 supplement. The number of cells with neuronal-like morphology and that were strongly positive for neural markers increased with sphere formation. Gene expression of neural markers also increased in hDFCs with sphere formation. Next, gene expression of neural markers was examined in hDFCs during neuronal differentiation after sphere formation. Expression of Musashi-1 and Musashi-2, MAP2, GFAP, MBP, and SOX10 was upregulated in hDFCs undergoing neuronal differentiation via neurospheres, whereas expression of nestin and β-III-tubulin was downregulated. In conclusion, hDFCs may be another optimal source of neural/glial cells for cell-based therapies to treat neurological diseases.

  14. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

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    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  15. An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

    Science.gov (United States)

    Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

    2008-01-01

    Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.

  16. β-Cell neogenesis: experimental considerations in adult stem cell differentiation.

    Science.gov (United States)

    Iskovich, Svetlana; Goldenberg-Cohen, Nitza; Stein, Jerry; Yaniv, Isaac; Farkas, Daniel L; Askenasy, Nadir

    2011-04-01

    The contribution of stem cells derived from adult tissues to the recovery of pancreatic islets from chemical injury is controversial. Analysis of nonhematopoietic differentiation of bone marrow-derived cells has yielded positive and negative results under different experimental conditions. Using the smallest subset of bone marrow cells lacking immuno-hematopoietic lineage markers, we have detected incorporation and conversion into insulin-producing cells. Donor cells identified by genomic markers silence green fluorescent protein (GFP) expression as a feature of differentiation, in parallel to expressing PDX-1 and proinsulin. Here we elaborate potential experimental difficulties that might result in false-negative results. The use of GFP as a reporter protein is suboptimal for differentiation experiments: (a) the bone marrow of GFP donors partially expresses the reporter protein, (b) differentiating bone marrow cells silence GFP expression, and (c) the endocrine pancreas is constitutively negative for GFP. In addition, design of the experiments, data analysis, and interpretation encounter numerous objective and subjective difficulties. Rigorous evaluation under optimized experimental conditions confirms the capacity of adult bone marrow-derived stem cells to adopt endocrine developmental traits, and demonstrates that GFP downregulation and silencing is a feature of differentiation.

  17. Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

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    Chen, Jong-Hang; Chou, Chin-Cheng

    2015-08-01

    This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling.

  18. Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

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    Angela Maria Cozzolino

    2016-01-01

    Full Text Available In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that, in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines as in vitro models of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

  19. Differentiation of Mesenchymal Stem Cells Into Dopaminergic Neuron-like Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LI GUO; HONG-XUE FAN; FEI YIN; HONG-QI MENG; LING LING; TA-NA HU-HE; PENG LI; CHUN-XIA ZHANG; SHUN YU; DE-SHENG DUAN

    2005-01-01

    Objective To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. Results MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032±2.489%, 41.580±5.101% and 34.958±5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. Conclusion MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.

  20. Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

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    Charles A. Easley, IV

    2012-09-01

    Full Text Available Human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been shown to differentiate into primordial germ cells (PGCs but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

  1. Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells

    Institute of Scientific and Technical Information of China (English)

    Claudia Lange; Helge Bruns; Dietrich Kluth; Axel R Zander; Henning C Fiegel

    2006-01-01

    AIM: To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells.METHODS: MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF),hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin,and alpha-fetoprotein (AFP) was performed in different cell populations.RESULTS: Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18,and AFP-RNA over two weeks. At wk 3, MSCs lost hepatooytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential.CONCLUSION: The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also prorides a beneficial environment for expansion and differentiation of FLC.

  2. In vitro differentiation of human adipose-derived mesenchymal stem cells into endothelial-like cells

    Institute of Scientific and Technical Information of China (English)

    GUAN Lidong; SHI Shuangshuang; PEI Xuetao; LI Shaoqing; WANG Yunfang; YUE Huimin; LIU Daqing; HE Lijuan; BAI Cixian; YAN Fang; NAN Xue

    2006-01-01

    The neovascularization of ischemic tissue is a crucial initial step for the functional rehabilitation and wound healing. However, the short of seed cell candidate for the foundation of vascular network is still a big issue. Human adipose tissue derived mesenchymal stem cells (hADSCs), which possess multilineage potential, are capable of adipogenic, osteogenic, and chondrogenic differentiation. We examined whether this kind of stem cells could differentiate into endothelial-like cells and participate in blood vessel formation, and whether they could be used as an ideal cell source for therapeutic angiogenesis in ischemic diseases or vascularization of tissue constructs. The results showed that hADSCs, grown under appropriately induced conditions, displayed characteristics similar to those of vessel endothelium. The differentiated cells expressed endothelial cell markers CD34 and vWF, and had high metabolism of acetylated low-density lipoprotein and prostacyclin. In addition, the induced cells were able to form tube-like structures when cultured on matrigel. Our data indicated that induced hADSCs could exhibit characteristics of endothelial cells. Therefore, these cells, as a source of human endothelial cells, may find many applications in such realms as engineering blood vessels, endothelial cell transplantation for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  3. Characterization of mitochondrial populations during stem cell differentiation.

    Science.gov (United States)

    Kerscher, Petra; Bussie, Blakely S; DeSimone, Katherine M; Dunn, David A; Lipke, Elizabeth A

    2015-01-01

    Mitochondrial dynamics play an important role in numerous physiological and pathophysiological phenomena in the developing and adult human heart. Alterations in structural aspects of cellular mitochondrial composition as a function of changes in physiology can easily be visualized using fluorescence microscopy. Commonly, mitochondrial location, number, and morphology are reported qualitatively due to the lack of automated and user-friendly computer-based analysis tools. Mitochondrial Quantification using MATLAB (MQM) is a computer-based tool to quantitatively assess these parameters by analyzing fluorescently labeled mitochondria within the cell; in particular, MQM provides numerical information on the number, area, and location of mitochondria within a cell in a time-efficient, automated, and unbiased way. This chapter describes the use of MQM's capabilities to quantify mitochondrial changes during human pluripotent stem cell (hPSC) differentiation into spontaneously contracting cardiomyocytes (SC-CMs), which follows physiological pathways of human heart development.

  4. Characterization of tumor cells and stem cells by differential nuclear methylation imaging

    Science.gov (United States)

    Tajbakhsh, Jian; Wawrowsky, Kolja A.; Gertych, Arkadiusz; Bar-Nur, Ori; Vishnevsky, Eugene; Lindsley, Erik H.; Farkas, Daniel L.

    2008-02-01

    DNA methylation plays a key role in cellular differentiation. Aberrant global methylation patterns are associated with several cancer types, as a result of changes in long-term activation status of up to 50% of genes, including oncogenes and tumor-suppressor genes, which are regulated by methylation and demethylation of promoter region CpG dinucleotides (CpG islands). Furthermore, DNA methylation also occurs in nonisland CpG sites (> 95% of the genome), present once per 80 dinucleotides on average. Nuclear DNA methylation increases during the course of cellular differentiation while cancer cells usually show a net loss in methylation. Given the large dynamic range in DNA methylation load, the methylation pattern of a cell can provide a valuable distinction as to its status during differentiation versus the disease state. By applying immunofluorescence, confocal microscopy and 3D image analysis we assessed the potential of differential nuclear distribution of methylated DNA to be utilized as a biomarker to characterize cells during development and when diseased. There are two major fields that may immediately benefit from this development: (1) the search for factors that contribute to pluripotency and cell fate in human embryonic stem cell expansion and differentiation, and (2) the characterization of tumor cells with regard to their heterogeneity in molecular composition and behavior. We performed topological analysis of the distribution of methylated CpG-sites (MeC) versus heterochromatin. This innovative approach revealed significant differences in colocalization patterns of MeC and heterochromatin-derived signals between undifferentiated and differentiated human embryonic stem cells, as well as untreated AtT20 mouse pituitary tumor cells compared to a subpopulation of these cells treated with 5-azacytidine for 48 hours.

  5. In Vitro Differentiation and Maturation of Human Embryonic Stem Cell into Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Amer Mahmood

    2011-01-01

    Full Text Available Human embryonic stem cells (hESCs, which have the potential to generate virtually any differentiated progeny, are an attractive cell source for transplantation therapy, regenerative medicine, and tissue engineering. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Basic science in the field of embryonic development, stem cell differentiation, and tissue engineering has offered important insights into key pathways and scaffolds that regulate hESC differentiation, which have produced advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, ectoderm, and many of their downstream derivatives. These findings have lead to identification of several pathways controlling the differentiation of hESCs into mesodermal derivatives such as myoblasts, mesenchymal cells, osteoblasts, chondrocytes, adipocytes, as well as hemangioblastic derivatives. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of bone and vascular diseases.

  6. Comparison of odontogenic differentiation of human dental follicle cells and human dental papilla cells.

    Directory of Open Access Journals (Sweden)

    Lijuan Guo

    Full Text Available Classical tooth development theory suggests that dental papilla cells (DPCs are the precursor cells of odontoblasts, which are responsible for dentin development. However, our previous studies have indicated that dental follicle cells (DFCs can differentiate into odontoblasts. To further our understanding of tooth development, and the differences in dentinogenesis between DFCs and DPCs, the odontogenic differentiation of DFCs and DPCs was characterized in vitro and in vivo. DFCs and DPCs were individually combined with treated dentin matrix (TDM before they were subcutaneously implanted into the dorsum of mice for 8 weeks. Results showed that 12 proteins were significantly differential, and phosphoserine aminotransferase 1 (PSAT1, Isoform 2 of hypoxia-inducible factor 1-alpha (HIF1A and Isoform 1 of annexin A2 (ANXA2, were the most significantly differential proteins. These proteins are related to regulation of bone balance, angiogenesis and cell survival in an anoxic environment. Both DFCs and DPCs express odontogenic, neurogenic and peridontogenic markers. Histological examination of the harvested grafts showed that both DFCs and DPCs form pulp-dentin/cementum-periodentium-like tissues in vivo. Hence, DFCs and DPCs have similar odontogenic differentiation potential in the presence of TDM. However, differences in glucose and amino acid metabolism signal transduction and protein synthesis were observed for the two cell types. This study expands our understanding on tooth development, and provides direct evidence for the use of alternative cell sources in tooth regeneration.

  7. Differentiation of human menstrual blood-derived endometrial mesenchymal stem cells into oocyte-like cells.

    Science.gov (United States)

    Lai, Dongmei; Guo, Ying; Zhang, Qiuwan; Chen, Yifei; Xiang, Charlie

    2016-11-01

    Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood are a unique stem cell source. Evidence suggests that EnSCs exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. However, the potential of EnSCs to differentiate into germline cells in vitro remains unclear. In this study, EnSCs were induced to differentiate into germ cells in a differentiation medium supplemented with 20% human follicular fluid. Our results demonstrated that EnSCs derived from human menstrual blood form oocyte-like cells and express germ cell markers. The induced cell aggregates contained not only oocyte-like structures but also cells expressing follicle stimulating hormone receptor and luteotropic hormone receptor, and produced estrogen and progesterone regulated by gonodatropin, suggesting that granulosa-like and theca-like cells were also induced. We further found that granulosa cells promote the development of oocyte-like cells and activate the induction of blastocyst-like structures derived from EnSCs. In conclusion, EnSCs may potentially represent an in vitro system for the investigation of human folliculogenesis.

  8. Ex vivo differentiation of multipotent adult progenitor cells to skin epidermal cells

    Institute of Scientific and Technical Information of China (English)

    Ji Kaihong; Xiong Jun; Fan Lixing; Hu Kaimeng; Liu Houqi

    2009-01-01

    Objective: By establishing the indirect contact co-culture system, we studied the in vitro condition for MAPCs differentiating into epidermal cells and the transformation of MAPCs into epidermal cell phenotype. Methods: Cell culture insert membrane was used for substitute basal membrane and MAPCs, fibroblast cells (FCs) and mixture of MAPCs and epidermal cells and FCs were separately implanted into 2 sides of it. PKH26 was used to label cloned MAPCs; type Ⅳ collagen rapid adhering method was used to isolate and culture the skin epidermal cells from 1-day-old SD rat. Results: Part of the MAPCs transformed into cells expressing keratin in the presence of peripheral epithelia and FCs. Type Ⅳ collagen rapid adhering method successfully selected rats' epidermal stem cells. The mixture of the 2 kinds of cells or indirect culture might promote the differentiation through mesenchymal factors secreted by dermis FC. Conclusion: We were the first to have established the in vitro model of MAPCs differentiation into epidermal cells, in which MAPCs were transformed into epithelium-like cells.

  9. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    Science.gov (United States)

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

  10. Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells.

    Science.gov (United States)

    Funakoshi, Tadanao; Spector, Myron

    2010-06-01

    Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones.

  11. Stem cell proliferation and differentiation a multitype branching process model

    CERN Document Server

    Macken, Catherine A

    1988-01-01

    The body contains many cellular systems that require the continuous production of new, fully functional, differentiated cells to replace cells lacking or having limited self-renewal capabilities that die or are damaged during the lifetime of an individual. Such systems include the epidermis, the epithelial lining of the gut, and the blood. For example, erythrocytes (red blood cells) lack nuclei and thus are incapable of self-replication. They have a life span in the circulation of about 120 days. Mature granulocytes, which also lack proliferative capacity, have a much shorter life span - typically 12 hours, though this may be reduced to only two or three hours in times of serious tissue infection. Perhaps a more familiar example is the outermost layer of the skin. This layer is composed of fully mature, dead epidermal cells that must be replaced by the descendants of stem cells lodged in lower layers of the epidermis (cf. Alberts et al. , 1983). In total, to supply the normal steady-state demands of cells, an...

  12. Transient expression of Olig1 initiates the differentiation of neural stem cells into oligodendrocyte progenitor cells

    NARCIS (Netherlands)

    Balasubramaniyan, [No Value; Timmer, N; Kust, B; Boddeke, E; Copray, S

    2004-01-01

    In order to develop an efficient strategy to induce the in vitro differentiation of neural stem cells (NSCs) into oligodendrocyte progenitor cells (OPCs), NSCs were isolated from E14 mice and grown in medium containing epidermal growth factor and fibroblast growth factor (FGF). Besides supplementing

  13. Efficient Differentiation of Embryonic Stem Cells into Neurons in Glial Cell-conditioned Medium under Attaching Conditions

    Institute of Scientific and Technical Information of China (English)

    Hai-Bin TIAN; Zeng-Liang BAI; Hong WANG; Jian-Quan CHEN; Guo-Xiang CHENG

    2005-01-01

    Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell- conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.

  14. Memory CD8+ T cell differentiation in viral infection: A cell for all seasons

    Institute of Scientific and Technical Information of China (English)

    Henry Radziewicz; Luke Uebelhoer; Bertram Bengsch; Arash Grakoui

    2007-01-01

    Chronic viral infections such as hepatitis B virus (HBV),hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are major global health problems affecting more than 500 million people worldwide. Virus-specific CD8+ T cells play an important role in the course and outcome of these viral infections and it is hypothesized that altered or impaired differentiation of virusspecific CD8+ T cells contributes to the development of persistence and/or disease progression. A deeper understanding of the mechanisms responsible for functional differentiation of CD8+ T cells is essential for the generation of successful therapies aiming to strengthen the adaptive component of the immune system.

  15. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    Science.gov (United States)

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  16. Differential cytotoxicity of copper ferrite nanoparticles in different human cells.

    Science.gov (United States)

    Ahmad, Javed; Alhadlaq, Hisham A; Alshamsan, Aws; Siddiqui, Maqsood A; Saquib, Quaiser; Khan, Shams T; Wahab, Rizwan; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Akhtar, Mohd Javed; Ahamed, Maqusood

    2016-10-01

    Copper ferrite nanoparticles (NPs) have the potential to be applied in biomedical fields such as cell labeling and hyperthermia. However, there is a lack of information concerning the toxicity of copper ferrite NPs. We explored the cytotoxic potential of copper ferrite NPs in human lung (A549) and liver (HepG2) cells. Copper ferrite NPs were crystalline and almost spherically shaped with an average diameter of 35 nm. Copper ferrite NPs induced dose-dependent cytotoxicity in both types of cells, evident by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and neutral red uptake assays. However, we observed a quite different susceptibility in the two kinds of cells regarding toxicity of copper ferrite NPs. Particularly, A549 cells showed higher susceptibility against copper ferrite NP exposure than those of HepG2 cells. Loss of mitochondrial membrane potential due to copper ferrite NP exposure was observed. The mRNA level as well as activity of caspase-3 enzyme was higher in cells exposed to copper ferrite NPs. Cellular redox status was disturbed as indicated by induction of reactive oxygen species (oxidant) generation and depletion of the glutathione (antioxidant) level. Moreover, cytotoxicity induced by copper ferrite NPs was efficiently prevented by N-acetylcysteine treatment, which suggests that reactive oxygen species generation might be one of the possible mechanisms of cytotoxicity caused by copper ferrite NPs. To the best of our knowledge, this is the first report showing the cytotoxic potential of copper ferrite NPs in human cells. This study warrants further investigation to explore the mechanisms of differential toxicity of copper ferrite NPs in different types of cells. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Enhancement of early cardiac differentiation of dedifferentiated fat cells by dimethyloxalylglycine via notch signaling pathway

    OpenAIRE

    Li, Fuhai; Li, Zongzhuang; Jiang, Zhi; Tian, Ye; Wang, Zhi; YI, WEI; Zhang, Chenyun

    2016-01-01

    Background: Hypoxia has been reported to possess the ability to induce mature lipid-filled adipocytes to differentiate into fibroblast-like multipotent dedifferentiated fat (DFAT) cells and stem cells such as iPSCs (interstitial pluripotent stem cells) and ESCs (embryonic stem cells) and then to differentiate into cardiomyocytes. However, the effect of hypoxia on cardiac differentiation of DFAT cells and its underlying molecular mechanism remains to be investigated. Objective: To investigate ...

  18. Two-step differentiation of mast cells from induced pluripotent stem cells.

    Science.gov (United States)

    Yamaguchi, Tomoko; Tashiro, Katsuhisa; Tanaka, Satoshi; Katayama, Sumie; Ishida, Waka; Fukuda, Ken; Fukushima, Atsuki; Araki, Ryoko; Abe, Masumi; Mizuguchi, Hiroyuki; Kawabata, Kenji

    2013-03-01

    Mast cells play important roles in the pathogenesis of allergic diseases. They are generally classified into 2 phenotypically distinct populations: connective tissue-type mast cells (CTMCs) and mucosal-type mast cells (MMCs). The number of mast cells that can be obtained from tissues is limited, making it difficult to study the function of mast cells. Here, we report the generation and characterization of CTMC-like mast cells derived from mouse induced pluripotent stem (iPS) cells. iPS cell-derived mast cells (iPSMCs) were generated by the OP9 coculture method or embryoid body formation method. The number of Safranin O-positive cells, expression levels of CD81 protein and histidine decarboxylase mRNA, and protease activities were elevated in the iPSMCs differentiated by both methods as compared with those in bone marrow-derived mast cells (BMMCs). Electron microscopic analysis revealed that iPSMCs contained more granules than BMMCs. Degranulation was induced in iPSMCs after stimulation with cationic secretagogues or vancomycin. In addition, iPSMCs had the ability to respond to stimulation with the IgE/antigen complex in vitro and in vivo. Moreover, when iPSMCs generated on OP9 cells were cocultured with Swiss 3T3 fibroblasts, protease activities as maturation index were more elevated, demonstrating that mature mast cells were differentiated from iPS cells. iPSMCs can be used as an in vitro model of CTMCs to investigate their functions.

  19. Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

    Science.gov (United States)

    Lu, Changming; Huguley, Samuel; Cui, Chun; Cabaniss, Lauren B; Waite, Peter D; Sarver, David M; Mamaeva, Olga A; MacDougall, Mary

    2016-01-01

    The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

  20. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

    Science.gov (United States)

    Sluch, Valentin M; Davis, Chung-ha O; Ranganathan, Vinod; Kerr, Justin M; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S; Mao, Hai-Quan; Zack, Donald J

    2015-11-13

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

  1. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  2. Biomaterials coated by dental pulp cells as substrate for neural stem cell differentiation.

    Science.gov (United States)

    Soria, Jose Miguel; Sancho-Tello, María; Esparza, M Angeles Garcia; Mirabet, Vicente; Bagan, Jose Vicente; Monleón, Manuel; Carda, Carmen

    2011-04-01

    This study is focused on the development of an in vitro hybrid system, consisting in a polymeric biomaterial covered by a dental pulp cellular stroma that acts as a scaffold offering a neurotrophic support for the subsequent survival and differentiation of neural stem cells. In the first place, the behavior of dental pulp stroma on the polymeric biomaterial based on ethyl acrylate and hydroxy ethyl acrylate copolymer was studied. For this purpose, cells from normal human third molars were grown onto 0.5-mm-diameter biomaterial discs. After cell culture, quantification of neurotrophic factors generated by the stromal cells was performed by means of an ELISA assay. In the second place, survival and differentiation of adult murine neural stem cells on the polymeric biomaterials covered by dental pulp stromal cells was studied. The results show the capacity of dental pulp cells to uniformly coat the majority of the material's surface and to secrete neurotrophic factors that become crucial for a subsequent differentiation of neural stem cells. The use of stromal cells cultured on scaffolding biomaterials provides neurotrophic pumps that may suggest new criteria for the design of cell therapy experiments in animal models to assist the repair of lesions in Central Nervous System.

  3. Differentiation of rat embryonic neural stem cells promoted by co-cultured Schwann cells

    Institute of Scientific and Technical Information of China (English)

    万虹; 安沂华; 张泽舜; 张亚卓; 王忠诚

    2003-01-01

    Objective To explore the factors which induce differentiation of embryonic neural stem cells. Methods Rat embryonic neural stem cells were co-cultured with newborn rat Schwann cells in serum-free medium. The phenotype and specific-markers including tubulin-β, glial fibrillary acidic protein (GFAP) and galactorcerebroside (GalC), were domonstrated by phase contrast microscopy and double immunofluorescence staining. Results Overall, 80%±5% of neural stem cells protruded several elongated processes and expressed tubulin-β antigen at high levels, while 20±3% of them protruded several short processes and were GalC or GFAP positive. Conclusion The factors secreted by Schwann cells could induce rat embryonic neural stem cell to differentiate.

  4. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    Full Text Available BACKGROUND: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. CONCLUSIONS/SIGNIFICANCE: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  5. Differential T helper cell response in tuberculous pleuritis

    Directory of Open Access Journals (Sweden)

    Prabha C

    2007-01-01

    Full Text Available Purpose: This study was conducted to understand the in vivo and in vitro immune responses and to find whether there exists any difference in the systemic and localized immune responses in tuberculous pleuritis. Methods: The in vivo levels of IFN-γ and IL-4 were compared in plasma (BL and pleural fluid (PF of 47 tuberculous (TB and 31 nontuberculous pleuritis (Non-TB patients. In vitro cytokine response to various mycobacterial antigens was studied in 19 TB patients by ELISA . Both ex vivo and in vitro cytokine responses were further ascertained by intracellular cytokine staining on purified CD4+ T cells from pleural fluid mononuclear cells (PFMC of 10 TB patients. Results: The ex vivo results showed a significant increase in IFN-γ levels and higher IFN-γ + T cells in PF. On the other hand, in vitro results showed simultaneous increase in both IFN-γ and IL-4 levels in the supernatants of antigen stimulated PFMC. Similarly antigen specific increase was observed in both IFN-γ + and IL-4+ T cells in all cultured conditions. However, the percentile increase was more in IL-4 secreting T cells, significant for PPD stimulation ( P < 0.05, indicating that in vitro cellular response was dominated by Th2 type. Conclusions: These results showed a differential T-helper response in TB pleuritis suggestive of predominant Th1 in vivo and mixed response (Th1 and Th2 under in vitro conditions.

  6. 3D surface topology guides stem cell adhesion and differentiation.

    Science.gov (United States)

    Viswanathan, Priyalakshmi; Ondeck, Matthew G; Chirasatitsin, Somyot; Ngamkham, Kamolchanok; Reilly, Gwendolen C; Engler, Adam J; Battaglia, Giuseppe

    2015-06-01

    Polymerized high internal phase emulsion (polyHIPE) foams are extremely versatile materials for investigating cell-substrate interactions in vitro. Foam morphologies can be controlled by polymerization conditions to result in either open or closed pore structures with different levels of connectivity, consequently enabling the comparison between 2D and 3D matrices using the same substrate with identical surface chemistry conditions. Additionally, here we achieve the control of pore surface topology (i.e. how different ligands are clustered together) using amphiphilic block copolymers as emulsion stabilizers. We demonstrate that adhesion of human mesenchymal progenitor (hES-MP) cells cultured on polyHIPE foams is dependent on foam surface topology and chemistry but is independent of porosity and interconnectivity. We also demonstrate that the interconnectivity, architecture and surface topology of the foams has an effect on the osteogenic differentiation potential of hES-MP cells. Together these data demonstrate that the adhesive heterogeneity of a 3D scaffold could regulate not only mesenchymal stem cell attachment but also cell behavior in the absence of soluble growth factors.

  7. Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

    Science.gov (United States)

    Fishman, Bettina; Segev, Hanna; Kopper, Oded; Nissenbaum, Jonathan; Schulman, Margarita; Benvenisty, Nissim; Itskovitz-Eldor, Joseph; Kitsberg, Danny

    2012-09-01

    New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

  8. miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

    OpenAIRE

    Gonzalez, Manuel A.; Tomé, María; López-Romero, Pedro; Albo, Carmen; Sepúlveda, Juan Carlos; Fernández-Gutierrez, Benjamin; Dopazo, Ana; Bernad, Antonio

    2010-01-01

    Abstract In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation pro...

  9. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...... is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...

  10. Directed differentiation of mouse embryonic stem cells into thyroid follicular cells.

    Science.gov (United States)

    Arufe, Maria C; Lu, Min; Kubo, Atsushi; Keller, Gordon; Davies, Terry F; Lin, Reigh-Yi

    2006-06-01

    Elucidating the molecular mechanisms leading to the induction and specification of thyroid follicular cells is important for our understanding of thyroid development. To characterize the key events in this process, we previously established an experimental embryonic stem (ES) cell model system, which shows that wild-type mouse CCE ES cells can give rise to thyrocyte-like cells in vitro. We extend our analysis in this report by using a genetically manipulated ES cell line in which green fluorescent protein (GFP) cDNA is targeted to the TSH receptor (TSHR) gene, linking GFP expression to the transcription of the endogenous TSHR gene. The appearance of GFP-positive cells was dependent on the formation of embryoid bodies from undifferentiated ES cells and was greatly enhanced by TSH treatment during the first 2-4 d of differentiation. With the support of Matrigel, highly enriched ES cell-derived GFP-positive cells formed thyroid follicle-like clusters in a serum-free medium supplemented with TSH. Importantly, these clusters display the characteristics of thyroid follicular cells. Immunofluorescent studies confirmed the colocalization of TSHR with the Na+/I- symporter in the clusters and indicated that Na+/I- symporter was expressed exclusively in the plasma membrane. In addition, I- uptake activity was observed in these cells. Our results indicate that ES cells can be induced to differentiate into thyroid follicular cells, providing a powerful tool to study embryonic thyroid development and function.

  11. Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

    Science.gov (United States)

    Goto, Yoshiyuki; Panea, Casandra; Nakato, Gaku; Cebula, Anna; Lee, Carolyn; Diez, Marta Galan; Laufer, Terri M; Ignatowicz, Leszek; Ivanov, Ivaylo I

    2014-04-17

    How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

  12. Effect of Rat Schwann Cell Secretion on Proliferation and Differentiation of Human Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and b-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were b-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.

  13. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Cedric Cortijo

    2012-12-01

    Full Text Available Planar cell polarity (PCP refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

  14. Sarcomatoid differentiation in renal cell carcinoma: prognostic implications

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall'Oglio

    2005-02-01

    Full Text Available INTRODUCTION: Renal cell carcinoma with sarcomatoid differentiation is a tumor with aggressive behavior that is poorly responsive to immunotherapy. The objective of this study is to report our experience in the treatment of 15 patients with this tumor. MATERIALS AND METHODS: We retrospectively analyzed 15 consecutive cases of renal cell carcinoma with sarcomatoid differentiation diagnosed between 1991 and 2003. The clinical presentation and the pathological stage were assessed, as were the tumor's pathological features, use of adjuvant immunotherapy and survival. The study's primary end-point was to assess survival of these individuals. RESULTS: The sample included 8 women and 7 men with mean age of 63 years (44 - 80; follow-up ranged from 1 to 100 months (mean 34. Upon presentation, 87% were symptomatic and 4 individuals had metastatic disease. Mean tumor size was 9.5 cm (4 - 24 with the following pathological stages: 7% pT1, 7% pT2, 33% pT3, and 53% pT4. The pathological features showed high-grade tumors with tumoral necrosis in 87% of the lesions and 80% of intratumoral microvascular invasion. Disease-free and cancer-specific survival rates were 40 and 46% respectively, with 2 cases responding to adjuvant immunotherapy. CONCLUSIONS: Patients with sarcomatoid tumors of the kidney have a low life expectancy, and sometimes surgical resection associated with immunotherapy can lead to a long-lasting therapeutic response.

  15. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes

    Science.gov (United States)

    Park, Yun-Jong; Koh, Jin; Kwon, Jin Teak; Park, Yong-Seok; Yang, Lijun; Cha, Seunghee

    2017-01-01

    Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren’s syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation. PMID:28158262

  16. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    DEFF Research Database (Denmark)

    Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel

    2012-01-01

    Planar cell polarity (PCP) refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.......5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...

  17. Differentiation of Neonatal Human-Induced Pluripotent Stem Cells to Prostate Epithelial Cells: A Model to Study Prostate Cancer Development

    Science.gov (United States)

    2014-06-01

    1 AD_________________ Award Number: W81XWH-12-1-0189 TITLE: Differentiation of Neonatal Human...CONTRACT NUMBER Differentiation of Neonatal Human Induced Pluripotent Stem Cells to Prostate Epithelial Cells: A Model to Study Prostate Cancer...13. SUPPLEMENTARY NOTES 14. ABSTRACT We set out to establish conditions for differentiation of human neonatal foreskin skin fibroblast

  18. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    Science.gov (United States)

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.

  19. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  20. Circadian Clock Genes Modulate Human Bone Marrow Mesenchymal Stem Cell Differentiation, Migration and Cell Cycle.

    Science.gov (United States)

    Boucher, Helene; Vanneaux, Valerie; Domet, Thomas; Parouchev, Alexandre; Larghero, Jerome

    2016-01-01

    Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2'E,3'Z-6-bromoindirubin-3'-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties.

  1. Basophil-derived IL-6 regulates TH17 cell differentiation and CD4 T cell immunity

    Science.gov (United States)

    Yuk, Chae Min; Park, Hyeung Ju; Kwon, Bo-In; Lah, Sang Joon; Chang, Jun; Kim, Ji-Young; Lee, Kyung-Mi; Park, Su-Hyung; Hong, Seokchan; Lee, Seung-Hyo

    2017-01-01

    Basophils are rare, circulating granulocytes proposed to be involved in T helper (TH) type 2 immunity, mainly through secretion of interleukin (IL)-4. In addition to IL-4, basophils produce IL-6 and tumor necrosis factor (TNF)-α in response to immunoglobulin E (IgE) crosslinking. Differentiation of TH17 cells requires IL-6 and transforming growth factor (TGF)-β, but whether basophils play a significant role in TH17 induction is unknown. Here we show a role for basophils in TH17 cell development by using in vitro T cell differentiation and in vivo TH17-mediated inflammation models. Bone marrow derived-basophils (BMBs) and splenic basophils produce significant amounts of IL-6 as well as IL-4 following stimulation with IgE crosslink or cholera toxin (CT). In addition, through IL-6 secretion, BMBs cooperate with dendritic cells to promote TH17 cell differentiation. In the TH17 lung inflammation model, basophils are recruited to the inflamed lungs following CT challenge, and TH17 responses are significantly reduced in the absence of basophils or IL-6. Furthermore, reconstitution with wild-type, but not IL-6-deficient, basophils restored CT-mediated lung inflammation. Lastly, basophil-deficient mice showed reduced phenotypes of TH17-dependent experimental autoimmune encephalomyelitis. Therefore, our results indicate that basophils are an important inducer of TH17 cell differentiation, which is dependent on IL-6 secretion. PMID:28134325

  2. Comparison Between Transepicardial Cell Transplantations: Autologous Undifferentiated Versus Differentiated Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Farid Azmoudeh Ardalan

    2007-06-01

    Full Text Available Background: Marrow-derived mesenchymal stem cells (MSCs have been heralded as a source of great promise for the regeneration of the infarcted heart. There are no clear data as to whether or not in vitro differentiation of MSCs into major myocardial cells can increase the beneficial effects of MSCs. The aim of this study was to address this issue.Methods: To induce MSCs to transdifferentiate into cardiomyocytes and endothelial cells, 5-Azacytidine and vascular endothelial growth factor (VEGF were used, respectively. Myocardial infarction in rabbits was generated by ligating the left anterior descending coronary artery. The animals were divided into three experimental groups: I control group, II undifferentiated mesenchymal stem cell transplantation group, and III differentiated mesenchymal stem cell transplantation group. The three groups received peri-infarct injections of culture media, autologous undifferentiated MSCs, and autologous differentiated MSCs, respectively. Echocardiography and pathology were performed in order to search for improvement in the cardiac function and reduction in the infarct size. Results: Improvements in the left ventricular function and reductions in the infarcted area were observed in both cell transplanted groups (Groups II and III to the same degree. Conclusions: There is no need for prior differentiation induction of marrow-derived MSCs before transplantation, and peri-infarct implantation of MSCs can effectively reduce the size of the infarct and improve the cardiac function.

  3. miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

    Institute of Scientific and Technical Information of China (English)

    Yuxin Ni; Kaizhi Zhang; Xuejuan Liu; Tingting Yang; Baixiang Wang; Li Fu; Lan A; Yanmin Zhou

    2014-01-01

    Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair folli-cles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regu-lating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA.

  4. Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHEN XU; MIN XIONG SHEN; DONG ZHU MA; LI YING WANG; XI LIANG ZHA

    2003-01-01

    Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 × l06 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

  5. Differentiation of dermis-derived multipotent cells into insulin-producing pancreatic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Chun-Meng Shi; Tian-Min Cheng

    2004-01-01

    AIM: To observe the plasticity of whether dermis-derived multipotent cells to differentiate into insulin-producing pancreatic cells in vitro.METHODS: A donal population of dermis-derived multipotent stem cells (DMCs) from newborn rat with the capacity to produce osteocytes, chondrocytes, adipocytes and neurons was used. The gene expression of cultured DMCs was assessed by DNA microarray using rat RGU34A gene expression probe arrays. DMCs were further cultured in the presence of insulin complex components (Insulintransferrin-selenium, ITS) to observe whether DMCs could be induced into insulin-producing pancreatic cells in vitro.RESULTS: DNA microarray analysis showed that cultured DMCs simultaneously expressed several genes associated with pancreatic cell, neural cell, epithelial cell and hepatocyte,widening its transcriptomic repertoire. When cultured in the specific induction medium containing ITS for pancreatic cells, DMCs differentiated into epithelioid cells that were positive for insulin detected by immunohistochemistry.CONCLUSION: Our data indicate that dermal multipotent cells may serve as a source of stem/progenitor cells for insulin-producing pancreatic cells.

  6. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Huang, Xiaohua [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044 (China); An, Yue [Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Ren, Feng [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); He, Xiaowen; Schachner, Melitta [Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, NJ (United States); Xiao, Zhicheng, E-mail: zhicheng.xiao@monash.edu [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); Ma, Keli, E-mail: makeli666@aliyun.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Li, Yali, E-mail: yalilipaper@gmail.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Anatomy, National University of Singapore, Singapore 119078 (Singapore)

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  7. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  8. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    Yanan Hou; Qin Huang; Tianjin Liu; Lihe Guo

    2008-01-01

    Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulinproducing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulinproducing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic --cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6,NK2 transcription factor-related locus 2, Islet 1, glucokinase,and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

  9. Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype.

    Science.gov (United States)

    Reyes-Martínez, Juana Elizabeth; Nieto-Patlán, Erik; Nieto-Patlán, Alejandro; Gonzaga-Bernachi, Job; Santos-Mendoza, Teresa; Serafín-López, Jeanet; Chávez-Blanco, Alma; Sandoval-Montes, Claudia; Flores-Romo, Leopoldo; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

    2014-01-01

    Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.

  10. Current concepts of hair cell differentiation and planar cell polarity in inner ear sensory organs.

    Science.gov (United States)

    Sienknecht, Ulrike J

    2015-07-01

    Phylogenetically and ontogenetically, vertebrate development led to the generation of several inner ear sensory organs. During embryogenesis, cell fate specification determines whether each progenitor cell differentiates into a sensory hair cell or a supporting cell within the common sensory primordium. Finally, all sensory epithelia of the inner ear consist of a hair cell/supporting cell mosaic, albeit with anatomical differences depending on the sensory organ type. Hair cells develop a polarized bundle of stereovilli that is of functional importance for mechanotransduction. After initiating stereovillar development, hair cells align their bundles in a coordinated fashion, generating a characteristic hair cell orientation pattern, a process referred to as planar cell polarity (PCP). The pathway that controls PCP in the inner ear needs both to establish the development of a polarized morphology of the stereovillar bundle of the hair cell and to organize a systematic hair cell alignment. Because the hair cell orientation patterns of the various inner ear organs and vertebrate species differ fundamentally, it becomes apparent that in vertebrates, different aspects of PCP need to be independently controlled. In spite of important progress recently gained in the field of PCP research, we still need to identify the mechanisms (1) that initiate molecular asymmetries in cells, (2) that guide the transmission of polarity information from cell to cell, and (3) that consistently translate such polarity information into morphological asymmetries of hair cells.

  11. Pancreas development and beta-cell differentiation of embryonic stem cells.

    Science.gov (United States)

    Rivas-Carrillo, Jorge David; Okitsu, Teru; Tanaka, Noriaki; Kobayashi, Naoya

    2007-01-01

    Embryonic stem (ES) cells may offer an unlimited cell source for the treatment of diabetes. However, a successful derivation of ES cells into islet-cells has proven to be more difficult than it was initially expected. Considering that the pancreas coordinates the global use of energy in the organism by secreting digestive enzymes and hormones, it is understandable that a sophisticated and tight regulation that lies on the pancreas itself to orchestrate its own tissue development and maturation. The complex process of endocrine cell differentiation can be better understood by analyzing the normal development of the pancreas. The proper detection of the signals provided in the pancreatic environment gives us a clue as to how the stem cells give rise to the whole pancreas. Careful and extensive screening of the natural or synthetic cytokines and growth factors and biochemical compounds that are essential in pancreatic development is required to properly mimic the process in vitro. Such a study would allow the researchers to achieve selective control of the differentiation and proliferation of the stem cells. The development and identification of the key molecules can provide us new insights into the pancreatic differentiation of the stem cells. We herein discuss the role of the microenvironment and transcriptional factors and cytokines, which have been recognized as important molecules during the major steps of the development of the pancreas. Finally, a more complete comprehension of the mechanisms that drive the pancreatic regeneration will provide us with new perspectives for future prophylactic and therapeutic interventions.

  12. Contribution of xanthine oxidoreductase to mammary epithelial and breast cancer cell differentiation in part modulates inhibitor of differentiation-1.

    Science.gov (United States)

    Fini, Mehdi A; Monks, Jenifer; Farabaugh, Susan M; Wright, Richard M

    2011-09-01

    Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

  13. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  14. Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Alkhalil

    2015-02-01

    Full Text Available Aim This study was focused on the isolation and characterization of mesenchymal stem cells (MSCs from human dental pulp (DPSC. Methods The study was performed in the Department for Oral and Cranio-Maxillo- Facial Surgey Hamad Medical Corporation, Doha, Qatar and Weill Cornell Medical Colleague Doha, Qatar, in period 2010-2011. Dental pulp was extracted from premolars and third molars of 19 healthy patients. The pulp was digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 1 hour at 37C. After filtration, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM Low Glucoses with 20% Fetal Bovine Serum (FBS, 2mM L-glutamine and antibiotics (100 U/mL penicillin, 100 ug/mL streptomycin at 37 °C under 5% CO2. Cultures were treated with osteoinductive medium for differentiation MSC in to the osteoblast cell line. Staining with Alizarin red were used for the detection of the osteoblast production and calcification new formed tissue. Results On the total of three out of 19 patients it was possible to isolate DPMSCs after 2 to 3 weeks: in one patient it was not possible to expand MSCs because of infection, and in other two patients positive Alizarin red staining reaction showed osteogenic differentiation capability and strong mineralization in vitro. Conclusion The main advantage of using DPSC is absence of morbidity. MSCs could be isolated noninvasively from teeth, routinely extracted in the clinic and discarded as medical waste. Standardization of clinical and laboratory protocols for DPMSCs isolation and team work coordination could lead to significantly improved result.

  15. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Jing [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Liu, Gexiu [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Yan, Guoyao [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); He, Dongmei [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Zhou, Ying [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Chen, Shengting, E-mail: shengtingchen@sina.cn [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China)

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  16. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  17. The Histone H2B Monoubiquitination Regulatory Pathway Is Required for Differentiation of Multipotent Stem Cells

    DEFF Research Database (Denmark)

    Karpiuk, Oleksandra; Najafova, Zeynab; Kramer, Frank;

    2012-01-01

    Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly und...

  18. Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Xuewei Xie; Zhouping Tang; Feng Xu; Na Liu; Zaiwang Li; Suiqiang Zhu; Wei Wang

    2009-01-01

    BACKGROUND: Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors.OBJECTIVE: To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells.DESIGN, TIME AND SETrlNG: Cytology was performed at the Department of Neurology, Tongji Medical College, Huazhong University of Science and Technology, China, from September 2007 to October 2008.MATERIALS: Mouse anti-nestin polyclonal antibody (Chemicon, USA), mouse anti-glial fibrillary acidic protein (GFAP) IgG1, mouse anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) IgG1, mouse anti-Tubulin Class-Ill IgG1 (Neo Markers, USA), Avidin-labeled Cy3 (KPL, USA), and goat anti-mouse IgG1: fluorescein isothiocyanate (FITC) (Serotec, UK) were used in this study.METHODS: Tissues were isolated from the embryonic olfactory bulb and subependymal region of Wistar rats. Serum-free DMEM/F12 culture media was used for co-culture experiments. Neural stem cells were incubated in serum-free or 5% fetal bovine serum-containing DMEM/F12 as controls.MAIN OUTCOME MEASURES: After 7 days of co-culture, neural stem cells and olfactory ensheathing cells underwent immunofluorescent staining for nestin, tubulin, glial fibrillary acidic protein, and CNPase.RESULTS: Olfactory ensheathing cells promoted proliferation and differentiation of neural stem cells into neuron-like cells, astrocytes and oligodendrocytes. The proportion of neuron-like cells was 78.2%, but the proportion of neurons in 5% fetal bovine serum DMEM/F12 was 48.3%. In the serum-free DMEM/F12, neural stem cells contracted, unevenly adhered to the glassware wall, or underwent apoptosis at 7 days.CONCLUSION: Olfactory ensheathing cells promote differentiation of neural stem cells mainly into neuron-like cells, and accelerate proliferation of neural stem cells. The outcome is better compared with serum-free medium or medium containing 5% fetal bovine

  19. The nuclear pore complex acts as a master switch for nuclear and cell differentiation

    Science.gov (United States)

    Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-01-01

    Cell differentiation is associated with the functional differentiation of the nucleus, in which alteration of the expression profiles of transcription factors occurs to destine cell fate. Nuclear transport machineries, such as importin-α, have also been reported as critical factors that induce cell differentiation. Using various fluorescence live cell imaging methods, including time-lapse imaging, FRAP analysis and live-cell imaging associated correlative light and electron microscopy (Live CLEM) of Tetrahymena, a unicellular ciliated protozoan, we have recently discovered that type switching of the NPC is the earliest detectable event of nuclear differentiation. Our studies suggest that this type switching of the NPC directs the fate of the nucleus to differentiate into either a macronucleus or a micronucleus. Our findings in this organism may provide new insights into the role of the NPC in controlling nuclear functions in general in eukaryotes, including controlling cell fate leading to cell differentiation in multicellular metazoa. PMID:26479399

  20. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    OpenAIRE

    2003-01-01

    Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plas...

  1. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    Donghui Zhang; Wei Jiang; Meng Liu; Xin Sui; Xiaolei Yin; Song Chen; Yan Shi; Hongkui Deng

    2009-01-01

    Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

  2. Forced expression of the Oct-4 gene influences differentiation of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists,forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.

  3. [Mouse teratocarcinoma: differentiation in cultures of a multipotential primitive cell line (author's transl)].

    Science.gov (United States)

    Nicolas, J F; Dubois, P; Jakob, H; Gaillard, J; Jacob, F

    1975-01-01

    A line of primitive cells of teratocarcinoma is able of differentiating during in vitro cultures. The differentiated cell types correspond to derivatives of the three germ layers. They appear according to a reproducible schedule. The differentiated cell types exhibit a normal karyotype. They have lost their tumorigenicity and have a limited life span. Agregates containing various numbers of primitive cells have been prepared and plated in culture dishes. Their study shows that the first differentiated cell types appear only when the agregates are greater than a certain critical mass. Primitive cells carry on their surface the F9 but not the H-2 antigen. During differentiation in culture, the H-2 antigen appears rapidly on certain cells. The fraction of H-2-carrying cells increases as a function of time while that of F9-carrying cells decreases.

  4. Expression of maturation-specific nuclear antigens in differentiating human myeloid leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Epstein, A.L.; Clevenger, C.V.; Huberman, E.

    1985-02-01

    The expression of three myeloid-specific nuclear antigens was studied by indirect immunofluorescence with murine monoclonal antibodies in human myeloid (HL-60, ML-2, KG-1, and B-II) leukemia cells treated with chemical inducers of cell differentiation. Treatment of the promyelocytic HL-60 cells with dimethyl sulfoxide or 1,25-dihydroxyvitamin DT induced the cells to acquire a phenotype that resembled that of granulocytes and monocytesmacrophages, respectively. These phenotypes were characterized by changes in cell growth, cell morphology, expression of specific cell surface antigens, and activities of lysozyme and nonspecific esterase enzymes. Induction of these differentiation markers in the HL-60 cells was associated with induction of the myeloid-specific nuclear antigens. The ML-2 cells, which are arrested at the myeloblast-promyelocyte stage, were also susceptible to the induction of cell differentiation and to changes in the expression of the nuclear antigens, but the degree of susceptibility was less than in the HL-60 cells. The less-differentiated KG-1 and B-II myeloid cells were either not responsive or responded only in a limited degree to the induction of cell differentiation or to changes in the expression of the nuclear antigens. The authors suggest that the reactivity of cells with monoclonal antibodies to specific nuclear antigens can be used as a maturational marker in cell differentiation studies. Furthermore, nuclear antigens expressed early in cellular differentiation may provide information about changes in regulatory elements in normal and malignant cells. 40 references, 2 figures, 1 table.

  5. Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH Pathway Genes.

    Directory of Open Access Journals (Sweden)

    Hui-Hung Tzeng

    Full Text Available Human mesenchymal stem cells (MSCs modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF and the expression of BDNF receptor tyrosine receptor kinase B (TrkB correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-ε and kinesin heavy chain (KIF5B increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1 decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signaling pathway.

  6. Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

    Science.gov (United States)

    Jones, Lindsay L; Alli, Rajshekhar; Li, Bofeng; Geiger, Terrence L

    2016-04-01

    How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

  7. WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

    Institute of Scientific and Technical Information of China (English)

    Isabelle Bisson; David M Prowse

    2009-01-01

    Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their abil-ity to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heteroge-neous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treat-ment with WNT inhibitors reduced both prostasphere size and self-renewal, In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are con-sistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen re-ceptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit am-plifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell charac-teristics and improve the therapeutic outcome.

  8. Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Juan Zhang; Hui Li; Zhao Wu; XiaoJun Tan; Fengying Liu; Xianghong Huang; Xiaoling Fang

    2013-01-01

    Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles before 40 years of age,representing one major cause of female infertility.Stem cells provide the possibility of a potential treatment for POF.In this study,rat embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were co-cultured with granulosa cells (GCs) to differentiate to GC-like cells.The level of estradiol (E2) analyzed by radioimmunoassay showed that the E2 concentration of the culture supernatant of co-cultured rat iPSCs and ESCs increased in a time-dependent manner,compared with the GCs group that has an opposite trend.The expression of follicle-stimulating hormone receptor (FSHR) was confirmed by immunostaining.These results indicated that rat iPSCs and ESCs were effectively induced to GC-like cells through indirect cell-to-cell contact.Real-time polymerase chain reaction was performed to analyze the expression level of marker genes in POF,including BMP15,FMR1,FSHR,INHA,AMH,NOBOX,FOXO3,EIF2B,FIGLA,and GDF9.The BMP15,FSHR,INHA,AMH,NOBOX,and GDF9 genes were significantly up-regulated in iPSCs and ESCs cocultured with GCs in comparison with ceils that were not co-cultured.Thus,here we demonstrated an available method to differentiate rat iPSCs and ESCs into GC-like cells in vitro for the possible cell therapy of POF.

  9. The role of sex chromosomes in mammalian germ cell differentiation: can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes?

    OpenAIRE

    Teruko Taketo

    2015-01-01

    The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resulta...

  10. Human hemarthrosis-derived progenitor cells can differentiate into osteoblast-like cells in vitro.

    Science.gov (United States)

    Niikura, Takahiro; Miwa, Masahiko; Sakai, Yoshitada; Lee, Sang Yang; Kuroda, Ryosuke; Fujishiro, Takaaki; Kubo, Seiji; Doita, Minoru; Kurosaka, Masahiro

    2005-11-04

    We hypothesized that intraarticular osteochondral fracture-induced hemarthrosis could be a useful cell source for bone regeneration, as it is thought to contain osteoprogenitor cells derived from bone marrow. Therefore, we investigated whether human hemarthrosis-derived cells have the potential to differentiate into osteoblast-like cells in vitro. We aspirated hemarthrosis from patients suffering from osteochondral fractures of knee joints, and cultured hemarthrosis-derived cells in a medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid, or without them as control. The morphology of the treated cells appeared to be cuboidal shape, differing from spindle-like shape observed in the control. Matrix mineralization was observed only in the treated culture. Alkaline phosphatase activity and gene expression of alkaline phosphatase, parathyroid hormone receptor, osteopontin, and osteocalcin were up-regulated compared with the control. These studies demonstrate that human hemarthrosis-derived cells can differentiate into osteoblast-like cells, i.e., they contain osteoprogenitor cells and are a useful cell source for bone regeneration.

  11. Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

    Institute of Scientific and Technical Information of China (English)

    Yi Peng WANG; Ze Fen WANG; Ying Chun ZHANG; Qing TIAN; Jian Zhi WANG

    2004-01-01

    Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation,we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

  12. A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation

    DEFF Research Database (Denmark)

    Shih, Hung Ping; Kopp, Janel L; Sandhu, Manbir

    2012-01-01

    In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch...... signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9......, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation...

  13. Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    During the last decade,increasing evidence suggested that bone marrow stromal cells(MSCs) have the potential to differentiate into neural lineages.Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions.However,no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported.In this study,we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions.By using two-dimensional gel electrophoresis(2-DE),we compared the protein profiles of MSCs before and after induced differentiation.We obtained 792 protein spots in the protein profile by 2-DE,and found that 74 spots changed significantly before and after the differentiation using PDQuest software,with 43 up-regulated and 31 down-regulated.We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) and by database searching,and found that they could be grouped into various classes,including cytoskeleton and structure proteins,growth factors,metabolic proteins,chaperone proteins,receptor proteins,cell cycle proteins,calcium binding proteins,and other proteins.These proteins also include neural and glial proteins,such as BDNF,CNTF and GFAP.The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

  14. Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

    Institute of Scientific and Technical Information of China (English)

    LI WenTing; SUN HuaLin; XU ZengLu; DING Fei; GU XiaoSong

    2009-01-01

    During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morpho-logical changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and in-duced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We ana-lyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

  15. Thymic Nurse Cells Support CD4-CD8+ Thymocytes to Differentiate into CD4+CD8+ Cells

    Institute of Scientific and Technical Information of China (English)

    Aidong Li; Xueli Liu; Baochun Duan; Jie Ma

    2005-01-01

    Thymic nurse cells (TNCs) represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4-CD8+ cells differentiated into CD4+CD8+ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4-CD8+ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4+CD8+ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4-CD8+ thymocytes to differentiate into CD4+CD8+ T cells by direct interaction.

  16. Spontaneous Differentiation of Dental Pulp stem cells on Dental polymers

    Science.gov (United States)

    Bherwani, Aneel; Suarato, Giulia; Qin, Sisi; Chang, Chung-Cheh; Akhavan, Aaron; Spiegel, Joseph; Jurukovski, Vladimir; Rafailovich, Miriam; Simon, Marcia

    2012-02-01

    Dental pulp stem cells were plated on two dentally relevant materials i.e. PMMA commonly used for denture and Titanium used for implants. In both cases, we probed for the role of surface interaction and substrate morphology. Different films of PMMA were spun cast directly onto Si wafers; PMMA fibers of different diameters were electro spun onto some of these substrates. Titanium metal was evaporated onto Si surfaces using an electron beam evaporator. In addition, on some surfaces, P4VP nanofibers were spun cast. DPSC were grown in alpha-MEM supplemented with 10% fetal bovine serum, 0.2mM L-ascorbic acid 2-phosphate, 2mm glutamine and 10mM beta-glycerol phosphate either with or without 10nM dexamethasone. After 21 days samples were examined using confocal microscopy of cells and by scanning electron microscopy (SEM) and Energy dispersive X-ray Analysis (EDAX). In the case of Titanium biomineralization was observed independent of dexamethasone, where the deposits were templated along the fibers. Minimal biomineralization was observed on flat Titanium and PMMA samples. Markers of osteogenesis and specific signaling pathways are being evaluated by RT-PCR, which are up regulated on each surface, to understand the fundamental manner in which surfaces interact with cell differentiation.

  17. Chondrogenic differentiation of mesenchymal stem cells: challenges and unfulfilled expectations.

    Science.gov (United States)

    Somoza, Rodrigo A; Welter, Jean F; Correa, Diego; Caplan, Arnold I

    2014-12-01

    Articular cartilage repair and regeneration provides a substantial challenge in Regenerative Medicine because of the high degree of morphological and mechanical complexity intrinsic to hyaline cartilage due, in part, to its extracellular matrix. Cartilage remains one of the most difficult tissues to heal; even state-of-the-art regenerative medicine technology cannot yet provide authentic cartilage resurfacing. Mesenchymal stem cells (MSCs) were once believed to be the panacea for cartilage repair and regeneration, but despite years of research, they have not fulfilled these expectations. It has been observed that MSCs have an intrinsic differentiation program reminiscent of endochondral bone formation, which they follow after exposure to specific reagents as a part of current differentiation protocols. Efforts have been made to avoid the resulting hypertrophic fate of MSCs; however, so far, none of these has recreated a fully functional articular hyaline cartilage without chondrocytes exhibiting a hypertrophic phenotype. We reviewed the current literature in an attempt to understand why MSCs have failed to regenerate articular cartilage. The challenges that must be overcome before MSC-based tissue engineering can become a front-line technology for successful articular cartilage regeneration are highlighted.

  18. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells

    Directory of Open Access Journals (Sweden)

    Gersende Caron

    2015-11-01

    Full Text Available Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxymethylation, and cell fate determination.

  19. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

    Science.gov (United States)

    Caron, Gersende; Hussein, Mourad; Kulis, Marta; Delaloy, Céline; Chatonnet, Fabrice; Pignarre, Amandine; Avner, Stéphane; Lemarié, Maud; Mahé, Elise A; Verdaguer-Dot, Núria; Queirós, Ana C; Tarte, Karin; Martín-Subero, José I; Salbert, Gilles; Fest, Thierry

    2015-11-03

    Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

  20. Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Sui Jing; Jiang Fangxu; Shi Bingyin

    2011-01-01

    Regenerative medicine,including cell-replacement strategies,may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date,significant progress has been made in generating insulin-secreting β cells from pluripotent mouse embryonic stem cells (ESCs).The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions.Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of β cells such as insulinl,insulin2,Islet1,MafA,insulinoma-associated antigen 1 (IA1) and so on,indicating a similar gene expression pattern to adult islet β cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion,this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro,but also leaded to the development of new strategies to derive transplantable islet-replacement β cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

  1. Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ionta, M. [Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas MG (Brazil); Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil); Rosa, M.C.; Almeida, R.B.; Freitas, V.M.; Rezende-Teixeira, P.; Machado-Santelli, G.M. [Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil)

    2012-05-25

    Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation [E-cadherin, connexin 26 (Cx26), and connexin 32 (Cx32)]. RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

  2. FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

    Science.gov (United States)

    Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

    2013-12-01

    The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

  3. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik;

    2013-01-01

    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize......Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...

  4. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    Science.gov (United States)

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  5. Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells.

    Science.gov (United States)

    Petrakova, O S; Ashapkin, V V; Shtratnikova, V Y; Kutueva, L I; Vorotelyak, E A; Borisov, M A; Terskikh, V V; Gvazava, I G; Vasiliev, A V

    2015-01-01

    The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

  6. Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation.

    Science.gov (United States)

    Park, Sung-Won; Do, Hyun-Jin; Ha, Woo Tae; Han, Mi-Hee; Song, Hyuk; Uhm, Sang-Jun; Chung, Hak-Jae; Kim, Jae-Hwan

    2014-01-01

    E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

  7. Characterization of protocadherin-1 expression in primary bronchial epithelial cells : association with epithelial cell differentiation

    NARCIS (Netherlands)

    Koning, Henk; Sayers, Ian; Stewart, Ceri E.; de Jong, Debora; ten Hacken, Nick H. T.; Postma, Dirkje S.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2012-01-01

    Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA an

  8. DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation.

    Science.gov (United States)

    Fernández-Nestosa, María José; Monturus, Estefanía; Sánchez, Zunilda; Torres, Francisco S; Fernández, Agustín F; Fraga, Mario F; Hernández, Pablo; Schvartzman, Jorge B; Krimer, Dora B

    2013-01-01

    In mice, the proviral integration of the Friend Spleen Focus Forming Virus (SFFV) within the PU.1 locus of erythroid precursors results in the development of erythroleukemia. SFFV integrates several kilobases upstream of the PU.1 transcription initiation start site leading to the constitutive activation of the gene which in turn results in a block of erythroid differentiation. In this study we have mapped and sequenced the exact location of the retroviral integration site. We have shown that SFFV integrates downstream of a previously described upstream regulatory element (URE), precisely 2,976 bp downstream of the URE-distal element. We have also found that SFFV persists integrated within the same location in resistant cell lines that have lost their differentiation capacity and in which case PU.1 remains silent. We have examined the methylation status of PU.1 and found that in resistant cells the nearby CpG islands remained methylated in contrast to a non-methylated status of the parental cell lines. Treatment with 5-aza-2'-deoxycytidine caused resistant cells to differentiate yet only when combined with HMBA. Altogether these results strongly suggest that methylation plays a crucial role with regard to PU.1 silencing. However, although demethylation is required, it is not sufficient to overcome the differentiation impasse. We have also showed that activation blockage of the Epo/Epo-R pathway remains despite of the absence of PU.1.

  9. Stochastic differential equation model for cerebellar granule cell excitability.

    Science.gov (United States)

    Saarinen, Antti; Linne, Marja-Leena; Yli-Harja, Olli

    2008-02-29

    Neurons in the brain express intrinsic dynamic behavior which is known to be stochastic in nature. A crucial question in building models of neuronal excitability is how to be able to mimic the dynamic behavior of the biological counterpart accurately and how to perform simulations in the fastest possible way. The well-established Hodgkin-Huxley formalism has formed to a large extent the basis for building biophysically and anatomically detailed models of neurons. However, the deterministic Hodgkin-Huxley formalism does not take into account the stochastic behavior of voltage-dependent ion channels. Ion channel stochasticity is shown to be important in adjusting the transmembrane voltage dynamics at or close to the threshold of action potential firing, at the very least in small neurons. In order to achieve a better understanding of the dynamic behavior of a neuron, a new modeling and simulation approach based on stochastic differential equations and Brownian motion is developed. The basis of the work is a deterministic one-compartmental multi-conductance model of the cerebellar granule cell. This model includes six different types of voltage-dependent conductances described by Hodgkin-Huxley formalism and simple calcium dynamics. A new model for the granule cell is developed by incorporating stochasticity inherently present in the ion channel function into the gating variables of conductances. With the new stochastic model, the irregular electrophysiological activity of an in vitro granule cell is reproduced accurately, with the same parameter values for which the membrane potential of the original deterministic model exhibits regular behavior. The irregular electrophysiological activity includes experimentally observed random subthreshold oscillations, occasional spontaneous spikes, and clusters of action potentials. As a conclusion, the new stochastic differential equation model of the cerebellar granule cell excitability is found to expand the range of dynamics

  10. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

    Directory of Open Access Journals (Sweden)

    Kanako Miyabayashi

    Full Text Available Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

  11. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid...

  12. Cdk4 functions in multiple cell types to control Drosophila intestinal stem cell proliferation and differentiation

    Directory of Open Access Journals (Sweden)

    Mojca Adlesic

    2016-03-01

    Full Text Available The proliferation of intestinal stem cells (ISCs and differentiation of enteroblasts to form mature enteroendocrine cells and enterocytes in the Drosophila intestinal epithelium must be tightly regulated to maintain homeostasis. We show that genetic modulation of CyclinD/Cdk4 activity or mTOR-dependent signalling cell-autonomously regulates enterocyte growth, which influences ISC proliferation and enteroblast differentiation. Increased enterocyte growth results in higher numbers of ISCs and defective enterocyte growth reduces ISC abundance and proliferation in the midgut. Adult midguts deficient for Cdk4 show severe disruption of intestinal homeostasis characterised by decreased ISC self-renewal, enteroblast differentiation defects and low enteroendocrine cell and enterocyte numbers. The ISC/enteroblast phenotypes result from a combination of cell autonomous and non-autonomous requirements for Cdk4 function. One non-autonomous consequence of Cdk4-dependent deficient enterocyte growth is high expression of Delta in ISCs and Delta retention in enteroblasts. We postulate that aberrant activation of the Delta–Notch pathway is a possible partial cause of lost ISC stemness. These results support the idea that enterocytes contribute to a putative stem cell niche that maintains intestinal homeostasis in the Drosophila anterior midgut.

  13. Diverse effects of cyclic AMP variants on osteogenic and adipogenic differentiation of human mesenchymal stromal cells

    NARCIS (Netherlands)

    Doorn, J.; Leusink, Maarten; Groen, N.; Peppel, van de J.; Leeuwen, van J.P.T.M.; Blitterswijk, van C.A.; Boer, de J.

    2012-01-01

    Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) may potentially be used in cell based bone tissue engineering applications to enhance the bone forming potential of these cells. Osteogenic and adipogenic differentiation are thought to be mutually exclusive and, although several

  14. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  15. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    Institute of Scientific and Technical Information of China (English)

    Xue-Jun Dong; Guo-Rong Zhang; Qing-Jun Zhou; Ruo-Lang Pan; Ye Chen; Li-Xin Xiang; Jian-Zhong Shao

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  16. Distribution and differentiation of mesenchymal stem cells in tumor tissue

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hai-feng; CHEN Jun; XU Zhi-shun; ZHANG Ke-qin

    2009-01-01

    Background Tumor has an ability to become enriched in mesenchymal stem cells (MSCs) and of guiding MSCs to migrate to tumor tissue. But there are lack of relevant reports on the distribution and differentiation of MSCs in tumor tissue and the effect on tumor growth after MSCs engrafted in tumor tissue. In this study, we observed the distribution of bone marrow MSCs in tumor tissue and the possibility of MSCs differentiating into myofibroblast under the induction of local tumor microenvironment.Methods Twenty-four New Zealand rabbits were randomly classified into the control group and the test group. MSCs were isolated and cultured for each animal, vx-2 tumor tissue was transplanted under the bladder mucosa of each animal. One week after the transplantation, the self F2 passage MSCs marked by 4',6-diamidino-2-phenylindole were transplanted into tumor tissue in the test group while only Dulbecco's modified Eagle's medium-low glucose was infused into the control group. Ultrasonography was performed for each animal 1,2, 3 and 4 week(s) after the vx-2 tumor mass was transplanted. The maximum bladder tumor diameter of each animal was recorded and the mean value of each group was calculated. One animal from each group was sacrificed in the third week and the remaining animals in the fourth week to observe the tumor development. Another animal treated the same as the test group was sacrificed to observe the distribution of MSCs in tumor tissue one week after self MSCs transplantation. Immunofluorescence was used to trace MSCs in tumor tissue. The double labeling immunofluorescence for α-smooth muscle actin (α-SMA) and vimentin was performed to identify whether the MSCs can differentiate into myofibroblast.Results The ultrasonography showed no tumor mass one week after the vx-2 tumor mass transplantation. The mean maximum tumor diameter of the control group and test group was (0.70±0.14) cm and (0.78±0.14) cm, respectively, and there was no significant difference (t=1

  17. Statins induce differentiation and cell death in neurons and astroglia.

    Science.gov (United States)

    März, Pia; Otten, Uwe; Miserez, André R

    2007-01-01

    Statins are potent inhibitors of the hydroxy-methyl-glutaryl-coenzyme A reductase, the rate limiting enzyme for cholesterol biosynthesis. Experimental and clinical studies with statins suggest that they have beneficial effects on neurodegenerative disorders. Thus, it was of interest to characterize the direct effects of statins on CNS neurons and glial cells. We have treated defined cultures of neurons and astrocytes of newborn rats with two lipophilic statins, atorvastatin and simvastatin, and analyzed their effects on morphology and survival. Treatment of astrocytes with statins induced a time- and dose-dependent stellation, followed by apoptosis. Similarly, statins elicited programmed cell death of cerebellar granule neurons but with a higher sensitivity. Analysis of different signaling cascades revealed that statins fail to influence classical pathways such as Akt or MAP kinases, known to be activated in CNS cells. In addition, astrocyte stellation triggered by statins resembled dibutryl-cyclic AMP (db-cAMP) induced morphological differentiation. However, in contrast to db-cAMP, statins induced upregulation of low-density lipoprotein receptors, without affecting GFAP expression, indicating separate underlying mechanisms. Analysis of the cholesterol biosynthetic pathway revealed that lack of mevalonate and of its downstream metabolites, mainly geranylgeranyl-pyrophosphate (GGPP), is responsible for the statin-induced apoptosis of neurons and astrocytes. Moreover, astrocytic stellation triggered by statins was inhibited by mevalonate and GGPP. Interestingly, neuronal cell death was significantly reduced in astrocyte/neuron co-cultures treated with statins. We postulate that under these conditions signals provided by astrocytes, e.g., isoprenoids play a key role in neuronal survival.

  18. Natural Killer Cell Differentiation From Hematopoietic Stem Cells: A Comparative Analysis of Heparin and Stromal Cell Supported Methods

    Science.gov (United States)

    Dezell, Steven A.; Ahn, Yong-Oon; Spanholtz, Jan; Wang, Hongbo; Weeres, Matthew; Jackson, Scott; Cooley, Sarah; Dolstra, Harry; Miller, Jeffrey S.; Verneris, Michael R.

    2011-01-01

    Natural killer (NK) cells differentiated from hematopietic stem cells (HSCs)may have significant clinical benefits over those from adult donors, including the ability to choose allo-reactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study NK differentiation from HSCs. Stroma and cytokines support NK differentiation, but may have considerable regulatory hurdles. Recently, a clinical grade heparin-based method has been reported and could serve as an alternative. How these two compare in NK generating efficiency or functionis unknown. We show that compared to heparin-based cultures, stromasignificantly increases the yield of HSC-derived NK cells by differentiating less committed progenitors into the NK lineage. NK cells generated by both approaches were similar for most NK activating or inhibitor receptors. While both approaches resulted in a phenotype consistent with CD56brightstage IV NK cells, heparin-based cultures favored the development of CD56+CD16+ cells, while stromaproducedmore KIR-expressing NK cells, both markers of terminal maturation. At day 21, stromal-based cultures showed significantly more IL-22 production and both methods yielded similar amounts of IFN-γ production and cytotoxicity (day 35). Thus, heparin-based cultures are an effective replacement for stroma and may facilitate clinical trials testing HSC-derived NK cells. PMID:22155502

  19. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

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    Fanny Ehret

    2015-11-01

    Full Text Available The niche concept of stem cell biology proposes a functional unit between the precursor cells and their local microenvironment, to which several cell types might contribute by cell–cell contacts, extracellular matrix, and humoral factors. We here established three co-culture models (with cell types separated by membrane for both adherent monolayers and neurospheres to address the potential influence of different niche cell types in the neurogenic zone of the adult hippocampus of mice. Astrocytes and endothelial cells enhanced precursor cell proliferation and neurosphere formation. Endothelial factors also led to a prolonged increase in proliferation after growth factor withdrawal, which otherwise induces differentiation. All niche cell types enhanced cell survival in monolayer cultures, endothelial cells also stimulated neuronal differentiation. A parallel trend elicited by astrocytes did not reach conventional statistical significance. Pericytes had variable effects here. We did not observe changes in differentiation in neurosphere co-cultures. In summary, our data indicate that in precursor cell culture protocols survival could be improved by adding as yet unknown factors physiologically contributed by astrocytes and endothelial cells. Our findings also underscore the complexity of the niche and the differential impact of factors from the different sources on distinct aspects of neuronal development. With the help of the models presented here, identification of these factors and their specific biological activity can now be initiated.

  20. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging

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    Wang Ruoning

    2010-05-01

    Full Text Available Abstract Background Dental pulp stem cells (DPSCs can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. However, the differentiation potential of naive DPSCs after serial passaging in the routine culture system has not been fully elucidated. Results DPSCs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on STRO-1 expression, cultured and passaged in the conventional culture media. The biological features of STRO-1+ DPSCs at the 1st and 9th passages were investigated. During the long-term passage, the proliferation ability of human STRO-1+ DPSCs was downregulated as indicated by the growth kinetics. When compared with STRO-1+ DPSCs at the 1st passage (DPSC-P1, the expression of mature osteoblast-specific genes/proteins (alkaline phosphatase, bone sialoprotein, osterix, and osteopontin, odontoblast-specific gene/protein (dentin sialophosphoprotein and dentin sialoprotein, and chondrocyte-specific gene/protein (type II collagen was significantly upregulated in human STRO-1+ DPSCs at the 9th passage (DPSC-P9. Furthermore, human DPSC-P9 cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day 3 and day 7 respectively, and produced more mineralized matrix than DPSC-P9 cells at day 14. In vivo transplantation results showed that rat DPSC-P1 cell pellets developed into dentin, bone and cartilage structures respectively, while DPSC-P9 cells can only generate bone tissues. Conclusions These findings suggest that STRO-1+ DPSCs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. The differentiation capacity of these DPSCs changes during cell passaging, and DPSCs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.

  1. Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting Cell Cycling and Differentiation.

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    Ichihara, Yoshinori; Doi, Toru; Ryu, Youngjae; Nagao, Motoshi; Sawada, Yasuhiro; Ogata, Toru

    2017-05-01

    Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC-rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU-positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate-mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α-cyano-4-hydroxy-cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986-995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  2. Differentiated NSC-34 cells as an in vitro cell model for VX.

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    Kanjilal, Baishali; Keyser, Brian M; Andres, Devon K; Nealley, Eric; Benton, Betty; Melber, Ashley A; Andres, Jaclynn F; Letukas, Valerie A; Clark, Offie; Ray, Radharaman

    2014-10-01

    The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17-23 h post-exposure) and dose (10-100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10-100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics.

  3. Generation of priming mesenchymal stem cells with enhanced potential to differentiate into specific cell lineages using extracellular matrix proteins.

    Science.gov (United States)

    Han, Na Rae; Yun, Jung Im; Park, Young Hyun; Ahn, Ji Yeon; Kim, Choonghyo; Choi, Jung Hoon; Lee, Eunsong; Lim, Jeong Mook; Lee, Seung Tae

    2013-07-01

    Poor understanding of the differentiation of mesenchymal stem cells (MSCs) has resulted in a low differentiation yield, and has hindered their application in medicine. As a solution, priming MSCs sensitive to signaling, thus stimulating differentiation into a specific cell lineage, may improve the differentiation yield. To demonstrate this, priming MSCs were produced by using a gelatin matrix for the isolation of primary MSCs from bone-marrow-derived primary cells. Subsequently, cellular characteristics and sensitivity to specific differentiation signals were analyzed at passage five. Compared to non-priming MSCs, priming MSCs showed no significant differences in cellular characteristics, but demonstrated a significant increase in sensitivity to neurogenic differentiation signals. These results demonstrate that generation of priming MSCs by specific extracellular signaling increases the rate of differentiation into a cell-specific lineage.

  4. Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells

    Institute of Scientific and Technical Information of China (English)

    MA Lian; FENG Xue-yong; CUI Bing-lin; Frieda Law; JIANG Xue-wu; YANG Li-ye; XIE Qing-dong; HUANG Tian-hua

    2005-01-01

    Background The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells.Methods MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and β-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM) .The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR). Results MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and β-mercaptoethanol also induced MSCs to express nestin, β-tubulinⅢ, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.Conclusions MSCs could be isolated from human umbilical

  5. Atmospheric-pressure plasma-irradiation inhibits mouse embryonic stem cell differentiation to mesoderm and endoderm but promotes ectoderm differentiation

    Science.gov (United States)

    Miura, Taichi; Hamaguchi, Satoshi; Nishihara, Shoko

    2016-04-01

    Recently, various effects of low-temperature atmospheric-pressure plasma irradiation on living cells have been demonstrated, such as tissue sterilization, blood coagulation, angiogenesis, wound healing, and tumor elimination. However, the effect of plasma-irradiation on the differentiation of mouse embryonic stem cells (mESCs) has not yet been clarified. A large number of reactive species are generated by plasma-irradiation in medium, of which hydrogen peroxide (H2O2) is one of the main species generated. Here, we investigated the effect of plasma-irradiation on the differentiation of mESCs using an embryoid body (EB) formation assay with plasma-irradiated medium or H2O2-supplemented non-irradiated medium. Our findings demonstrated that plasma-irradiated medium potently inhibits the differentiation from mESCs to mesoderm and endoderm by inhibiting Wnt signaling as determined by quantitative polymerase chain reaction and immunoblotting analyses. In contrast, both the plasma-irradiated medium and H2O2-supplemented non-irradiated medium enhanced the differentiation to epiblastoid, ectodermal, and neuronal lineages by activation of fibroblast growth factor 4 (FGF4) signaling, suggesting that these effects are caused by the H2O2 generated by plasma-irradiation in medium. However, in each case, the differentiation to glial cells remained unaffected. This study is the first demonstration that plasma-irradiation affects the differentiation of mESCs by the regulation of Wnt and FGF4 signaling pathways.

  6. Isolation and Multiple Differentiation Potential Assessment of Human Gingival Mesenchymal Stem Cells

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    Yuan Gao

    2014-11-01

    Full Text Available The aim of this study was to isolate human mesenchymal stem cells (MSCs from the gingiva (GMSCs and confirm their multiple differentiation potentials, including the odontogenic lineage. GMSCs, periodontal ligament stem cells (PDLSCs and dermal stem cells (DSCs cultures were analyzed for cell shape, cell cycle, colony-forming unit-fibroblast (CFU-F and stem cell markers. Cells were then induced for osteogenic and adipogenic differentiation and analyzed for differentiation markers (alkaline phosphatase (ALP activity, mineralization nodule formation and Runx2, ALP, osteocalcin (OCN and collagen I expressions for the osteogenic differentiation, and lipid vacuole formation and PPARγ-2 expression for the adipogenic differentiation. Besides, the odontogenic differentiation potential of GMSCs induced with embryonic tooth germ cell-conditioned medium (ETGC-CM was observed. GMSCs, PDLSCs and DSCs were all stromal origin. PDLSCs showed much higher osteogenic differentiation ability but lower adipogenic differentiation potential than DSCs. GMSCs showed the medial osteogenic and adipogenic differentiation potentials between those of PDLSCs and DSCs. GMSCs were capable of expressing the odontogenic genes after ETGC-CM induction. This study provides evidence that GMSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source.

  7. Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

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    Biernat Wojciech

    2009-02-01

    Full Text Available Abstract Background Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. Methods Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA: exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. Results In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP. Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+ and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8, as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. Conclusion Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

  8. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  9. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    XIE Feng; ZHANG WenJie; CHEN FanFan; ZHOU GuangDong; CUI Lei; LIU Wei; CAO YiLin

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. FIk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type Ⅱ collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class Ⅰ molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  10. Notch signalling inhibits CD4 expression during initiation and differentiation of human T cell lineage.

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    Stephen M Carlin

    Full Text Available The Delta/Notch signal transduction pathway is central to T cell differentiation from haemopoietic stem cells (HSCs. Although T cell development is well characterized using expression of cell surface markers, the detailed mechanisms driving differentiation have not been established. This issue becomes central with observations that adult HSCs exhibit poor differentiation towards the T cell lineage relative to neonatal or embryonic precursors. This study investigates the contribution of Notch signalling and stromal support cells to differentiation of adult and Cord Blood (CB human HSCs, using the Notch signalling OP9Delta co-culture system. Co-cultured cells were assayed at weekly intervals during development for phenotype markers using flow cytometry. Cells were also assayed for mRNA expression at critical developmental stages. Expression of the central thymocyte marker CD4 was initiated independently of Notch signalling, while cells grown with Notch signalling had reduced expression of CD4 mRNA and protein. Interruption of Notch signalling in partially differentiated cells increased CD4 mRNA and protein expression, and promoted differentiation to CD4(+ CD8(+ T cells. We identified a set of genes related to T cell development that were initiated by Notch signalling, and also a set of genes subsequently altered by Notch signal interruption. These results demonstrate that while Notch signalling is essential for establishment of the T cell lineage, at later stages of differentiation, its removal late in differentiation promotes more efficient DP cell generation. Notch signalling adds to signals provided by stromal cells to allow HSCs to differentiate to T cells via initiation of transcription factors such as HES1, GATA3 and TCF7. We also identify gene expression profile differences that may account for low generation of T cells from adult HSCs.

  11. Differentiation-inducing and anti-proliferative activities of lupeol on canine melanoma cells.

    Science.gov (United States)

    Ogihara, Kikumi; Naya, Yuko; Okamoto, Yoshiharu; Hata, Keishi

    2014-01-01

    Canine melanoma is the most common oral malignant tumor reported in the field of veterinary medicine. We found that lupeol, a lupine triterpene, inhibited mouse melanoma cell growth in vitro and in vivo by inducing cell differentiation. In the present study, we examined the differentiation-inducing activities of lupeol on 4 canine melanoma cells in vitro and in vivo. The induction of canine melanoma cell differentiation by lupeol was confirmed by evaluating some differentiation markers such as tyrosinase with real-time RT-PCR. Furthermore, we transplanted canine melanoma cells into a severe combined immunodeficiency mouse, and studied the anti-progressive effects of lupeol on tumor tissue. The gene expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein-2, which are markers of pigment cell differentiation, was induced in 4 canine oral malignant melanoma cells by lupeol, and the agent markedly inhibited tumor progression in canine melanoma-bearing mice.

  12. What understanding tendon cell differentiation can teach us about pathological tendon ossification.

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    Magne, D; Bougault, C

    2015-08-01

    Tendons are the structures that attach muscles to bones and transmit mechanical forces. Tendon cells are composed of mature tenocytes and a rare population of tendon stem cells. Both cell types ensure homeostasis and repair of tendon extracellular matrix to guarantee its specific mechanical properties. Moreover, tendon cells seem to present a marked potential for trans-differentiation, predominantly into the chondrocyte and osteoblast lineages. In this review article, we first present chronic tendon pathologies associated with abnormal ossification, such as spondyloarthritis and calcifying tendinopathy, and discuss how tendon cell differentiation and trans-differentiation may participate in these diseases. We moreover present the factors known to influence tendon cell differentiation and trans-differentiation, with a particular emphasis on extracellular environment, mechanical stimulation and several soluble factors that can tip the balance toward one or another lineage. A better understanding of the neglected tendon cell biology may be extremely useful to understand the pathological mechanisms of spondyloarthritis and calcifying tendinopathy.

  13. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

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    Kentaro Kikuchi

    2003-01-01

    Full Text Available Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.

  14. Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

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    Zhang, Kai; Pang, Kunpeng; Wu, Xinyi

    2014-06-15

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

  15. Rigidity of silicone substrates controls cell spreading and stem cell differentiation

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    Vertelov, Grigory; Gutierrez, Edgar; Lee, Sin-Ae; Ronan, Edward; Groisman, Alex; Tkachenko, Eugene

    2016-09-01

    The dependences of spreading and differentiation of stem cells plated on hydrogel and silicone gel substrates on the rigidity and porosity of the substrates have recently been a subject of some controversy. In experiments on human mesenchymal stem cells plated on soft, medium rigidity, and hard silicone gels we show that harder gels are more osteogenic, softer gels are more adipogenic, and cell spreading areas increase with the silicone gel substrate rigidity. The results of our study indicate that substrate rigidity induces some universal cellular responses independently of the porosity or topography of the substrate.

  16. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lei [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Wang, Huaxi [Southern Medical University, 510515 Guangzhou (China); Yang, Yan [College of Pharmacy, Jinan University, 510632 Guangzhou (China); Liu, Hui [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Zhang, Qihao; Xiang, Qi [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China); Ge, Renshan [Population Council, Rockefeller University, 10065 New York (United States); Su, Zhijian, E-mail: tjnuszj@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Huang, Yadong, E-mail: tydhuang@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China)

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  17. Effector T cell differentiation: are master regulators of effector T cells still the masters?

    Science.gov (United States)

    Wang, Chao; Collins, Mary; Kuchroo, Vijay K

    2015-12-01

    Effector CD4 T cell lineages have been implicated as potent inducers of autoimmune diseases. Tbet, Gata3 and Rorgt are master transcriptional regulators of Th1, Th2 and Th17 lineages respectively and promote the distinct expression of signature cytokines. Significant progress has been made in understanding the transcriptional network that drives CD4 T cell differentiation, revealing novel points of regulation mediated by transcription factors, cell surface receptors, cytokines and chemokines. Epigenetic modifications and metabolic mediators define the transcriptional landscape in which master transcription factors operate and collaborate with a network of transcriptional modifiers to guide lineage specification, plasticity and function.

  18. Mesenchymal Stem Cells for Cardiac Regenerative Therapy: Optimization of Cell Differentiation Strategy.

    Science.gov (United States)

    Shen, Han; Wang, Ying; Zhang, Zhiwei; Yang, Junjie; Hu, Shijun; Shen, Zhenya

    2015-01-01

    With the high mortality rate, coronary heart disease (CHD) has currently become a major life-threatening disease. The main pathological change of myocardial infarction (MI) is the induction of myocardial necrosis in infarction area which finally causes heart failure. Conventional treatments cannot regenerate the functional cell efficiently. Recent researches suggest that mesenchymal stem cells (MSCs) are able to differentiate into multiple lineages, including cardiomyocyte-like cells in vitro and in vivo, and they have been used for the treatment of MI to repair the injured myocardium and improve cardiac function. In this review, we will focus on the recent progress on MSCs derived cardiomyocytes for cardiac regeneration after MI.

  19. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    Science.gov (United States)

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  20. In vitro study of cell differentiation by two type mouse embryo stem cells on mono- and multilayer nanocarbon tubes

    Science.gov (United States)

    Imai, Koichi; Akasaka, Tsukasa; Watari, Fumio; Tanoue, Akito; Nakamura, Kazuaki; Suese, Kazuhiko; Takashima, Hiromasa; Nishikawa, Tetsunari; Tanaka, Akio; Takeda, Shoji

    2012-09-01

    The effects of nanomaterials on human reproduction and development remain unknown. The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We previously investigated the myocardial cell differentiation of ES-D3 cells using monolayer (SWCNTs) and multilayer (MWCNTs) nanocarbon tubes. As a result, in spite of having the same carbon composition, the effects on the cell differentiation levels differed between the tubes. We investigated their cell differentiation and cytotoxic effects on EL M3 and ES-R1-EGFP B2/EGFP cells, which require feeder cells. As a result, myocardial pulse rates differed between the presence of SWCNTs and MWCNTs even when feeder cells existed between the samples and cells. The different surface structures of SWCNTs and MWCNTs may have influenced ES cell differentiation.

  1. Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells.

    Science.gov (United States)

    Shi, Wei; Liao, Yang; Willis, Simon N; Taubenheim, Nadine; Inouye, Michael; Tarlinton, David M; Smyth, Gordon K; Hodgkin, Philip D; Nutt, Stephen L; Corcoran, Lynn M

    2015-06-01

    When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.

  2. Epigenetic remodeling of chromatin architecture: exploring tumor differentiation therapies in mesenchymal stem cells and sarcomas.

    Science.gov (United States)

    Siddiqi, Sara; Mills, Joslyn; Matushansky, Igor

    2010-03-01

    Sarcomas are the mesenchymal-derived malignant tumors of connective tissues (e.g., fat, bone, and cartilage) presumed to arise from aberrant development or differentiation of mesenchymal stem cells (MSCs). Appropriate control of stem cell maintenance versus differentiation allows for normal connective tissue development. Current theories suggest that loss of this control--through accumulation of genetic lesions in MSCs at various points in the differentiation process--leads to development of sarcomas, including undifferentiated, high grade sarcoma tumors. The initiation of stem cell differentiation is highly associated with alteration of gene expression, which depends on chromatin remodeling. Epigenetic chromatin modifying agents have been shown to induce cancer cell differentiation and are currently being used clinically to treat cancer. This review will focus on the importance of epigenetic chromatin remodeling in the context of mesenchymal stem cells, sarcoma tumorigenesis and differentiation therapy.

  3. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    Science.gov (United States)

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  4. Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

    Science.gov (United States)

    Barwick, Benjamin G; Scharer, Christopher D; Bally, Alexander P R; Boss, Jeremy M

    2016-10-01

    The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

  5. Differential proteome analysis of chikungunya virus infection on host cells.

    Directory of Open Access Journals (Sweden)

    Christina Li-Ping Thio

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

  6. Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Gandhimathi, Chinnasamy [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Venugopal, Jayarama Reddy [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Tham, Allister Yingwei [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Ramakrishna, Seeram [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Kumar, Srinivasan Dinesh, E-mail: dineshkumar@ntu.edu.sg [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore)

    2015-04-01

    Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(L-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228 ± 62–320 ± 22 nm and 1.5–6.9 μm respectively. Resulting nanofibrous scaffolds are highly porous (87–94%) with ultimate tensile strength observed in the range of 1.51–4.86 MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering. - Highlights: • We fabricated and characterized hybrid porous nanofibrous scaffolds. • PLACL/SF/AA/TC/n-HA scaffolds promote cell differentiation and mineralization. • Porous nanofibrous scaffolds initiate MSC differentiation into osteogenic cells. • Biomimetic nanofibrous scaffolds have good potential for bone tissue engineering.

  7. Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

    Science.gov (United States)

    Li, Chunhe; Wang, Jin

    2013-01-01

    Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

  8. Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation.

    Science.gov (United States)

    Azedi, Fereshteh; Kazemnejad, Somaieh; Zarnani, Amir Hassan; Soleimani, Masoud; Shojaei, Amir; Arasteh, Shaghayegh

    2017-02-01

    In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.

  9. Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells.

    Science.gov (United States)

    Kourouklis, Andreas P; Kaylan, Kerim B; Underhill, Gregory H

    2016-08-01

    Recent approaches have utilized microfabricated platforms to examine combinations of microenvironmental signals that regulate stem and progenitor cell differentiation. However, the majority of these efforts have focused on the biochemical properties of extracellular matrix (ECM) or soluble factors without simultaneously exploring the biomechanical effects of cell-substrate interactions. To address this need, we combined a high-throughput approach for the analysis of combinatorial ECM cues with substrates of modular stiffness and traction force microscopy. This integrated approach enabled the characterization of cell-generated traction stress and phenotypic expression in response to ECM cues. We investigated the impact of substrate stiffness and ECM composition on the differentiation of bipotential mouse embryonic liver (BMEL) progenitor cells. We observed that hepatocyte differentiation was primarily regulated by ECM composition, and cholangiocyte differentiation was cooperatively influenced by ECM proteins and stiffness properties. In particular, stiffness-mediated cholangiocyte differentiation was observed for cells cultured on fibronectin, while collagen IV promoted differentiation independent of substrate stiffness. We demonstrated the influence of cell contractility and traction stress in early cholangiocyte specification and further uncovered the roles of ERK and ROCK in this differentiation process. Overall, these findings illustrate the involvement of biomechanical signals in liver progenitor differentiation. Further, this approach could enable investigations for a broad range of cell types and ECM proteins, providing an integrated platform for evaluating the combinatorial effects of biochemical and biophysical signals in cell differentiation.

  10. A Lin28 homologue reprograms differentiated cells to stem cells in the moss Physcomitrella patens

    Science.gov (United States)

    Li, Chen; Sako, Yusuke; Imai, Akihiro; Nishiyama, Tomoaki; Thompson, Kari; Kubo, Minoru; Hiwatashi, Yuji; Kabeya, Yukiko; Karlson, Dale; Wu, Shu-Hsing; Ishikawa, Masaki; Murata, Takashi; Benfey, Philip N.; Sato, Yoshikatsu; Tamada, Yosuke; Hasebe, Mitsuyasu

    2017-01-01

    Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3′-untranslated region (3′-UTR). Removal of the 3′-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28. PMID:28128346

  11. Differential gene expression in stromal cells of human giant cell tumor of bone.

    Science.gov (United States)

    Wuelling, M; Delling, G; Kaiser, E

    2004-12-01

    Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.

  12. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  13. Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Yanhui Lu; Xiaodong Yuan; Qiaoyu Sun; Ya Ou

    2013-01-01

    Preliminary research from our group found altered autophagy intensity during adipose-derived stromal cell differentiation into neuronal-like cells, and that this change was associated with morphological changes in differentiated cells. This study aimed to verify the role of rapamycin, an autophagy activator, in the process of adipose-derived stromal cell differentiation into neuronal-like cells. Immunohistochemical staining showed that expression of neuron-specific enolase and neurofilament-200 were gradually upregulated in adipose-derived stromal cells after 5 mM β-mercaptoethanol induction, and the differentiation rate gradually increased with induction time. Using transmission electron microscopy, induced cells were shown to exhibit cytoplasmic autophagosomes, with bilayer membranes, and autolysosomes. After rapamycin (200μg/L) induction for 1 hour, adipose-derived stromal cells began to extend long processes, similar to the morphology of neuronal-like cells, while untreated cells did not exhibit similar morphologies until 3 hours after induction. Moreover, the differentiation rate was significantly increased after rapamycin treatment. Compared with untreated cells, expression of LC3, an autophagy protein, was also significantly upregulated. Positive LC3 expression tended to concentrate at cell nuclei with increasing induction times. Our experimental findings indicate that autophagy can significantly increase the speed of adipose-derived stromal cell differentiation into neuronal-like cells.

  14. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells.

    Science.gov (United States)

    Kuroda, Yasumasa; Wakao, Shohei; Kitada, Masaaki; Murakami, Toru; Nojima, Makoto; Dezawa, Mari

    2013-01-01

    Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

  15. The Effect of Bone Marrow Mesenchymal Stem Cells on Vitamin D3 Induced Monocytic Differentiation of U937 Cells

    OpenAIRE

    Molaeipour, Zahra; Shamsasanjan, karim; Movassaghpour, Ali Akbari; Akbarzadehlaleh, Parvin; Sabaghi, Fatemeh; Saleh, Mahshid

    2016-01-01

    Purpose: Mesenchymal stem cells (MSCs) are key components of the hematopoietic stem cells (HSCs) niche. They control the process of hematopoiesis by secreting regulatory cytokines, growth factors and expression of important cell adhesion molecules for cell-tocell interactions. In this research, we have investigated the effect of bone marrow derived MSCs on monocytic differentiation of U937 cells line.

  16. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism

    Directory of Open Access Journals (Sweden)

    J Kang

    2014-11-01

    Full Text Available Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP-linked basic fibroblast growth factor (FGF2-immobilised polystyrene surface (PS-MBP-FGF2 was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC behaviour in response to two different mechanisms of cell adhesion; (i FGF2-heparan sulphate proteoglycan (HSPG-mediated adhesion vs. (ii fibronectin (FN-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  17. Control of mesenchymal stem cell phenotype and differentiation depending on cell adhesion mechanism.

    Science.gov (United States)

    Kang, J; Park, H M; Kim, Y W; Kim, Y H; Varghese, S; Seok, H K; Kim, Y G; Kim, S H

    2014-11-25

    Control of cell-matrix adhesion has become an important issue in the regulation of stem cell function. In this study, a maltose-binding protein (MBP)-linked basic fibroblast growth factor (FGF2)-immobilised polystyrene surface (PS-MBP-FGF2) was applied as an artificial matrix to regulate integrin-mediated signalling. We sought to characterise human mesenchymal-stem cell (hMSC) behaviour in response to two different mechanisms of cell adhesion; (i) FGF2-heparan sulphate proteoglycan (HSPG)-mediated adhesion vs. (ii) fibronectin (FN)-integrin-mediated adhesion. Heparin inhibited hMSC adhesion to PS-MBP-FGF2 but not to FN-coated surface. The phosphorylation of focal adhesion kinase, cytoskeletal re-organisation, and cell proliferation were restricted in hMSCs adhering to PS-MBP-FGF2 compared to FN-coated surface. Expression of MSC markers, such as CD105, CD90 and CD166, decreased in hMSCs expanded on PS-MBP-FGF2 compared to expression in cells expanded on FN-coated surface. hMSCs that were expanded on FN-coated surface differentiated into osteogenic and adipogenic cells more readily than those that were expanded on PS-MBP-FGF2. Furthermore, we characterised the N-linked glycan structures of hMSCs depending on the cell adhesion mechanism using mass spectrometry (MS)-based quantitative techniques. MS analysis revealed that 2,3-sialylated glycans, a potential marker of stem cell function, were more abundant on hMSCs expanded on FN-coated surface than on those expanded on PS-MBP-FGF2. Thus, the differentiation potential of hMSCs is controlled by the type of adhesion substrate that might provide an idea for the design of biomaterials to control stem cell fate. Elucidation of the glycan structure on the cell membrane may help characterise hMSC function.

  18. Platelet-rich fibrin-induced bone marrow mesenchymal stem cell differentiation into osteoblast-like cells and neural cells

    Institute of Scientific and Technical Information of China (English)

    Qi Li; Yajun Geng; Lei Lu; Tingting Yang; Mingrui Zhang; Yanmin Zhou

    2011-01-01

    Bone marrow mesenchymal stem cells were allowed to develop for 14 days in a platelet-rich fibrin environment. Results demonstrated that platelet-rich fibrin significantly promoted bone marrow mesenchymal stem cell proliferation. In addition, there was a dose-dependent increase in Runt-related transcription factor-2 and bone morphogenetic protein-2 mRNA expression, as well as neuron-specific enolase and glial acidic protein. Results showed that platelet-rich fibrin promoted bone marrow mesenchymal stem cell proliferation and differentiation of osteoblastlike cells and neural cells in a dose-dependent manner.

  19. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model.

    Directory of Open Access Journals (Sweden)

    Seyed Jamaleddin Mousavi

    Full Text Available Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa, intermediate (20-25 kPa or hard (30-45 kPa substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis.

  20. Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

    Science.gov (United States)

    Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

    2003-01-01

    In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

  1. Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

    Science.gov (United States)

    Mousavi, Seyed Jamaleddin; Hamdy Doweidar, Mohamed

    2015-01-01

    Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis. PMID:25933372

  2. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals.

    Directory of Open Access Journals (Sweden)

    Hiroko Mochizuki-Kawai

    Full Text Available In the petals of some species of flowers, programmed cell death (PCD begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP and S1/P1 nuclease (LoNUC genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12 drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.

  3. Reverse engineering life: physical and chemical mimetics for controlled stem cell differentiation into cardiomyocytes.

    Science.gov (United States)

    Skuse, Gary R; Lamkin-Kennard, Kathleen A

    2013-01-01

    Our ability to manipulate stem cells in order to induce differentiation along a desired developmental pathway has improved immeasurably in recent years. That is in part because we have a better understanding of the intracellular and extracellular signals that regulate differentiation. However, there has also been a realization that stem cell differentiation is not regulated only by chemical signals but also by the physical milieu in which a particular stem cell exists. In this regard we are challenged to mimic both chemical and physical environments. Herein we describe a method to induce stem cell differentiation into cardiomyocytes using a combination of chemical and physical cues. This method can be applied to produce differentiated cells for research and potentially for cell-based therapy of cardiomyopathies.

  4. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg;

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation......-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which...... despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different...

  5. Neural cell co-culture induced differentiation of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Nailong Yang; Lili Xu; Fen Yang

    2008-01-01

    BACKGROUND: It has been previously demonstrated that the neural cell microenvironment has the ability to induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into the neural cells.OBJECTIVE: To establish a co-culture system of human BMSCs and neural cells, and to observe effects of this co-culture system on differentiation of human BMSCs into neural cells.DESIGN, TIME AND SETTING: A comparative observation experiment, performed at the Center Laboratory of the Affiliated Hospital of Medical College Qingdao University from October 2006 to December 2007.MATERIALS: Neural cells were obtained from human fetal brain tissue. BMSCs were harvested from female patients that underwent autonomous stem cell transplantation.METHODS: BMSCs in the co-culture group consisted of BMSCs and third passage neural cells. BMSCs in the control group were solely cultured in vitro.MAIN OUTCOME MEASURES: Morphological changes of BMSCs were observed, and expression of the neuronal specific marker, neuron-specific enolase (NSE), was analyzed by immunofluorescence staining after4-5-day co-culture.RESULTS: The number of neural cells in the co-culture group increased and the cells spread on the culture bottle surface. Radial dendrite formed and connected with each other. NSE-immunoreactive cells were also detected. The positive ratio of NSE-positive cells reached (32.7±11.5)%, with morphological characteristics similar to neuronal cells. Human BMSCs did not express NSE in the control group.CONCLUSION: The microenvironment provided by neurons induced differentiation of BMSCs into neuronal-like cells.

  6. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa;

    2008-01-01

    Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

  7. Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

    Directory of Open Access Journals (Sweden)

    Rina Wassermann

    2012-01-01

    Full Text Available Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D. However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA and silibinin (SIL synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR and retinoid X receptor alpha (RXRα protein levels and vitamin D response element (VDRE transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

  8. Stochastic differentiation into an osteoclast lineage from cloned macrophage-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Shin-Ichi, E-mail: shayashi@med.tottori-u.ac.jp [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Murata, Akihiko; Okuyama, Kazuki; Shimoda, Yuhki; Hikosaka, Mari [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Yasuda, Hisataka [Planning and Development, Bioindustry Division, Oriental Yeast Co., Ltd, Itabashi-Ku, Tokyo 174-8505 (Japan); Yoshino, Miya [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer The frequency of C7 differentiation into osteoclast was low and constant. Black-Right-Pointing-Pointer Only extended C7 cell cultures exponentially increased osteoclast+ cultures. Black-Right-Pointing-Pointer C7 cell differentiation into committed osteoclast precursors is on 'autopilot'. Black-Right-Pointing-Pointer The system may maintain the stem cell self-renewal and differentiation. -- Abstract: Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor {kappa}B ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

  9. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells.

    Science.gov (United States)

    Farber, D L; Acuto, O; Bottomly, K

    1997-08-01

    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

  10. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    Science.gov (United States)

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  11. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    Science.gov (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  12. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen;

    2005-01-01

    One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...... or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another...

  13. Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

    OpenAIRE

    Bauwens, Celine L.; Toms, Derek; Ungrin, Mark

    2016-01-01

    Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization l...

  14. PROPERTIES OF PROLIFERATION AND DIFFERENTIATION OF NEONATAL RAT RETINAL PROGENITOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    Kang Qianyan; Liu Yong; Zhao Jianjun; Qiu Fen; Chen Xinlin; Tian Yumei; Hu Ming

    2006-01-01

    Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.

  15. Lipopolysaccharide-activated microglial-induced neuroglial cell differentiation in bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoguang Luo; Chunlin Ge; Yan Ren; Hongmei Yu; Zhe Wu; Qiushuang Wang; Chaodong Zhang

    2008-01-01

    BACKGROUND: Microglia are very sensitive to environmental changes, often becoming activated by pathological conditions. Activated microglia can exert a dual role in injury and repair in various diseases of the central nervous system, including cerebral ischemia, Parkinson's disease, and Alzheimer's disease. OBJECTIVE: An immortal microglial cell line, BV2, was treated with varying concentrations of lipopolysaccharide (LPS) to induce a pathological situation. Supernatant was harvested and incubated with bone marrow mesenchymal stem cells and, concomitantly, bone marrow mesenchymal stem cell differentiation was observed. DESIGN: A controlled observation, in vitro experiment. SETTING: Department of Neurology, First Affiliated Hospital of China Medical University. MATERIALS: Five male 2-3-week-old Sprague Dawley rats were purchased from Animal Laboratory Center of China Medical University and included in this study. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The microglial cell line BV2 was produced by Cell Research Institute of Chinese Academy of Sciences. LPS was produced by Sigma Company, USA. METHODS: This study was performed in the Central Laboratory of China Medical University from September 2006 to March 2007. Rat femoral and tibial bone marrow was collected for separation and primary culture of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cell cultures were divided into 5 groups: control group, non-activated group, as well as low-, medium-, and high-dose LPS groups. In the control group, bone marrow mesenchymal stem cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (volume fraction 0.1). In the non-activated group, bone marrow mesenchymal stem cells were incubated with non-activated BV2 supernatant. In the low-, medium-, and high-dose LPS groups, bone marrow mesenchymal stem cells were incubated with LPS (0.01, 0.1 and 1

  16. Molecular and environmental cues in cardiac differentiation of mesenchymal stem cells

    NARCIS (Netherlands)

    Ramkisoensing, Arti Anushka

    2014-01-01

    In this thesis molecular and environmental cues in cardiac differentiation of mesenchymal stem cells were investigated. The main conclusions were that the cardiac differentiation potential of human mesenchymal stem cells negatively correlates with donor age. This in its own shows a negative relation

  17. Argonaute-2-null embryonic stem cells are retarded in self-renewal and differentiation

    Indian Academy of Sciences (India)

    P Chandra Shekar; Adnan Naim; D Partha Sarathi; Satish Kumar

    2011-09-01

    RNA interference (RNAi) pathways regulate self-renewal and differentiation of embryonic stem (ES) cells. Argonaute 2 (Ago2) is a vital component of RNA-induced silencing complex (RISC) and the only Ago protein with slicer activity. We generated Ago2-deficient ES cells by conditional gene targeting. Ago2-deficient ES cells are defective in the small-RNA-mediated gene silencing and are significantly compromised in biogenesis of mature microRNA. The self-renewal rate of Ago2-deficient ES cells is affected due to failure of silencing of Cdkn1a by ES-cell-specific microRNAs (miRNA) in the absence of Ago2. Interestingly, unlike Dicer- and Dgcr8-deficient ES cells, they differentiate to all three germ layers both in vivo and in vitro. However, early differentiation of Ago2-deficient ES cells is delayed by 2–4 days as indicated by persistence of higher levels of self-renewal/ pluripotency markers during differentiation. Further, appearance of morphological and differentiation markers is also delayed during the differentiation. In this study we show that Ago2 is essential for normal self-renewal and differentiation. Also, our data suggest that self-renewal and differentiation of ES cells are regulated by both siRNA and miRNA pathways.

  18. Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chad M. Teven

    2011-01-01

    Full Text Available Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES cells. Mesenchymal stem cells (MSCs are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

  19. Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow

    Directory of Open Access Journals (Sweden)

    Huang Hai-Yan

    2010-05-01

    Full Text Available Abstract Background Adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A, revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPβ, C/EBPα and PPARγ. However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs, remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPβ, C/EBPα and PPARγ during adipocyte differentiation from hBMSCs. Results Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. Conclusions The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.

  20. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract Background Embryonic stem cells (ESCs can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs. However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC

  1. Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status.

    Directory of Open Access Journals (Sweden)

    Timothy M LeJeune

    Full Text Available Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2 and the pancreas (Panc28, whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116 and pancreas (MIA PaCa. Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT.

  2. Increased expression of the matrix metalloproteinase 2 in differentiating Tera 2 human embryonal carcinoma cells.

    Science.gov (United States)

    Tienari, J; Pertovaara, L; Saksela, O; Lehtonen, E; Vartio, T

    1994-01-15

    Secretion of proteolytic enzymes by cells has been implicated in tissue remodeling during embryonic development as well as in invasive neoplastic diseases. We studied the regulation of type-IV-collagenase activity in Tera 2 human embryonal carcinoma cells, which in the undifferentiated state proliferate rapidly and are tumorigenic. The undifferentiated cells produced relatively low levels of matrix-metalloproteinase-2 (MMP-2) activity. This activity was not markedly affected by exogenous basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though the plasminogen activator activity of the cells was increased by these agents. Tera 2 cells can be induced by retinoic acid to differentiate into quiescent cells, of which many express neuronal characteristics. The type-IV-collagenase activity of the cells increased markedly during the differentiation. This increase was mainly due to increased expression of MMP-2. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was not markedly affected by the differentiation of Tera 2 cells. The results show that in the Tera 2 cell system, increased expression of MMP-2 is characteristic of the differentiated derivatives. This is in contrast with many other model systems, where increased type-IV-collagenase activity is associated with the malignant phenotype. This pattern of regulation may reflect the facts that Tera 2 cells resemble early embryonic cells and that their differentiation mimics related cell-differentiation processes in the developing embryo.

  3. Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells

    Science.gov (United States)

    Fang, Yongxiang; Wang, Bin; Zhao, Yannan; Xiao, Zhifeng; Li, Jing; Cui, Yi; Han, Sufang; Wei, Jianshu; Chen, Bing; Han, Jin; Meng, Qingyuan; Hou, Xianglin; Luo, Jianxun; Dai, Jianwu; Jing, Zhizhong

    2017-01-01

    The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs. PMID:28169322

  4. Viral infection triggers rapid differentiation of human blood monocytes into dendritic cells.

    Science.gov (United States)

    Hou, Wanqiu; Gibbs, James S; Lu, Xiuju; Brooke, Christopher B; Roy, Devika; Modlin, Robert L; Bennink, Jack R; Yewdell, Jonathan W

    2012-03-29

    Surprisingly little is known about the interaction of human blood mononuclear cells with viruses. Here, we show that monocytes are the predominant cell type infected when peripheral blood mononuclear cells are exposed to viruses ex vivo. Remarkably, infection with vesicular stomatitis virus, vaccinia virus, and a variety of influenza A viruses (including circulating swine-origin virus) induces monocytes to differentiate within 18 hours into CD16(-)CD83(+) mature dendritic cells with enhanced capacity to activate T cells. Differentiation into dendritic cells does not require cell division and occurs despite the synthesis of viral proteins, which demonstrates that monocytes counteract the capacity of these highly lytic viruses to hijack host cell biosynthetic capacity. Indeed, differentiation requires infectious virus and viral protein synthesis. These findings demonstrate that monocytes are uniquely susceptible to viral infection among blood mononuclear cells, with the likely purpose of generating cells with enhanced capacity to activate innate and acquired antiviral immunity.

  5. The Effect of Bone Marrow Mesenchymal Stem Cells on Vitamin D3 Induced Monocytic Differentiation of U937 Cells

    OpenAIRE

    Zahra Molaeipour; Karim Shamsasanjan; Ali Akbari Movassaghpour; Parvin Akbarzadehlaleh; Fatemeh Sabaghi; Mahshid Saleh

    2016-01-01

    Purpose: Mesenchymal stem cells (MSCs) are key components of the hematopoietic stem cells (HSCs) niche. They control the process of hematopoiesis by secreting regulatory cytokines, growth factors and expression of important cell adhesion molecules for cell-to-cell interactions. In this research, we have investigated the effect of bone marrow derived MSCs on monocytic differentiation of U937 cells line. Methods: U937 cells were cultured in both direct co-culture with...

  6. Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

    Directory of Open Access Journals (Sweden)

    Benson Heather L

    2012-03-01

    Full Text Available Abstract Background Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs and plasmacytoid (pDCs are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown. Methods Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d prior to transplanting into C57BL/6 mice (H-2b, followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+. Results Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production. Conclusion Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

  7. Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

    Institute of Scientific and Technical Information of China (English)

    Han-Tso Lin; Hung-Hai Ku; Chung-Lan Kao; Kun-Hsiung Lee; Yuh-Lih Chang; Shih-Hwa Chiou; Fu-Ting Tsai; Tung-Hu Tsai; Dey-Chyi Sheu; Larry LT Ho

    2007-01-01

    AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro.METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of Nucleofector- Electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectin coated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation.RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18

  8. Differential Effects of Methyl-4-Phenylpyridinium Ion, Rotenone, and Paraquat on Differentiated SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    João Barbosa Martins

    2013-01-01

    Full Text Available Paraquat (PQ, a cationic nonselective bipyridyl herbicide, has been used as neurotoxicant to modulate Parkinson’s disease in laboratory settings. Other compounds like rotenone (ROT, a pesticide, and 1-methyl-4-phenylpyridinium ion (MPP+ have been widely used as neurotoxicants. We compared the toxicity of these three neurotoxicants using differentiated dopaminergic SH-SY5Y human cells, aiming to elucidate their differential effects. PQ-induced neurotoxicity was shown to be concentration and time dependent, being mitochondrial dysfunction followed by neuronal death. On the other hand, cells exposure to MPP+ induced mitochondrial dysfunction, but not cellular lyses. Meanwhile, ROT promoted both mitochondrial dysfunction and neuronal death, revealing a biphasic pattern. To further elucidate PQ neurotoxic mechanism, several protective agents were used. SH-SY5Y cells pretreatment with tiron (TIR and 2-hydroxybenzoic acid sodium salt (NaSAL, both antioxidants, and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, a nitric oxide synthase inhibitor, partially protected against PQ-induced cell injury. Additionally, 1-(2-[bis(4-fluorophenylmethoxy]ethyl-4-(3-phenyl-propylpiperazine (GBR 12909, a dopamine transporter inhibitor, and cycloheximide (CHX, a protein synthesis inhibitor, also partially protected against PQ-induced cell injury. In conclusion, we demonstrated that PQ, MPP+, and ROT exerted differential toxic effects on dopaminergic cells. PQ neurotoxicity occurred through exacerbated oxidative stress, with involvement of uptake through the dopamine transporter and protein synthesis.

  9. Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

    Science.gov (United States)

    Tapia-Limonchi, Rafael; Cahuana, Gladys M; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Beltran-Povea, Amparo; Hitos, Ana B; Hmadcha, Abdelkrim; Martin, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R

    2016-09-01

    Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

  10. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    Science.gov (United States)

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

  11. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture.

    Science.gov (United States)

    Pineda, Emma T; Nerem, Robert M; Ahsan, Tabassum

    2013-01-01

    Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.

  12. A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels.

    Science.gov (United States)

    Luo, Wei; Mayeux, Jessica; Gutierrez, Toni; Russell, Lisa; Getahun, Andrew; Müller, Jennifer; Tedder, Thomas; Parnes, Jane; Rickert, Robert; Nitschke, Lars; Cambier, John; Satterthwaite, Anne B; Garrett-Sinha, Lee Ann

    2014-07-15

    Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine k