WorldWideScience

Sample records for multipotent adult germline

  1. Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yoon Hee [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Gupta, Mukesh Kumar, E-mail: goops@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Oh, Shin Hye [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of)

    2010-03-10

    This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W{sup v} mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

  2. Perivascular ancestors of adult multipotent stem cells

    NARCIS (Netherlands)

    M. Corselli (Mirko); C.W. Chen; M. Crisan (Mihaela); L. Lazzari (Lorenza); B. Péault (Bruno)

    2010-01-01

    textabstractIndependent studies by numerous investigators have shown that it is possible to harvest multipotent progenitor cells from diverse dissociated and cultured fetal, perinatal, and principally adult developed tissues. Despite the increasingly recognized medical value of these progenitor cell

  3. Multipotent adult progenitor cells : their role in wound healing and the treatment of dermal wounds

    NARCIS (Netherlands)

    Herdrich, B. J.; Lind, R. C.; Liechty, K. W.

    2008-01-01

    The use of cellular therapy in the treatment of dermal wounds is currently an active area of investigation. Multipotent adult progenitor cells (MAPC) are an attractive choice for cytotherapy because they have a large proliferative potential, the ability to differentiate into different cell types and

  4. Multipotent (adult) and pluripotent stem cells for heart regeneration: what are the pros and cons?

    Science.gov (United States)

    Liao, Song-Yan; Tse, Hung-Fat

    2013-12-24

    Heart failure after myocardial infarction is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the loss of cardiomyocytes during myocardial infarction but are unable to replenish the permanent loss of cardiomyocytes after the insult, which contributes to progressive pathological left ventricular remodeling and progressive heart failure. As a result, cell-based therapies using multipotent (adult) stem cells and pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) have been explored as potential therapeutic approaches to restore cardiac function in heart failure. Nevertheless, the optimal cell type with the best therapeutic efficacy and safety for heart regeneration is still unknown. In this review, the potential pros and cons of different types of multipotent (adult) stem cells and pluripotent stem cells that have been investigated in preclinical and clinical studies are reviewed, and the future perspective of stem cell-based therapy for heart regeneration is discussed.

  5. Adult Vascular Wall Resident Multipotent Vascular Stem Cells, Matrix Metalloproteinases, and Arterial Aneurysms

    Directory of Open Access Journals (Sweden)

    Bruno Amato

    2015-01-01

    Full Text Available Evidences have shown the presence of multipotent stem cells (SCs at sites of arterial aneurysms: they can differentiate into smooth muscle cells (SMCs and are activated after residing in a quiescent state in the vascular wall. Recent studies have implicated the role of matrix metalloproteinases in the pathogenesis of arterial aneurysms: in fact the increased synthesis of MMPs by arterial SMCs is thought to be a pivotal mechanism in aneurysm formation. The factors and signaling pathways involved in regulating wall resident SC recruitment, survival, proliferation, growth factor production, and differentiation may be also related to selective expression of different MMPs. This review explores the relationship between adult vascular wall resident multipotent vascular SCs, MMPs, and arterial aneurysms.

  6. File list: Pol.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.10.AllAg.Germline_containing_young_adult ce10 RNA polymerase Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  7. File list: NoD.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adl.05.AllAg.Germline_containing_young_adult ce10 No description Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/NoD.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  8. File list: NoD.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adl.20.AllAg.Germline_containing_young_adult ce10 No description Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/NoD.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  9. File list: Unc.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adl.10.AllAg.Germline_containing_young_adult ce10 Unclassified Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Unc.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  10. File list: Unc.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adl.05.AllAg.Germline_containing_young_adult ce10 Unclassified Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Unc.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  11. File list: NoD.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adl.50.AllAg.Germline_containing_young_adult ce10 No description Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/NoD.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  12. File list: NoD.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adl.10.AllAg.Germline_containing_young_adult ce10 No description Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/NoD.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  13. File list: Unc.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adl.20.AllAg.Germline_containing_young_adult ce10 Unclassified Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Unc.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  14. File list: Pol.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.50.AllAg.Germline_containing_young_adult ce10 RNA polymerase Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  15. File list: Pol.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.20.AllAg.Germline_containing_young_adult ce10 RNA polymerase Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  16. File list: Unc.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adl.50.AllAg.Germline_containing_young_adult ce10 Unclassified Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Unc.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  17. File list: Pol.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adl.05.AllAg.Germline_containing_young_adult ce10 RNA polymerase Adult Germline containing young... adult http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  18. Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways.

    Science.gov (United States)

    Götherström, Cecilia; Lundqvist, Andreas; Duprez, Ida Rasmusson; Childs, Richard; Berg, Louise; le Blanc, Katarina

    2011-03-01

    Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ. Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.

  19. Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues.

    Science.gov (United States)

    Shen, Jie-fei; Sugawara, Atsunori; Yamashita, Joe; Ogura, Hideo; Sato, Soh

    2011-07-01

    When adipose-derived stem cells (ASCs) are retrieved from the stromal vascular portion of adipose tissue, a large amount of mature adipocytes are often discarded. However, by modified ceiling culture technique based on their buoyancy, mature adipocytes can be easily isolated from the adipose cell suspension and dedifferentiated into lipid-free fibroblast-like cells, named dedifferentiated fat (DFAT) cells. DFAT cells re-establish active proliferation ability and undertake multipotent capacities. Compared with ASCs and other adult stem cells, DFAT cells showed unique advantages in their abundance, isolation and homogeneity. In this concise review, the establishment and culture methods of DFAT cells are introduced and the current profiles of their cellular nature are summarized. Under proper induction culture in vitro or environment in vivo, DFAT cells could demonstrate adipogenic, osteogenic, chondrogenic and myogenic potentials. In angiogenic conditions, DFAT cells could exhibit perivascular characteristics and elicit neovascularization. Our preliminary findings also suggested the pericyte phenotype underlying such cell lineage, which supported a novel interpretation about the common origin of mesenchymal stem cells and tissue-specific stem cells within blood vessel walls. Current research on DFAT cells indicated that this alternative source of adult multipotent cells has great potential in tissue engineering and regenerative medicine.

  20. File list: ALL.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adl.10.AllAg.Germline_containing_young_adult ce10 All antigens Adult Germline containing young...4938,SRX466563,SRX495065,SRX495061,SRX494933,SRX495102,SRX494887 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/ALL.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  1. File list: Oth.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.20.AllAg.Germline_containing_young_adult ce10 TFs and others Adult Germline containing young....jp/kyushu-u/ce10/assembled/Oth.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  2. File list: His.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adl.10.AllAg.Germline_containing_young_adult ce10 Histone Adult Germline containing young...archive.biosciencedbc.jp/kyushu-u/ce10/assembled/His.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  3. File list: His.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adl.50.AllAg.Germline_containing_young_adult ce10 Histone Adult Germline containing young...osciencedbc.jp/kyushu-u/ce10/assembled/His.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  4. File list: Oth.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.10.AllAg.Germline_containing_young_adult ce10 TFs and others Adult Germline containing young....jp/kyushu-u/ce10/assembled/Oth.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  5. File list: Oth.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.05.AllAg.Germline_containing_young_adult ce10 TFs and others Adult Germline containing young....jp/kyushu-u/ce10/assembled/Oth.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  6. File list: InP.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adl.50.AllAg.Germline_containing_young_adult ce10 Input control Adult Germline containing young...p/kyushu-u/ce10/assembled/InP.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  7. File list: Oth.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.50.AllAg.Germline_containing_young_adult ce10 TFs and others Adult Germline containing young....jp/kyushu-u/ce10/assembled/Oth.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  8. File list: ALL.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adl.20.AllAg.Germline_containing_young_adult ce10 All antigens Adult Germline containing young...6563,SRX495065,SRX495061,SRX495102,SRX495062,SRX494887,SRX495066 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/ALL.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  9. File list: InP.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adl.20.AllAg.Germline_containing_young_adult ce10 Input control Adult Germline containing young...X495061,SRX495102,SRX495062,SRX495066 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/InP.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  10. File list: His.Adl.20.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adl.20.AllAg.Germline_containing_young_adult ce10 Histone Adult Germline containing young...archive.biosciencedbc.jp/kyushu-u/ce10/assembled/His.Adl.20.AllAg.Germline_containing_young_adult.bed ...

  11. File list: ALL.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adl.05.AllAg.Germline_containing_young_adult ce10 All antigens Adult Germline containing young...4871,SRX494938,SRX495065,SRX495061,SRX494933,SRX495042,SRX495102 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/ALL.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  12. File list: ALL.Adl.50.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adl.50.AllAg.Germline_containing_young_adult ce10 All antigens Adult Germline containing young...e.biosciencedbc.jp/kyushu-u/ce10/assembled/ALL.Adl.50.AllAg.Germline_containing_young_adult.bed ...

  13. File list: His.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adl.05.AllAg.Germline_containing_young_adult ce10 Histone Adult Germline containing young...archive.biosciencedbc.jp/kyushu-u/ce10/assembled/His.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  14. File list: InP.Adl.10.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adl.10.AllAg.Germline_containing_young_adult ce10 Input control Adult Germline containing young...X466563,SRX495065,SRX495061,SRX495102 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/InP.Adl.10.AllAg.Germline_containing_young_adult.bed ...

  15. File list: InP.Adl.05.AllAg.Germline_containing_young_adult [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adl.05.AllAg.Germline_containing_young_adult ce10 Input control Adult Germline containing young...X495065,SRX495061,SRX495042,SRX495102 http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/InP.Adl.05.AllAg.Germline_containing_young_adult.bed ...

  16. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

    Science.gov (United States)

    Serafini, Marta; Dylla, Scott J.; Oki, Masayuki; Heremans, Yves; Tolar, Jakub; Jiang, Yuehua; Buckley, Shannon M.; Pelacho, Beatriz; Burns, Terry C.; Frommer, Sarah; Rossi, Derrick J.; Bryder, David; Panoskaltsis-Mortari, Angela; O'Shaughnessy, Matthew J.; Nelson-Holte, Molly; Fine, Gabriel C.; Weissman, Irving L.; Blazar, Bruce R.; Verfaillie, Catherine M.

    2007-01-01

    For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs. PMID:17227908

  17. Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells.

    Science.gov (United States)

    Li, Tianqing; Lewallen, Michelle; Chen, Shuyi; Yu, Wei; Zhang, Nian; Xie, Ting

    2013-06-01

    Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin(+)Sox2(+)Pax6(+) multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.

  18. Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells

    Institute of Scientific and Technical Information of China (English)

    Tianqing Li; Michelle Lewallen; Shuyi Chen; Wei Yu; Nian Zhang; Ting Xie

    2013-01-01

    Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases,such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD).Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptordeficient mice,but there is still some concern of tumor formation.In this study,we have successfully cultured Nestin+Sox2+Pax6+ multipotent retinal stem cells (RSCs) from the adult mouse retina,which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation.After they have been expanded for over 35 passages in the presence of FGF and EGF,the cultured RSCs still maintain stable proliferation and differentiation potential.Under proper differentiation conditions,they can differentiate into all the major retinal cell types found in the adult retina.More importantly,they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions.Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes,RSC-derived photoreceptor cells integrate into the retina,morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons.When transplanted into eyes of photoreceptor-deficient rd1 mutant mice,a RP model,RSC-derived photoreceptors can partially restore light response,indicating that those RSC-derived photoreceptors are functional.Finally,there is no evidence for tumor formation in the photoreceptor-transplanted eyes.Therefore,this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.

  19. Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells.

    Science.gov (United States)

    Chen, Pei-Min; Yen, Men-Luh; Liu, Ko-Jiunn; Sytwu, Huey-Kang; Yen, B-Linju

    2011-07-18

    In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.

  20. Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

    Science.gov (United States)

    Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

    2016-12-01

    : MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8(+) cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8(-)CD69(+) T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune

  1. Intravenous multipotent adult progenitor cell therapy after traumatic brain injury: modulation of the resident microglia population

    Directory of Open Access Journals (Sweden)

    Walker Peter A

    2012-09-01

    Full Text Available Abstract Introduction We have demonstrated previously that the intravenous delivery of multipotent adult progenitor cells (MAPC after traumatic brain injury affords neuroprotection via interaction with splenocytes, leading to an increase in systemic anti-inflammatory cytokines. We hypothesize that the observed modulation of the systemic inflammatory milieu is related to T regulatory cells and a subsequent increase in the locoregional neuroprotective M2 macrophage population. Methods C57B6 mice were injected with intravenous MAPC 2 and 24 hours after controlled cortical impact injury. Animals were euthanized 24, 48, 72, and 120 hours after injury. In vivo, the proportion of CD4+/CD25+/FOXP3+ T-regulatory cells were measured in the splenocyte population and plasma. In addition, the brain CD86+ M1 and CD206+ M2 macrophage populations were quantified. A series of in vitro co-cultures were completed to investigate the need for direct MAPC:splenocyte contact as well as the effect of MAPC therapy on M1 and M2 macrophage subtype apoptosis and proliferation. Results Significant increases in the splenocyte and plasma T regulatory cell populations were observed with MAPC therapy at 24 and 48 hours, respectively. In addition, MAPC therapy was associated with an increase in the brain M2/M1 macrophage ratio at 24, 48 and 120 hours after cortical injury. In vitro cultures of activated microglia with supernatant derived from MAPC:splenocyte co-cultures also demonstrated an increase in the M2/M1 ratio. The observed changes were secondary to an increase in M1 macrophage apoptosis. Conclusions The data show that the intravenous delivery of MAPC after cortical injury results in increases in T regulatory cells in splenocytes and plasma with a concordant increase in the locoregional M2/M1 macrophage ratio. Direct contact between the MAPC and splenocytes is required to modulate activated microglia, adding further evidence to the central role of the spleen in MAPC

  2. Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues

    OpenAIRE

    Shen, Jie-fei; Sugawara, Atsunori; Yamashita, Joe; Ogura, Hideo; Sato, Soh

    2011-01-01

    When adipose-derived stem cells (ASCs) are retrieved from the stromal vascular portion of adipose tissue, a large amount of mature adipocytes are often discarded. However, by modified ceiling culture technique based on their buoyancy, mature adipocytes can be easily isolated from the adipose cell suspension and dedifferentiated into lipid-free fibroblast-like cells, named dedifferentiated fat (DFAT) cells. DFAT cells re-establish active proliferation ability and undertake multipotent capaciti...

  3. Induced multipotency in adult keratinocytes through down-regulation of ΔNp63 or DGCR8

    Science.gov (United States)

    Chakravarti, Deepavali; Su, Xiaohua; Cho, Min Soon; Bui, Ngoc Hoang Bao; Coarfa, Cristian; Venkatanarayan, Avinashnarayan; Benham, Ashley L.; Flores González, Ramón E.; Alana, Jennifer; Xiao, Weimin; Leung, Marco L.; Vin, Harina; Chan, Io Long; Aquino, Arianexys; Müller, Nicole; Wang, Hongran; Cooney, Austin J.; Parker-Thornburg, Jan; Tsai, Kenneth Y.; Gunaratne, Preethi H.; Flores, Elsa R.

    2014-01-01

    The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63−/− epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63−/− epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells. PMID:24449888

  4. Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

    Science.gov (United States)

    He, Xiaoli; H'ng, Shiau-Chen; Leong, David T; Hutmacher, Dietmar W; Melendez, Alirio J

    2010-08-01

    High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

  5. Oligodendrocyte differentiation from adult multipotent stem cells is modulated by glutamate.

    Science.gov (United States)

    Cavaliere, F; Urra, O; Alberdi, E; Matute, C

    2012-02-02

    We used multipotent stem cells (MSCs) derived from the young rat subventricular zone (SVZ) to study the effects of glutamate in oligodendrocyte maturation. Glutamate stimulated oligodendrocyte differentiation from SVZ-derived MSCs through the activation of specific N-methyl-D-aspartate (NMDA) receptor subunits. The effect of glutamate and NMDA on oligodendrocyte differentiation was evident in both the number of newly generated oligodendrocytes and their morphology. In addition, the levels of NMDAR1 and NMDAR2A protein increased during differentiation, whereas NMDAR2B and NMDAR3 protein levels decreased, suggesting differential expression of NMDA receptor subunits during maturation. Microfluorimetry showed that the activation of NMDA receptors during oligodendrocyte differentiation elevated cytosolic calcium levels and promoted myelination in cocultures with neurons. Moreover, we observed that stimulation of MSCs by NMDA receptors induced the generation of reactive oxygen species (ROS), which were negatively modulated by the NADPH inhibitor apocynin, and that the levels of ROS correlated with the degree of differentiation. Taken together, these findings suggest that ROS generated by NADPH oxidase by the activation of NMDA receptors promotes the maturation of oligodendrocytes and favors myelination.

  6. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

    Science.gov (United States)

    Marie, Pauline P.; Ronsseray, Stéphane; Boivin, Antoine

    2016-01-01

    In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the “ping-pong” amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development. PMID:27932388

  7. From Embryo to Adult: piRNA-Mediated Silencing throughout Germline Development in Drosophila

    Directory of Open Access Journals (Sweden)

    Pauline P. Marie

    2017-02-01

    Full Text Available In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs. Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development has not been addressed. Here, we show that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, we detect rhino-dependent piRNAs indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the “ping-pong” amplification step. Moreover, we show that Heterochromatin Protein 1a is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal, and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, data presented here show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

  8. Multipotent adult progenitor cells decrease cold ischemic injury in ex vivo perfused human lungs: an initial pilot and feasibility study.

    Science.gov (United States)

    La Francesca, Saverio; Ting, Anthony E; Sakamoto, Jason; Rhudy, Jessica; Bonenfant, Nicholas R; Borg, Zachary D; Cruz, Fernanda F; Goodwin, Meagan; Lehman, Nicholas A; Taggart, Jennifer M; Deans, Robert; Weiss, Daniel J

    2014-01-01

    Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality following lung transplantation. Improved organ preservation techniques will decrease ischemia-reperfusion injury (IRI) contributing to PGD. Adult bone marrow-derived adherent stem cells, including mesenchymal stromal (stem) cells (MSCs) and multipotent adult progenitor cells (MAPCs), have potent anti-inflammatory actions, and we thus postulated that intratracheal MAPC administration during donor lung processing would decrease IRI. The goal of the study was therefore to determine if intratracheal MAPC instillation would decrease lung injury and inflammation in an ex vivo human lung explant model of prolonged cold storage and subsequent reperfusion. Four donor lungs not utilized for transplant underwent 8 h of cold storage (4°C). Following rewarming for approximately 30 min, non-HLA-matched allogeneic MAPCs (1 × 10(7) MAPCs/lung) were bronchoscopically instilled into the left lower lobe (LLL) and vehicle comparably instilled into the right lower lobe (RLL). The lungs were then perfused and mechanically ventilated for 4 h and subsequently assessed for histologic injury and for inflammatory markers in bronchoalveolar lavage fluid (BALF) and lung tissue. All LLLs consistently demonstrated a significant decrease in histologic and BALF inflammation compared to vehicle-treated RLLs. These initial pilot studies suggest that use of non-HLA-matched allogeneic MAPCs during donor lung processing can decrease markers of cold ischemia-induced lung injury.

  9. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

    Directory of Open Access Journals (Sweden)

    Kee Hang Lee

    Full Text Available Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs immortalized by the human telomerase reverse transcriptase (hTERT gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM cells were injected into adult (4-6-week-old Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL, they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.

  10. Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2

    Science.gov (United States)

    Reading, James L; Vaes, Bart; Hull, Caroline; Sabbah, Shereen; Hayday, Thomas; Wang, Nancy S; DiPiero, Anthony; Lehman, Nicholas A; Taggart, Jen M; Carty, Fiona; English, Karen; Pinxteren, Jef; Deans, Robert; Ting, Anthony E; Tree, Timothy I M

    2015-01-01

    T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1β-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients. PMID:26216515

  11. In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation

    Directory of Open Access Journals (Sweden)

    Chiara Garrovo

    2013-01-01

    Full Text Available Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

  12. Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.

    Science.gov (United States)

    Bedi, Supinder S; Hetz, Robert; Thomas, Chelsea; Smith, Philippa; Olsen, Alex B; Williams, Stephen; Xue, Hasen; Aroom, Kevin; Uray, Karen; Hamilton, Jason; Mays, Robert W; Cox, Charles S

    2013-12-01

    We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation.

  13. Pesticide methoxychlor promotes the epigenetic transgenerational inheritance of adult-onset disease through the female germline.

    Directory of Open Access Journals (Sweden)

    Mohan Manikkam

    Full Text Available Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14 and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring generation progeny for control (vehicle exposed and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

  14. Pesticide methoxychlor promotes the epigenetic transgenerational inheritance of adult-onset disease through the female germline.

    Science.gov (United States)

    Manikkam, Mohan; Haque, M Muksitul; Guerrero-Bosagna, Carlos; Nilsson, Eric E; Skinner, Michael K

    2014-01-01

    Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.

  15. Hippocampal stem cells: so they are multipotent!

    Institute of Scientific and Technical Information of China (English)

    Verdon Taylor

    2011-01-01

    Although neurogenesis continues throughout life in the mammalian brain, the issue of whether the stem cells that drive the process in vivo are self-renewing and multipotent remains unclear. In a recent landmark paper by Bonaguidi et al. (2011) published in Cell, the authors provide clonal evidence that neural stem cells in the dentate gyrus of the adult hippocampus are indeed multipotent and undergo symmetric cell divisions.%Although neurogenesis continues throughout life in the mammalian brain,the issue of whether the stem cells that drive the process in vivo are self-renewing and multipotent remains unclear.In a recent landmark paper by Bonaguidi et al.(2011) pubiished in Cell,the authors provide cional evidence that neural stem cells in the dentate gyrus of the adult hippocampus are indeed multipotent and undergo symmetric cell divisions.

  16. Assessment of Effects of Si-Ca-P Biphasic Ceramic on the Osteogenic Differentiation of a Population of Multipotent Adult Human Stem Cells

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    Patricia Ros-Tárraga

    2016-11-01

    Full Text Available A new type of bioceramic with osteogenic properties, suitable for hard tissue regeneration, was synthesised. The ceramic was designed and obtained in the Nurse’s A-phase-silicocarnotite subsystem. The selected composition was that corresponding to the eutectoid 28.39 wt % Nurse’s A-phase-71.61 wt % silicocarnotite invariant point. We report the effect of Nurse’s A-phase-silicocarnotite ceramic on the capacity of multipotent adult human mesenchymal stem cells (ahMSCs cultured under experimental conditions, known to adhere, proliferate and differentiate into osteoblast lineage cells. The results at long-term culture (28 days on the material confirmed that the undifferentiated ahMSCs cultured and in contact with the material surface adhered, spread, proliferated, and produced a mineralised extracellular matrix on the studied ceramic, and finally acquired an osteoblastic phenotype. These findings indicate that it underwent an osteoblast differentiation process. All these findings were more significant than when cells were grown on plastic, in the presence and absence of this osteogenic supplement, and were more evident when this supplement was present in the growth medium (GM. The ceramic evaluated herein was bioactive, cytocompatible and capable of promoting the proliferation and differentiation of undifferentiated ahMSCs into osteoblasts, which may be important for bone integration into the clinical setting.

  17. Migration and differentiation of bone marrow-derived multipotent adult progenitor cells through tail vein injection in a rat model of cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Lei Lei; Ruixiang Zhou

    2009-01-01

    BACKGROUND: Multipotent adult progenitor cells (MAPCs) from the bone marrow have been shown to differentiate into neurons.OBJECTIVE: To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection.DESIGN, TIME AND SETTING: Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007.MATERIALS: Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups (n = 40).METHODS: Mononuclear cells were harvested from bone marrow using the Ficoll-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells (GLYA+) with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). A total of 1 mL cell suspension, containing 5 ×106 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats.MAIN OUTCOME MEASURES: After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests.RESULTS: Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses, Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs (P < 0.05). In addition, rats in the MAPCs

  18. 大鼠多能成体祖细胞体外成软骨的诱导和鉴定%Chondrogenesis of Rats’Bone Marrow-derived Multipotent Adult Progenitor Cells in vitro and Related Identification

    Institute of Scientific and Technical Information of China (English)

    徐海艇; 潘骏; 李永领; 朱烈烈

    2016-01-01

    [Objective] To explore chondrogenic differentiation of rats bone marrow-derived multipotent adult progenitor cells(MAPCs) in experimental culture medium.[Methods] MAPCs were isolated from rats bone marrow and expanded to passage 2. The morphology of MAPCs was observed in vitro. Then the cell groups were cultured in chondrogenic media of H-DMEM containing TGF-β3, BMP-6, IGF-1, the control one was made natural differentiation with routine fluid 10% FB5, and dexamethasone forming cell pellet and assessed by Safranin O and collagen Ⅱ staining. And Alcian blue chromatomery of glycosaminoglycan(GAG) was used to determine the differentiation. [Results] MAPCs demonstrated fibroblast-like morphology and showed good proliferation ability. After chondrogenic induction for 4 weeks, the pellets formed by MAPCs had a transparent white cartilage-like morphology in chondrogenic medium while a less shape in control medium. Histologically, the group showed a homogeneous cartilage-like structure with strong safranin O staining and strong collagen 2 staining. Alcian blue staining showed the content of GAG formatting were higher than the control group(P<0.05), with difference of statistical meaning. [Conclusion]MAPCs have high chondrogenic potential and could be an ideal seed in cartilage tissue engineering.%[目的]探讨大鼠多能成体祖细胞(multipotent adult progenitor cells,MAPCs)在特定培养条件下向软骨细胞定向分化的能力。[方法]分离培养大鼠MAPCs,扩增至第2代,观察其形态学特点,并将其离心成团后分为2组,诱导组应用转化生长因子-β3(transforming growth factor-β3,TGF-β3)、骨形态发生蛋白-6(bone morphogenetic protein-6,BMP-6)、胰岛素样生长因子-1(insulin-like growth factors-1,IGF-1)及地塞米松等进行成软骨诱导分化,对照组用含10%胎牛血清(fetal bovine serum, FBS)的高糖DMEM培养液(dulbecco's modifiedeagle medium high-glucose, H-DMEM)常规

  19. Fate restriction and multipotency in retinal stem cells.

    Science.gov (United States)

    Centanin, Lázaro; Hoeckendorf, Burkhard; Wittbrodt, Joachim

    2011-12-02

    Stem cells have the capacity to both self-renew and generate postmitotic cells. Long-term tracking of individual clones in their natural environment constitutes the ultimate way to validate postembryonic stem cells. We identify retinal stem cells (RSCs) using the spatiotemporal organization of the fish retina and follow the complete offspring of a single cell during the postnatal life. RSCs generate two tissues of the adult fish retina, the neural retina (NR) and the retinal-pigmented epithelium (RPE). Despite their common embryonic origin and tight coordination during continuous organ growth, we prove that NR and RPE are maintained by dedicated RSCs that contribute in a fate-restricted manner to either one or the other tissue. We show that in the NR, RSCs are multipotent and generate all neuron types and glia. The clonal origin of these different cell types from a multipotent NSC has far-reaching implications for cell type and tissue homeostasis.

  20. Multipotent glia-like stem cells mediate stress adaptation.

    Science.gov (United States)

    Rubin de Celis, Maria F; Garcia-Martin, Ruben; Wittig, Dierk; Valencia, Gabriela D; Enikolopov, Grigori; Funk, Richard H; Chavakis, Triantafyllos; Bornstein, Stefan R; Androutsellis-Theotokis, Andreas; Ehrhart-Bornstein, Monika

    2015-06-01

    The neural crest-derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. Here, we show that a defined pool of glia-like nestin-expressing progenitor cells in the adult adrenal medulla contributes to this plasticity. These glia-like cells have features of adrenomedullary sustentacular cells, are multipotent, and are able to differentiate into chromaffin cells and neurons. The adrenal is central to the body's response to stress making its proper adaptation critical to maintaining homeostasis. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia-like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general.

  1. Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.

    Science.gov (United States)

    Kopp, Janel L; Dubois, Claire L; Schaffer, Ashleigh E; Hao, Ergeng; Shih, Hung Ping; Seymour, Philip A; Ma, Jenny; Sander, Maike

    2011-02-01

    One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.

  2. The oogenic germline starvation response in C. elegans.

    Directory of Open Access Journals (Sweden)

    Hannah S Seidel

    Full Text Available Many animals alter their reproductive strategies in response to environmental stress. Here we have investigated how L4 hermaphrodites of Caenorhabditis elegans respond to starvation. To induce starvation, we removed food at 2 h intervals from very early- to very late-stage L4 animals. The starved L4s molted into adulthood, initiated oogenesis, and began producing embryos; however, all three processes were severely delayed, and embryo viability was reduced. Most animals died via 'bagging,' because egg-laying was inhibited, and embryos hatched in utero, consuming their parent hermaphrodites from within. Some animals, however, avoided bagging and survived long term. Long-term survival did not rely on embryonic arrest but instead upon the failure of some animals to produce viable progeny during starvation. Regardless of the bagging fate, starved animals showed two major changes in germline morphology: All oogenic germlines were dramatically reduced in size, and these germlines formed only a single oocyte at a time, separated from the remainder of the germline by a tight constriction. Both changes in germline morphology were reversible: Upon re-feeding, the shrunken germlines regenerated, and multiple oocytes formed concurrently. The capacity for germline regeneration upon re-feeding was not limited to the small subset of animals that normally survive starvation: When bagging was prevented ectopically by par-2 RNAi, virtually all germlines still regenerated. In addition, germline shrinkage strongly correlated with oogenesis, suggesting that during starvation, germline shrinkage may provide material for oocyte production. Finally, germline shrinkage and regeneration did not depend upon crowding. Our study confirms previous findings that starvation uncouples germ cell proliferation from germline stem cell maintenance. Our study also suggests that when nutrients are limited, hermaphrodites scavenge material from their germlines to reproduce. We discuss

  3. Effects of Dermal Multipotent Cell Transplantation on Skin Wound Healing

    Institute of Scientific and Technical Information of China (English)

    ShiChunmeng; ChengTianmin; SuYongping; RanXinze; MaiYue; QuJifu; LouShufen; XuHui; LuoChengji

    2005-01-01

    There is increasing evidence that dermis contains adult multipotent stem cells. To investigate the effects of dermis-derived multipotent cells on wound healing, we transplanted a clonal population of dermis-derived multipotent cells (termed as DMCs) by topical and systemic application into the skin wound of rats with simple wounds and rats with combined wound and radiation injury. Our results suggest that both topical and systemic transplantation of DMCs accelerate the healing process in rats with a simple wound; the promoting effect by topical transplantation occurs earlier than systemic transplantation. However, systemic transplantation of DMCs promotes the healing process in irradiated rats, while topical transplantation of DMCs fails. Further studies on the mechanisms of DMCs to promote wound healing indicate that the supernatant of DMCs could promote the proliferation of fibroblasts and epidermal cells; DMCs expressed transcripts of a serics of cytokincs and cxtraccllular matrix molecules, including VEGF, PDGF, HGF, TGF-β, ICAM-1, VCAM-1, and Fibronectin, which were closely related to the wound healing by DNA microarray analysis. The implanted DMCs can engraft into recipient skin wounded tissues after transplantation by the FISH analysis with Y-chromosome-specific probe. Systemic transplantation of DMCs also promotes the recovery of peripheral white blood cells in irradiated rats. These results demonstrate the different effects of DMCs on wound healing in nonirradiated and irradiated rats and illustrate the importance of optimizing wound healing via the topical or systemic transplantation of stem cells.

  4. Modeling sarcomagenesis using multipotent mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Rene Rodriguez; Ruth Rubio; Pablo Menendez

    2012-01-01

    Because of their unique properties,multipotent mesenchymal stem cells (MSCs) represent one of the most promising adult stem cells being used worldwide in a wide array of clinical applications.Overall,compelling evidence supports the long-term safety of ex vivo expanded human MSCs,which do not seem to transform spontaneously.However,experimental data reveal a link between MSCs and cancer,and MSCs have been reported to inhibit or promote tumor growth depending on yet undefined conditions.Interestingly,solid evidence based on transgenic mice and genetic intervention of MSCs has placed these cells as the most likely cell of origin for certain sarcomas.This research area is being increasingly explored to develop accurate MSC-based models of sarcomagenesis,which will be undoubtedly valuable in providing a better understanding about the etiology and pathogenesis of mesenchymal cancer,eventually leading to the development of more specific therapies directed against the sarcoma-initiating cell.Unfortunately,still little is known about the mechanisms underlying MSC transformation and further studies are required to develop bona fide sarcoma models based on human MSCs.Here,we comprehensively review the existing MSC-based models of sarcoma and discuss the most common mechanisms leading to tumoral transformation of MSCs and sarcomagenesis.

  5. Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood

    OpenAIRE

    Martinello, Tiziana; Bronzini, Ilaria; Maccatrozzo, Lisa; Iacopetti, Ilaria; Sampaolesi, Maurilio; Mascarello, Francesco; Patruno, Marco

    2010-01-01

    Mammalian adult stem cells show, in vitro, extensive differentiative ability and may represent a versatile tool for tissue regenerative purposes, even after long term storage. Multipotent stem cells isolated from horse blood have been shown to possess the capacity to differentiate into diverse mesenchymal lineages although their full characterization is still at an early stage. The aim of this study was to examine the effects of cryopreservation on stemness characteristics of adult equine mes...

  6. Transplantation of bone marrow multipotent adult progenitor cells for treating Parkinson disease in rats%骨髓基质多能成体祖细胞移植治疗大鼠帕金森病

    Institute of Scientific and Technical Information of China (English)

    周瑞祥; 孙圣刚

    2007-01-01

    various characteristics of bone marrow derived-multipotent adult progenitor cells (MAPCs) enable them to become one the ideal sources of cells for cell transplantation.OBJECTIVE: To explore the hypothesis that MAPCs were able to enter the brain and reduce the neurological functional deficits in rats by injecting intravenously.DESTGN: A randomized controlled experiment.ETTING: Department of Neurology, Wuhan First Hospital.MATERIALS: The experiments were performed in the laboratory of Department of Neurology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from October 2003 to March 2005. Eighty healthy Sprague-Dawley (SD) rats of 180-200 g were provided by the experimental animal center of Tongji Medical College,Huazhong University of Science and Technology.METHODS: The rats were made into models of Parkinson disease, the bone marrow-derived MAPCs, which were in vitro purified, proliferated and treated with 5-bromo-2-deoxyuridine (BrdU), were injected via caudal vein. After three months,the immunohistochemical technique, reverse transcription-polymerase chain reaction (RT-PCR), electron microscopy and behavioral tests were used to identify the MAPCs or neuron-like cells derived from MAPCs in brain and their functions.MAIN OUTCOME MEASURES: ① Results of behavioral observation; ② Results of immunihistochemical staining.RESULTS: After implantation, MAPCs could survive and differentiate into neuron-like cells in substantia nigra and striatum. MAPCs-derived dopaminergic neurons caused gradual and sustained behavioral restoration of 6-hydroxydopamine (6-OHDA)-mediated motor asymmetry. The levels of dopamine beta-hydroxylase (DBH), nerve growth factor (NGF) or dopamine transporter (DAT) mRNA were up-regulated significantly. It was observed under electron microscope that immature synapse implicated MAPCs- derived neuron should play an important role in the reconstruction of neural circuitry.CONCLUSION: Transplanted bone marrow derived

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

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

  9. Multipotent pancreas progenitors: Inconclusive but pivotaltopic

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    The establishment of multipotent pancreas progenitors(MPP) should have a significant impact not only on theontology of the pancreas, but also for the translationalresearch of glucose-responding endocrine b-cells.Deficiency of the latter may lead to the pandemic type1 or type 2 diabetes mellitus, a metabolic disorder.An ideal treatment of which would potentially be thereplacement of destroyed or failed b-cells, by restoringfunction of endogenous pancreatic endocrine cells orby transplantation of donor islets or in vitro generatedinsulin-secreting cells. Thus, considerable research effortshave been devoted to identify MPP candidates in the preandpost-natal pancreas for the endogenous neogenesisor regeneration of endocrine insulin-secreting cells. Inorder to advance this inconclusive but critical field, wehere review the emerging concepts, recent literatureand newest developments of potential MPP and proposemeasures that would assist its forward progression.

  10. Calcification of multipotent prostate tumor endothelium.

    Science.gov (United States)

    Dudley, Andrew C; Khan, Zia A; Shih, Shou-Ching; Kang, Soo-Young; Zwaans, Bernadette M M; Bischoff, Joyce; Klagsbrun, Michael

    2008-09-01

    Solid tumors require new blood vessels for growth and metastasis, yet the biology of tumor-specific endothelial cells is poorly understood. We have isolated tumor endothelial cells from mice that spontaneously develop prostate tumors. Clonal populations of tumor endothelial cells expressed hematopoietic and mesenchymal stem cell markers and differentiated to form cartilage- and bone-like tissues. Chondrogenic differentiation was accompanied by an upregulation of cartilage-specific col2a1 and sox9, whereas osteocalcin and the metastasis marker osteopontin were upregulated during osteogenic differentiation. In human and mouse prostate tumors, ectopic vascular calcification was predominately luminal and colocalized with the endothelial marker CD31. Thus, prostate tumor endothelial cells are atypically multipotent and can undergo a mesenchymal-like transition.

  11. Caenorhabditis elegans MES-3 is a target of GLD-1 and functions epigenetically in germline development.

    Science.gov (United States)

    Xu, L; Paulsen, J; Yoo, Y; Goodwin, E B; Strome, S

    2001-11-01

    The maternal-effect sterile (MES) proteins are maternally supplied regulators of germline development in Caenorhabditis elegans. In the hermaphrodite progeny from mes mutant mothers, the germline dies during larval development. On the basis of the similarities of MES-2 and MES-6 to known transcriptional regulators and on the basis of the effects of mes mutations on transgene expression in the germline, the MES proteins are predicted to be transcriptional repressors. One of the MES proteins, MES-3, is a novel protein with no recognizable motifs. In this article we show that MES-3 is localized in the nuclei of embryos and germ cells, consistent with its predicted role in transcriptional regulation. Its distribution in the germline and in early embryos does not depend on the wild-type functions of the other MES proteins. However, its nuclear localization in midstage embryos and its persistence in the primordial germ cells depend on wild-type MES-2 and MES-6. These results are consistent with biochemical data showing that MES-2, MES-3, and MES-6 associate in a complex in embryos. The distribution of MES-3 in the adult germline is regulated by the translational repressor GLD-1: MES-3 is absent from the region of the germline where GLD-1 is known to be present, MES-3 is overexpressed in the germline of gld-1 mutants, and GLD-1 specifically binds the mes-3 3' untranslated region (3' UTR). Analysis of temperature-shifted mes-3(bn21ts) worms and embryos indicates that MES-3 function is required in the mother's germline and during embryogenesis to ensure subsequent normal germline development. We propose that MES-3 acts epigenetically to induce a germline state that is inherited through both meiosis and mitosis and that is essential for survival of the germline.

  12. Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering.

    Science.gov (United States)

    Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki; Hamada, Takashi; Ono, Yohei; Nakashima, Motoshige; Ishizuka, Shinya; Matsukawa, Tetsuya; Yamashita, Satoshi; Tsuchiya, Saho; Ishiguro, Naoki

    2016-10-21

    The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Lessons for Inductive Germline Determination

    Science.gov (United States)

    Seervai, Riyad N.H.; Wessel, Gary M.

    2015-01-01

    SUMMARY Formation of the germline in an embryo marks a fresh round of reproductive potential, yet the developmental stage and location within the embryo where the primordial germ cells (PGCs) form differs wildly among species. In most animals, the germline is formed either by an inherited mechanism, in which maternal provisions within the oocyte drive localized germ-cell fate once acquired in the embryo, or an inductive mechanism that involves signaling between cells that directs germ-cell fate. The inherited mechanism has been widely studied in model organisms such as Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, and Danio rerio. Given the rapid generation time and the effective adaptation for laboratory research of these organisms, it is not coincidental that research on these organisms has led the field in elucidating mechanisms for germline specification. The inductive mechanism, however, is less well understood and is studied primarily in the mouse (Mus musculus). In this review, we compare and contrast these two fundamental mechanisms for germline determination, beginning with the key molecular determinants that play a role in the formation of germ cells across all animal taxa. We next explore the current understanding of the inductive mechanism of germ-cell determination in mice, and evaluate the hypotheses for selective pressures on these contrasting mechanisms. We then discuss the hypothesis that the transition between these determination mechanisms, which has happened many times in phylogeny, is more of a continuum than a binary change. Finally, we propose an analogy between germline determination and sex determination in vertebrates—two of the milestones of reproduction and development—in which animals use contrasting strategies to activate similar pathways. PMID:23450642

  14. Lessons for inductive germline determination.

    Science.gov (United States)

    Seervai, Riyad N H; Wessel, Gary M

    2013-08-01

    Formation of the germline in an embryo marks a fresh round of reproductive potential, yet the developmental stage and location within the embryo where the primordial germ cells (PGCs) form differs wildly among species. In most animals, the germline is formed either by an inherited mechanism, in which maternal provisions within the oocyte drive localized germ-cell fate once acquired in the embryo, or an inductive mechanism that involves signaling between cells that directs germ-cell fate. The inherited mechanism has been widely studied in model organisms such as Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, and Danio rerio. Given the rapid generation time and the effective adaptation for laboratory research of these organisms, it is not coincidental that research on these organisms has led the field in elucidating mechanisms for germline specification. The inductive mechanism, however, is less well understood and is studied primarily in the mouse (Mus musculus). In this review, we compare and contrast these two fundamental mechanisms for germline determination, beginning with the key molecular determinants that play a role in the formation of germ cells across all animal taxa. We next explore the current understanding of the inductive mechanism of germ-cell determination in mice, and evaluate the hypotheses for selective pressures on these contrasting mechanisms. We then discuss the hypothesis that the transition between these determination mechanisms, which has happened many times in phylogeny, is more of a continuum than a binary change. Finally, we propose an analogy between germline determination and sex determination in vertebrates-two of the milestones of reproduction and development-in which animals use contrasting strategies to activate similar pathways.

  15. Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs.

    Science.gov (United States)

    McEwen, Tamara J; Yao, Qiuming; Yun, Sijung; Lee, Chin-Yung; Bennett, Karen L

    2016-10-15

    Over four hundred different microRNAs (miRNAs) have been identified in the genome of the model organism the nematode Caenorhabditis elegans. As the germline is dedicated to the preservation of each species, and almost half of all the cells in an adult nematode are germline, it is likely that regulatory miRNAs are important for germline development and maintenance. In C. elegans the miR35 family has strong maternal effects, contributing to normal embryogenesis and to adult fecundity. To determine whether any particular miRNAs are greatly enriched in the C. elegans germline we used RNA-seq to compare the miRNA populations in several germline-defective strains of adult C. elegans worms, including glp-4(germline proliferation-4), glh-1(germline helicase-1) and dcr-1(dicer-1). Statistical analyses of RNA-seq comparisons identified 13 miRNAs that are germline-enriched, including seven members of the well-studied miR35 family that were reduced as much as 1000-fold in TaqMan qRT PCR miRNA assays. Along with the miR35s, six others: miR-56 (a member of the miR51 family),-70, -244, -260 , -788 and -4813, none of which previously considered as such, were also identified by RNA-seq as germline-enriched candidates. We went on to develop a successful miRNA in situ hybridization protocol for C. elegans, revealing miR35s specifically concentrate during oogenesis in the pachytene region of the gonad, and persist throughout early embryogenesis, while in adult animals neither let-7 nor miR-228 has a germline-bias.

  16. Whole-genome sequencing of spermatocytic tumors provides insights into the mutational processes operating in the male germline

    DEFF Research Database (Denmark)

    Giannoulatou, Eleni; Maher, Geoffrey J; Ding, Zhihao

    2017-01-01

    Adult male germline stem cells (spermatogonia) proliferate by mitosis and, after puberty, generate spermatocytes that undertake meiosis to produce haploid spermatozoa. Germ cells are under evolutionary constraint to curtail mutations and maintain genome integrity. Despite constant turnover...

  17. Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Eric F Joyce

    Full Text Available Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.

  18. Lgr5 Marks Neural Crest Derived Multipotent Oral Stromal Stem Cells.

    Science.gov (United States)

    Boddupally, Keerthi; Wang, Guangfang; Chen, Yibu; Kobielak, Agnieszka

    2016-03-01

    It has been suggested that multipotent stem cells with neural crest (NC) origin persist into adulthood in oral mucosa. However their exact localization and role in normal homeostasis is unknown. In this study, we discovered that Lgr5 is expressed in NC cells during embryonic development, which give rise to the dormant stem cells in the adult tongue and oral mucosa. Those Lgr5 positive oral stromal stem cells display properties of NC stem cells including clonal growth and multipotent differentiation. RNA sequencing revealed that adult Lgr5+ oral stromal stem cells express high number of neural crest related markers like Sox9, Twist1, Snai1, Myc, Ets1, Crabp1, Epha2, and Itgb1. Using lineage-tracing experiments, we show that these cells persist more than a year in the ventral tongue and some areas of the oral mucosa and give rise to stromal progeny. In vivo transplantation demonstrated that these cells reconstitute the stroma. Our studies show for the first time that Lgr5 is expressed in the NC cells at embryonic day 9.5 (E9.5) and is maintained during embryonic development and postnataly in the stroma of the ventral tongue, and some areas of the oral mucosa and that Lgr5+ cells participate in the maintenance of the stroma.

  19. Cartilage Regeneration by Chondrogenic Induced Adult Stem Cells in Osteoarthritic Sheep Model: e98770

    National Research Council Canada - National Science Library

    Chinedu C Ude; Shamsul B Sulaiman; Ng Min-Hwei; Chen Hui-Cheng; Johan Ahmad; Norhamdan M Yahaya; Aminuddin B Saim; Ruszymah B H Idrus

    2014-01-01

    ...), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model...

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  20. Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation

    Directory of Open Access Journals (Sweden)

    S Dimitrova-Nakov

    2012-05-01

    Full Text Available Upon in vitro induction or in vivo implantation, the stem cells of the dental pulp display hallmarks of odontoblastic, osteogenic, adipogenic or neuronal cells. However, whether these phenotypes result from genuine multipotent cells or from coexistence of distinct progenitors is still an open question. Furthermore, determining whether a single cell-derived progenitor is capable of undergoing a differentiation cascade leading to tissue repair in situ is important for the development of cell therapy strategies. Three clonal pulp precursor cell lines (A4, C5, H8, established from embryonic ED18 first molars of mouse transgenic for a recombinant plasmid adeno-SV40, were induced to differentiate towards the odonto/osteogenic, chondrogenic or adipogenic programme. Expression of phenotypic markers of each lineage was evaluated by RT-PCR, histochemistry or immunocytochemistry. The clones were implanted into mandibular incisors or calvaria of adult mice. The A4 clone was capable of being recruited towards at least 3 mesodermal lineages in vitro and of contributing to dentin-like or bone formation, in vivo, thus behaving as a multipotent cell. In contrast, the C5 and H8 clones displayed a more restricted potential. Flow cytometric analysis revealed that isolated monopotent and multipotent clones could be distinguished by a differential expression of CD90. Altogether, isolation of these clonal lines allowed demonstrating the coexistence of multipotential and restricted-lineage progenitors in the mouse pulp. These cells may further permit unravelling specificities of the different types of pulp progenitors, hence facilitating the development of cell-based therapies of the dental pulp or other cranio-facial tissues.

  1. IN VITRO PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM A BULGE REGION OF WHISKER FOLLICLE

    Directory of Open Access Journals (Sweden)

    R. G. Vasyliev

    2014-08-01

    Full Text Available A culture method for multipotent neural crest-derived stem cell isolated from the bulge region of the hair follicle of whisker pad of adult mice has been described and their biological properties have been studied. It was shown that the cells possess a fibroblast-like morphology, they are nestin-positive and cytokeratin-negative, and also express the following surface markers: CD44, CD73, CD90 and Sca-1. This cell type shows the functional properties of stem cells in culture: clonogenicity, self-renewal, sphere-forming capacity and the ability to the directed multilineage differentiation. Due to these properties, neural crest-derived multipotent stem cells are promising for application in the regenerative medicine

  2. CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

    Science.gov (United States)

    Campbell, Anne C; Updike, Dustin L

    2015-05-15

    Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription.

  3. Characteristics and multipotency of equine dedifferentiated fat cells.

    Science.gov (United States)

    Murata, Daiki; Yamasaki, Atsushi; Matsuzaki, Shouta; Sunaga, Takafumi; Fujiki, Makoto; Tokunaga, Satoshi; Misumi, Kazuhiro

    2016-01-01

    Dedifferentiated fat (DFAT) cells have been shown to be multipotent, similar to mesenchymal stem cells (MSCs). In this study, we aimed to establish and characterize equine DFAT cells. Equine adipocytes were ceiling cultured, and then dedifferentiated into DFAT cells by the seventh day of culture. The number of DFAT cells was increased to over 10 million by the fourth passage. Flow cytometry of DFAT cells showed that the cells were strongly positive for CD44, CD90, and major histocompatibility complex (MHC) class I; moderately positive for CD11a/18, CD105, and MHC class II; and negative for CD34 and CD45. Moreover, DFAT cells were positive for the expression of sex determining region Y-box 2 as a marker of multipotency. Finally, we found that DFAT cells could differentiate into osteogenic, chondrogenic, and adipogenic lineages under specific nutrient conditions. Thus, DFAT cells could have clinical applications in tissue regeneration, similar to MSCs derived from adipose tissue.

  4. Epigenetic modifications and self-renewal regulation of mouse germline stem cells

    Institute of Scientific and Technical Information of China (English)

    Jiyoung Lee; Takashi Shinohara

    2011-01-01

    Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.

  5. Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

    Science.gov (United States)

    Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

    2015-01-01

    The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in

  6. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    Science.gov (United States)

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  7. Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes

    DEFF Research Database (Denmark)

    Elabd, Christian; Chiellini, Chiara; Carmona, Mamen

    2009-01-01

    adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to beta......In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent......-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one...

  8. Germline modification of domestic animals.

    Science.gov (United States)

    Tang, L; González, R; Dobrinski, I

    2015-01-01

    Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change

  9. Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

    DEFF Research Database (Denmark)

    Ritz, Caitlin; Ruminski, Wojciech; Hougaard, Karin S.

    2011-01-01

    (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates...... and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females...

  10. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  11. Achieving immortality in the C. elegans germline.

    Science.gov (United States)

    Smelick, Chris; Ahmed, Shawn

    2005-01-01

    Germline immortality is a topic that has intrigued theoretical biologists interested in aging for over a century. The germ cell lineage can be passed from one generation to the next, indefinitely. In contrast, somatic cells are typically only needed for a single generation and are then discarded. Germ cells may, therefore, harbor rejuvenation mechanisms that enable them to proliferate for eons. Such processes are thought to be either absent from or down-regulated in somatic cells, although cell non-autonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that foster eternal youth in germ cells is lacking. The mysteries of germline immortality are being addressed in the nematode Caenorhabditis elegans by studying mutants that reproduce normally for several generations but eventually become sterile. The mortal germline mutants probably become sterile as a consequence of accumulating various forms of heritable cellular damage. Such mutants are abundant, indicating that several different biochemical pathways are required to rejuvenate the germline. Thus, forward genetics should help to define mechanisms that enable the germline to achieve immortality.

  12. Multipotent adult progenitor cell conditions enhance human adipose-derived stem cells’ ability to protect retinal neurons%多能成体祖细胞培养条件促进人脂肪干细胞的视网膜神经保护功能

    Institute of Scientific and Technical Information of China (English)

    娄慧; 徐国旭

    2014-01-01

    目的:检测多能成体祖细胞(MAPC)的培养条件对猴骨髓间充质细胞(BMMSCs)和人脂肪干细胞(hADSCs)生长的影响,旨在获得更适合治疗视网膜变性疾病的供体细胞。方法通过细胞形态观察、MTT实验、克隆形成率、PCR检测、以及成脂、成骨、成软骨分化潜能检测等,研究MAPC培养条件下猴BMMSCs和hADSCs的特征,并用DMEM/LG和MAPC培养条件培养的hADSCs进行RCS大鼠视网膜下腔移植,通过视网膜电图(ERG)和TUNEL检测,判断细胞移植治疗对视功能及视网膜细胞凋亡的影响。结果与常规培养基相比,MAPC培养条件能促进猴BMMSCs增殖,细胞变小,但传2代后,细胞变得宽大扁平,出现衰老征象;然而,MAPC培养条件下的hADSCs细胞增殖能力及克隆形成率均增强,形成的克隆较大可稳定传10代以上,且具有成脂、成骨、成软骨的多向分化潜能,细胞表面标记物及细胞因子出现差异表达:CD140b、CD90、CD47、HGF和PEDF显著上调,CD73、CD105和IL-6显著下调。与对照组相比,移植DMEM/LG和MAPC培养条件培养的hADSCs(P4)3周后,RCS大鼠的B波波幅明显升高,外核层细胞凋亡明显减少。结论 MAPC培养条件培养的hADSCs显示出更好的视网膜神经保护作用,适合用于治疗视网膜退行性疾病。%Objective To investigate the effects of multipotent adult progenitor cell (MAPC) conditions on biological characteristics and therapeutic effects of monkey bone marrow mesenchymal stem cells (BMMSCs) and human adipose-derived stem cells (hADSCs), thus to obtain optimal donor cells for treatment of retinal degeneration (RD). Methods The cell morphology, MTT assay, clone formation, adipogenic, osteogenic, chondrogenic differentiation potential and qRT-PCR technique were used to detect biological characteristics of monkey BMMSCs and hADSCs. The hADSCs were cultured under MAPC condition medium and transplanted into

  13. Both asymmetric mitotic segregation and cell-to-cell invasion are required for stable germline transmission of Wolbachia in filarial nematodes

    Directory of Open Access Journals (Sweden)

    Frédéric Landmann

    2012-04-01

    Parasitic filarial nematodes that belong to the Onchocercidae family live in mutualism with Wolbachia endosymbionts. We developed whole-mount techniques to follow the segregation patterns of Wolbachia through the somatic and germline lineages of four filarial species. These studies reveal multiple evolutionarily conserved mechanisms that are required for Wolbachia localization to the germline. During the initial embryonic divisions, Wolbachia segregate asymmetrically such that they concentrate in the posteriorly localized P2 blastomere, a precursor to the adult germline and hypodermal lineages. Surprisingly, in the next division they are excluded from the germline precursor lineage. Rather, they preferentially segregate to the C blastomere, a source of posterior hypodermal cells. Localization to the germline is accomplished by a distinct mechanism in which Wolbachia invade first the somatic gonadal cells close to the ovarian distal tip cell, the nematode stem cell niche, from the hypodermis. This tropism is associated with a cortical F-actin disruption, suggesting an active engulfment. Significantly, germline invasion occurs only in females, explaining the lack of Wolbachia in the male germline. Once in the syncytial environment of the ovaries, Wolbachia rely on the rachis to multiply and disperse into the germ cells. The utilization of cell-to-cell invasion for germline colonization may indicate an ancestral mode of horizontal transfer that preceded the acquisition of the mutualism.

  14. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

    Directory of Open Access Journals (Sweden)

    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  15. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans.

    Science.gov (United States)

    Andralojc, Karolina M; Campbell, Anne C; Kelly, Ashley L; Terrey, Markus; Tanner, Paige C; Gans, Ian M; Senter-Zapata, Michael J; Khokhar, Eraj S; Updike, Dustin L

    2017-02-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.

  16. File list: NoD.Oth.50.AllAg.Multipotent_otic_progenitor [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: InP.Oth.10.AllAg.Multipotent_otic_progenitor [Chip-atlas[Archive

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  4. Autologous Germline Mitochondrial Energy Transfer (AUGMENT) in Human Assisted Reproduction.

    Science.gov (United States)

    Woods, Dori C; Tilly, Jonathan L

    2015-11-01

    Ovarian aging is characterized by a decline in both the total number and overall quality of oocytes, the latter of which has been experimentally tied to mitochondrial dysfunction. Clinical studies in the late 1990s demonstrated that transfer of cytoplasm aspirated from eggs of young female donors into eggs of infertile women at the time of intracytoplasmic sperm injection improved pregnancy success rates. However, donor mitochondria were identified in offspring, and the United States Food and Drug Administration raised questions about delivery of foreign genetic material into human eggs at the time of fertilization. Accordingly, heterologous cytoplasmic transfer, while promising, was in effect shut down as a clinical protocol. The recent discovery of adult oogonial (oocyte-generating) stem cells in mice, and subsequently in women, has since re-opened the prospects of delivering a rich source of pristine and patient-matched germline mitochondria to boost egg health and embryonic developmental potential without the need for young donor eggs to obtain cytoplasm. Herein we overview the science behind this new protocol, which has been patented and termed autologous germline mitochondrial energy transfer, and its use to date in clinical studies for improving pregnancy success in women with a prior history of assisted reproduction failure.

  5. Epigenetic inheritance and plasticity: The responsive germline.

    Science.gov (United States)

    Jablonka, Eva

    2013-04-01

    Developmental plasticity, the capacity of a single genotype to give rise to different phenotypes, affects evolutionary dynamics by influencing the rate and direction of phenotypic change. It is based on regulatory changes in gene expression and gene products, which are partially controlled by epigenetic mechanisms. Plasticity involves not just epigenetic changes in somatic cells and tissues; it can also involve changes in germline cells. Germline epigenetic plasticity increases evolvability, the capacity to generate heritable, selectable, phenotypic variations, including variations that lead to novel functions. I discuss studies that show that some complex adaptive responses to new challenges are mediated by germline epigenetic processes, which can be transmitted over variable number of generations, and argue that the heritable variations that are generated epigenetically have an impact on both small-scale and large-scale aspects of evolution. First, I review some recent ecological studies and models that show that germline (gametic) epigenetic inheritance can lead to cumulative micro-evolutionary changes that are rapid and semi-directional. I suggest that "priming" and "epigenetic learning" may be of special importance in generating heritable, fine-tuned adaptive responses in populations. Second, I consider work showing how genomic and environmental stresses can also lead to epigenome repatterning, and produce changes that are saltational.

  6. Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

    Science.gov (United States)

    Centanin, Lázaro; Ander, Janina-J; Hoeckendorf, Burkhard; Lust, Katharina; Kellner, Tanja; Kraemer, Isabel; Urbany, Cedric; Hasel, Eva; Harris, William A; Simons, Benjamin D; Wittbrodt, Joachim

    2014-09-01

    The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.

  7. Identification of a distinct small cell population from human bone marrow reveals its multipotency in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    James Wang

    Full Text Available Small stem cells, such as spore-like cells, blastomere-like stem cells (BLSCs, and very-small embryonic-like stem cells (VSELs have been described in recent studies, although their multipotency in human tissues has not yet been confirmed. Here, we report the discovery of adult multipotent stem cells derived from human bone marrow, which we call StemBios (SB cells. These isolated SB cells are smaller than 6 ìm and are DAPI+ and Lgr5+ (Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5. Because Lgr5 has been characterized as a stem cell marker in the intestine, we hypothesized that SB cells may have a similar function. In vivo cell tracking assays confirmed that SB cells give rise to three types of cells, and in vitro studies demonstrated that SB cells cultured in proprietary media are able to grow to 6-25 ìm in size. Once the SB cells have attached to the wells, they differentiate into different cell lineages upon exposure to specific differentiation media. We are the first to demonstrate that stem cells smaller than 6 ìm can differentiate both in vivo and in vitro. In the future, we hope that SB cells will be used therapeutically to cure degenerative diseases.

  8. Dorsomorphin promotes survival and germline competence of zebrafish spermatogonial stem cells in culture.

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    Ten-Tsao Wong

    Full Text Available Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo;Tg(piwil1:DsRed transgenic fish using a zebrafish ovarian feeder cell line (OFC3 that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.

  9. Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.

    Science.gov (United States)

    Seidel, Hannah S; Kimble, Judith

    2015-11-09

    Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells--including germline stem cells--become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions--GLP-1/Notch signaling--becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance.

  10. Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver.

    Directory of Open Access Journals (Sweden)

    Reto M Baertschiger

    Full Text Available Multipotent mesenchymal stromal cells (MSC are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments when compared to adult MSC (2/10 experiments. However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an

  11. Multipotent natural agents to combat Alzheimer's disease. Functional spectrum and structural features

    Institute of Scientific and Technical Information of China (English)

    Hong-fang JI; Hong-yu ZHANG

    2008-01-01

    With the accelerated aging of human society, Alzheimer's disease (AD) is becoming one of the biggest threats to human health. Since multiple pathogenetic factors are implicated in the disease, the current hitting-one-target therapeutic strategy has proved inefficient to AD. As a result, finding multipotent agents that aim at multiple targets is attracting more and more attention. Although multifunc-tional anti-AD agents can be created by incorporating two or more pharmacophores in one scaffold, naturally occurring multipotent agents also attracted much attention. In this review, we first describe the functions of some typical naturally originated multipotent anti-AD compounds, then summarize their structural fea-tures and reveal that phenolics with certain flexibility predominate in these agents, which are of significance to find novel multipotent drugs to combat AD and other neurodegenerative diseases as well.

  12. Angiogenic Potential of Multipotent Stromal Cells from the Umbilical Cord: an In Vitro Study.

    Science.gov (United States)

    Arutyunyan, I V; Kananykhina, E Yu; Fatkhudinov, T Kh; El'chaninov, A V; Makarov, A V; Raimova, E Sh; Bol'shakova, G B; Sukhikh, G T

    2016-05-01

    The mechanisms of proangiogenic activity of multipotent stromal cells from human umbilical cord were analyzed in vitro. The absence of secreted forms of proangiogenic growth factor VEGF-A in the culture medium conditioned by umbilical cord-derived multipotent stromal cells was shown by ELISA. However, the possibility of paracrine stimulation of cell proliferation, mobility, and directed migration of endothelial EA.hy926 cells was demonstrated by using MTT test, Transwell system, and monolayer wound modeling. The capacity of multipotent stromal cells to acquire the phenotype of endothelium-like cells was analyzed using differentiation media of three types. It was found that VEGF-A is an essential but not sufficient inductor of differentiation of umbilical cord-derived multipotent stromal cells into CD31+ cells.

  13. Identification of Multipotent Progenitors that Emerge Prior to Hematopoietic Stem Cells in Embryonic Development

    Directory of Open Access Journals (Sweden)

    Matthew A. Inlay

    2014-04-01

    Full Text Available Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS than the aorta-gonad-mesonephros (AGM or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.

  14. Harvesting multipotent progenitor cells from a small sample of tonsillar biopsy for clinical applications.

    Science.gov (United States)

    Khatri, Raju; Arad, Michal; Ortlip, Timothy; Portney, Benjamin A; Meltzer, W Alex; Diaconu, Silviu; Silipino, Lorna E; Wang, Ying; Kaetzel, David M; Taylor, Rodney J; Zalzman, Michal

    2017-07-27

    Human adult stem cells hold the potential for the cure of numerous conditions and degenerative diseases. They possess major advantages over pluripotent stem cells as they can be derived from donors at any age, and therefore pose no ethical concerns or risk of teratoma tumor formation in vivo. Furthermore, they have a natural ability to differentiate and secrete factors that promote tissue healing without genetic manipulation. However, at present, clinical applications of adult stem cells are limited by a shortage of a reliable, standardized, and easily accessible tissue source which does not rely on specimens discarded from unrelated surgical procedures. Human tonsil-derived mesenchymal progenitor cells (MPCs) were isolated from a small sample of tonsillar tissue (average 0.88 cm(3)). Our novel procedure poses a minimal mechanical and enzymatic insult to the tissue, and therefore leads to high cell viability and yield. We characterized these MPCs and demonstrated robust multipotency in vitro. We further show that these cells can be propagated and maintained in xeno-free conditions. We have generated tonsillar biopsy-derived MPC (T-MPC) lines from multiple donors across a spectrum of age, sex, and race, and successfully expanded them in culture. We characterized them by cell surface markers, as well as in vitro expansion and differentiation potential. Our procedure provides a robust yield of tonsillar biopsy-derived T-MPCs. Millions of MPCs can be harvested from a sample smaller than 1 g, which can be collected from a fully awake donor in an outpatient setting without the need for general anesthesia or hospitalization. Our study identifies tonsillar biopsy as an abundant source of adult MPCs for regenerative medicine.

  15. Monitoring the Bystander Killing Effect of Human Multipotent Stem Cells for Treatment of Malignant Brain Tumors

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    Cindy Leten

    2016-01-01

    Full Text Available Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683 in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI and magnetic resonance imaging (MRI. Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1 outliers can be detected earlier, (2 GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3 a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.

  16. Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.

    Science.gov (United States)

    Wystrychowski, Wojciech; Patlolla, Bhagat; Zhuge, Yan; Neofytou, Evgenios; Robbins, Robert C; Beygui, Ramin E

    2016-06-13

    Acute myocardial infarction (MI) leads to an irreversible loss of proper cardiac function. Application of stem cell therapy is an attractive option for MI treatment. Adipose tissue has proven to serve as a rich source of stem cells (ADSCs). Taking into account the different morphogenesis, anatomy, and physiology of adipose tissue, we hypothesized that ADSCs from different adipose tissue depots may exert a diverse multipotency and cardiogenic potential. The omental, pericardial, and epicardial adipose tissue samples were obtained from organ donors and patients undergoing heart transplantation at our institution. Human foreskin fibroblasts were used as the control group. Isolated ADSCs were analyzed for adipogenic and osteogenic differentiation capacity and proliferation potential. The immunophenotype and constitutive gene expression of alkaline phosphatase (ALP), GATA4, Nanog, and OCT4 were analyzed. DNA methylation inhibitor 5-azacytidine was exposed to the cells to stimulate the cardiogenesis. Finally, reprogramming towards cardiomyocytes was initiated with exogenous overexpression of seven transcription factors (ESRRG, GATA4, MEF2C, MESP1, MYOCD, TBX5, ZFPM2) previously applied successfully for fibroblast transdifferentiation toward cardiomyocytes. Expression of cardiac troponin T (cTNT) and alpha-actinin (Actn2) was analyzed 3 weeks after initiation of the cardiac differentiation. The multipotent properties of isolated plastic adherent cells were confirmed with expression of CD29, CD44, CD90, and CD105, as well as successful differentiation toward adipocytes and osteocytes; with the highest osteogenic and adipogenic potential for the epicardial and omental ADSCs, respectively. Epicardial ADSCs demonstrated a lower doubling time as compared with the pericardium and omentum-derived cells. Furthermore, epicardial ADSCs revealed higher constitutive expression of ALP and GATA4. Increased Actn2 and cTNT expression was observed after the transduction of seven

  17. Germline genome-editing research and its socioethical implications.

    Science.gov (United States)

    Ishii, Tetsuya

    2015-08-01

    Genetically modifying eggs, sperm, and zygotes ('germline' modification) can impact on the entire body of the resulting individual and on subsequent generations. With the advent of genome-editing technology, human germline gene modification is no longer theoretical. Owing to increasing concerns about human germline gene modification, a voluntary moratorium on human genome-editing research and/or the clinical application of human germline genome editing has recently been called for. However, whether such research should be suspended or encouraged warrants careful consideration. The present article reviews recent research on mammalian germline genome editing, discusses the importance of public dialogue on the socioethical implications of human germline genome-editing research, and considers the relevant guidelines and legislation in different countries.

  18. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

    NARCIS (Netherlands)

    C.E. Wong (Christine); S. Paratore (Sabrina); M.T. Dours-Zimmermann (María); T. Rochat (Thierry); T. Pietri (Thomas); U. Suter (Ueli); D. Zimmermann (Dieter); S. Dufour (Sylvie); J.P. Thiery (Joachim); D.N. Meijer (Dies); C. Beermann (Christopher); Y. Barrandon (Yann); L. Sommer (Lukas)

    2006-01-01

    textabstractGiven their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and

  19. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

    NARCIS (Netherlands)

    C.E. Wong (Christine); S. Paratore (Sabrina); M.T. Dours-Zimmermann (María); T. Rochat (Thierry); T. Pietri (Thomas); U. Suter (Ueli); D. Zimmermann (Dieter); S. Dufour (Sylvie); J.P. Thiery (Joachim); D.N. Meijer (Dies); C. Beermann (Christopher); Y. Barrandon (Yann); L. Sommer (Lukas)

    2006-01-01

    textabstractGiven their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and di

  20. The molecular anatomy of spontaneous germline mutations in human testes.

    Science.gov (United States)

    Qin, Jian; Calabrese, Peter; Tiemann-Boege, Irene; Shinde, Deepali Narendra; Yoon, Song-Ro; Gelfand, David; Bauer, Keith; Arnheim, Norman

    2007-09-01

    The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3) to >10(4) times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6)) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an

  1. The molecular anatomy of spontaneous germline mutations in human testes.

    Directory of Open Access Journals (Sweden)

    Jian Qin

    2007-09-01

    Full Text Available The frequency of the most common sporadic Apert syndrome mutation (C755G in the human fibroblast growth factor receptor 2 gene (FGFR2 is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3 to >10(4 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6 the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model. This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation

  2. Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

    DEFF Research Database (Denmark)

    Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole;

    2009-01-01

    Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... that tumorigenic transformation of hMSC-TERT20 cells induced the expression of members of several cancer-germline antigen gene families (ie, GAGE, MAGE-A, and XAGE-1), with promoter hypomethylation and histone acetylation of the corresponding genes. Both in vitro cultures and tumor xenografts derived from...

  3. Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates

    Directory of Open Access Journals (Sweden)

    Mohammad Moad

    2017-08-01

    Full Text Available Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1 enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

  4. Derivation of multipotent progenitors from human circulating CD14+ monocytes.

    Science.gov (United States)

    Seta, Noriyuki; Kuwana, Masataka

    2010-07-01

    Circulating CD14(+) monocytes are originated from hematopoietic stem cells in the bone marrow and believed to be committed precursors for phagocytes, such as macrophages. Recently, we have reported a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14(+) monocytes, but circulating precursors for MOMCs still remain undetermined. Comparative analysis of gene expression profiles of MOMCs and other monocyte-derived cells has revealed that embryonic stem cell markers, Nanog and Oct-4, are specifically expressed by MOMCs. In vitro generation of MOMCs requires binding to fibronectin and exposure to soluble factors derived from activated platelets. MOMCs contain progenitors with capacity to differentiate into a variety of nonphagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium, indicating that circulating monocytes are more multipotent than previously thought. In addition, MOMCs are capable of promoting ex vivo expansion of human hematopoietic progenitor cells through direct cell-to-cell contact and secretion of a variety of hematopoietic growth factors. These findings obtained from the research on MOMCs indicate that CD14(+) monocytes in circulation are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue.

  5. Translational control in germline stem cell development.

    Science.gov (United States)

    Slaidina, Maija; Lehmann, Ruth

    2014-10-13

    Stem cells give rise to tissues and organs during development and maintain their integrity during adulthood. They have the potential to self-renew or differentiate at each division. To ensure proper organ growth and homeostasis, self-renewal versus differentiation decisions need to be tightly controlled. Systematic genetic studies in Drosophila melanogaster are revealing extensive regulatory networks that control the switch between stem cell self-renewal and differentiation in the germline. These networks, which are based primarily on mutual translational repression, act via interlocked feedback loops to provide robustness to this important fate decision.

  6. Translational activation maintains germline tissue homeostasis during adulthood.

    Science.gov (United States)

    Nousch, Marco; Eckmann, Christian R

    2015-01-01

    Adult tissue maintenance is achieved through a tightly controlled equilibrium of 2 opposing cell fates: stem cell proliferation and differentiation. In recent years, the germ line emerged as a powerful in vivo model tissue to investigate the underlying gene expression mechanisms regulating this balance. Studies in numerous organisms highlighted the prevalence of post-transcriptional mRNA regulation, which relies on RNA-targeting factors that influence mRNA fates (e.g. decay or translational efficiency). Conserved translational repressors were identified that build negative feedback loops to ensure one or the other cell fate. However, to facilitate a fast and efficient transition between 2 opposing cell fates, translational repression per se appears not to be sufficient, suggesting the involvement of additional modes of gene expression regulation. Cytoplasmic poly(A) polymerases (cytoPAPs) represent a unique class of post-transcriptional mRNA regulators that modify mRNA 3' ends and positively influence cytoplasmic mRNA fates. We recently discovered that the 2 main cytoPAPs, GLD-2 and GLD-4, use distinct mechanisms to promote gene expression and that cytoPAP-mediated mRNA activation is important for regulating the size of the proliferative germ cell pool in the adult Caenorhabditis elegans gonad. Here, we comment on the different mechanisms of the 2 cytoPAPs as translational activators in germ cell development and focus on their biological roles in maintaining the balance between germline stem cell proliferation and differentiation in the Caenorhabditis elegans gonad.

  7. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

    Science.gov (United States)

    Andralojc, Karolina M.; Kelly, Ashley L.; Tanner, Paige C.

    2017-01-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline. PMID:28182654

  8. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Karolina M Andralojc

    2017-02-01

    Full Text Available Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3. The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules. ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.

  9. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Quanwen Liu

    2016-01-01

    Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

  10. Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth.

    Directory of Open Access Journals (Sweden)

    Catherine To

    Full Text Available Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs, but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

  11. Methods in Molecular Biology: Germline Stem Cells | Center for Cancer Research

    Science.gov (United States)

    The protocols in Germline Stem Cells are intended to present selected genetic, molecular, and cellular techniques used in germline stem cell research. The book is divided into two parts. Part I covers germline stem cell identification and regulation in model organisms. Part II covers current techniques used in in vitro culture and applications of germline stem cells.

  12. Germline mosaicism at the fragile X locus

    Energy Technology Data Exchange (ETDEWEB)

    Papp, A.C.; Snyder, P.J.; Sedra, M.S. [Ohio State Univ., Columbus, OH (United States)] [and others

    1994-09-01

    The fragile X full mutation, which is associated with the phenotypic expression of the disorder, is characterized by an expansion of CGG repeat and hypermethylation of the CpG island adjacent to the FMR1 gene. New mutations leading to amplification of the CGG repeat have not been reported. We have identified a fragile X syndrome pedigree where the disorder is associated with a molecular deletion. The deletion was present in the DNA of two affected sons but was absent in the mother`s somatic cell (lymphocyte) DNA. This was confirmed by dosage analysis of the Southern blot using StB12-3 and an additional probe against the dystrophin gene and by PCR analysis of DXS548 alleles. The results are consistent with the deletion arising as a postzygotic event in the mother, who therefore is germinally mosaic. The case reported here clearly demonstrates that FMR1 deletions, unlike the expansions, are not always inherited and the finding of heterozygosity or normal dosage from lymphocyte DNA in the mother of a deletion case does not necessarily rule out the possibility of having a second affected child. The deletion of FMR1 gene may be responsible for a small but significant number of fragile X cases. Therefore, it is imperative that those involved in genetic counseling recognize this diagnostic pitfall. Since it depends upon the size of the mutant clone in the mosaic mother, the exact recurrence risk in germline carriers is unknown. However, prenatal and carrier testing should be performed independently of the outcome of the mother. Furthermore, it is possible that the deletion may not be restricted to the germline, and therefore the mother may actually be a somatic mosaic.

  13. Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates.

    Science.gov (United States)

    Kawamura, Kazuo; Sugino, Yasuo; Sunanaga, Takeshi; Fujiwara, Shigeki

    2008-01-01

    The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.

  14. Identification of germline transcriptional regulatory elements in Aedes aegypti

    Science.gov (United States)

    Akbari, Omar S.; Papathanos, Philippos A.; Sandler, Jeremy E.; Kennedy, Katie; Hay, Bruce A.

    2014-02-01

    The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

  15. Developmental expression of "germline"- and "sex determination"-related genes in the ctenophore Mnemiopsis leidyi.

    Science.gov (United States)

    Reitzel, Adam M; Pang, Kevin; Martindale, Mark Q

    2016-01-01

    An essential developmental pathway in sexually reproducing animals is the specification of germ cells and the differentiation of mature gametes, sperm and oocytes. The "germline" genes vasa, nanos and piwi are commonly identified in primordial germ cells, suggesting a molecular signature for the germline throughout animals. However, these genes are also expressed in a diverse set of somatic stem cells throughout the animal kingdom leaving open significant questions for whether they are required for germline specification. Similarly, members of the Dmrt gene family are essential components regulating sex determination and differentiation in bilaterian animals, but the functions of these transcription factors, including potential roles in sex determination, in early diverging animals remain unknown. The phylogenetic position of ctenophores and the genome sequence of the lobate Mnemiopsis leidyi motivated us to determine the compliment of these gene families in this species and determine expression patterns during development. Our phylogenetic analyses of the vasa, piwi and nanos gene families show that Mnemiopsis has multiple genes in each family with multiple lineage-specific paralogs. Expression domains of Mnemiopsis nanos, vasa and piwi, during embryogenesis from fertilization to the cydippid stage, were diverse, with little overlapping expression and no or little expression in what we think are the germ cells or gametogenic regions. piwi paralogs in Mnemiopsis had distinct expression domains in the ectoderm during development. We observed overlapping expression domains in the apical organ and tentacle apparatus of the cydippid for a subset of "germline genes," which are areas of high cell proliferation, suggesting that these genes are involved with "stem cell" specification and maintenance. Similarly, the five Dmrt genes show diverse non-overlapping expression domains, with no clear evidence for expression in future gametogenic regions of the adult. We also

  16. Clinical Report: Germline Mosaicism in Cornelia de Lange Syndrome

    Science.gov (United States)

    Slavin, Thomas P.; Lazebnik, Noam; Clark, Dinah M.; Vengoechea, Jaime; Cohen, Leslie; Kaur, Maninder; Konczal, Laura; Crowe, Carol A.; Corteville, Jane E.; Nowaczyk, Malgorzata J.; Byrne, Janice L.; Jackson, Laird G.; Krantz, Ian D.

    2012-01-01

    Cornelia de Lange syndrome (CdLS) is a genetic disorder associated with delayed growth, intellectual disability, limb reduction defects and characteristic facial features. Germline mosaicism has been a described mechanism for CdLS when there are several affected offspring of apparently unaffected parents. Presently, the recurrence risk for CdLS has been estimated to be as high as 1.5%; however, this figure may be an underrepresentation. We report on the molecularly defined germline mosaicism cases from a large CdLS database, representing the first large case series on germline mosaicism in CdLS. Of the 12 families, eight have been previously described; however, four have not. No one specific gene mutation, either in the NIPBL or the SMC1A gene, was associated with an increased risk for germline mosaicism. Suspected or confirmed cases of germline mosaicism in our database range from a conservative 3.4% up to 5.4% of our total cohort. In conclusion, the potential reproductive recurrence risk due to germline mosiacism should be addressed in prenatal counseling for all families who have had a previously affected pregnancy or child with CdLS. PMID:22581668

  17. Pharmaceutical induction of ApoE secretion by multipotent mesenchymal stromal cells (MSCs

    Directory of Open Access Journals (Sweden)

    Whitney Mandolin J

    2008-09-01

    Full Text Available Abstract Background Apolipoprotein E (ApoE is a molecular scavenger in the blood and brain. Aberrant function of the molecule causes formation of protein and lipid deposits or "plaques" that characterize Alzheimer's disease (AD and atherosclerosis. There are three human isoforms of ApoE designated ε2, ε3, and ε4. Each isoform differentially affects the structure and function of the protein and thus the development of disease. Homozygosity for ApoE ε4 is associated with atherosclerosis and Alzheimer's disease whereas ApoE ε2 and ε3 tend to be protective. Furthermore, the ε2 form may cause forms of hyperlipoproteinemia. Therefore, introduction of ApoE ε3 may be beneficial to patients that are susceptible to or suffering from these diseases. Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs are adult progenitor cells found in numerous tissues. They are easily expanded in culture and engraft into host tissues when administered appropriately. Furthermore, MSCs are immunosuppressive and have been reported to engraft as allogeneic transplants. In our previous study, mouse MSCs (mMSCs were implanted into the brains of ApoE null mice, resulting in production of small amounts of ApoE in the brain and attenuation of cognitive deficits. Therefore human MSCs (hMSCs are a promising vector for the administration of ApoE ε3 in humans. Results Unlike mMSCs, hMSCs were found not to express ApoE in culture; therefore a molecular screen was performed for compounds that induce expression. PPARγ agonists, neural stem cell conditioned medium, osteo-inductive media, dexamethasone, and adipo-inductive media (AIM were tested. Of the conditions tested, only AIM or dexamethasone induced sustained secretion of ApoE in MSCs and the duration of secretion was only limited by the length of time MSCs could be sustained in culture. Upon withdrawal of the inductive stimuli, the ApoE secretion persisted for a further 14 days. Conclusion The data

  18. L-MYC Expression Maintains Self-Renewal and Prolongs Multipotency of Primary Human Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhongqi Li

    2016-09-01

    Full Text Available Pre-clinical studies indicate that neural stem cells (NSCs can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008 that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI. These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases.

  19. Crestospheres: Long-Term Maintenance of Multipotent, Premigratory Neural Crest Stem Cells

    Directory of Open Access Journals (Sweden)

    Laura Kerosuo

    2015-10-01

    Full Text Available Premigratory neural crest cells comprise a transient, embryonic population that arises within the CNS, but subsequently migrates away and differentiates into many derivatives. Previously, premigratory neural crest could not be maintained in a multipotent, adhesive state without spontaneous differentiation. Here, we report conditions that enable maintenance of neuroepithelial “crestospheres” that self-renew and retain multipotency for weeks. Moreover, under differentiation conditions, these cells can form multiple derivatives in vitro and in vivo after transplantation into chick embryos. Similarly, human embryonic stem cells directed to a neural crest fate can be maintained as crestospheres and subsequently differentiated into several derivatives. By devising conditions that maintain the premigratory state in vitro, these results demonstrate that neuroepithelial neural crest precursors are capable of long-term self-renewal. This approach will help uncover mechanisms underlying their developmental potential, differentiation and, together with the induced pluripotent stem cell techniques, the pathology of human neurocristopathies.

  20. The Characterisation of Pluripotent and Multipotent Stem Cells Using Fourier Transform Infrared Microspectroscopy

    Directory of Open Access Journals (Sweden)

    Mark J. Tobin

    2013-08-01

    Full Text Available Fourier transform infrared (FTIR microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods.

  1. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp

    DEFF Research Database (Denmark)

    Pierdomenico, Laura; Bonsi, Laura; Calvitti, Mario

    2005-01-01

    BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potent...... characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation....

  2. Organotins Are Potent Activators of PPARγ and Adipocyte Differentiation in Bone Marrow Multipotent Mesenchymal Stromal Cells

    OpenAIRE

    2011-01-01

    Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator–activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazo...

  3. Migrating neural crest cells in the trunk of the avian embryo are multipotent

    OpenAIRE

    Fraser, Scott E.; Bronner-Fraser, Marianne

    1991-01-01

    Trunk neural crest cells migrate extensively and give rise to diverse cell types, including cells of the sensory and autonomic nervous systems. Previously, we demonstrated that many premigratory trunk neural crest cells give rise to descendants with distinct phenotypes in multiple neural crest derivatives. The results are consistent with the idea that neural crest cells are multipotent prior to their emigration from the neural tube and become restricted in phenotype after leaving the neural t...

  4. Second Malignant Neoplasms in Patients With Cowden Syndrome With Underlying Germline PTEN Mutations

    Science.gov (United States)

    Ngeow, Joanne; Stanuch, Kim; Mester, Jessica L.; Barnholtz-Sloan, Jill S.; Eng, Charis

    2014-01-01

    Purpose Patients with Cowden syndrome (CS) with underlying germline PTEN mutations are at increased risk of breast, thyroid, endometrial, and renal cancers. To our knowledge, risk of subsequent cancers in these patients has not been previously explored or quantified. Patients and Methods We conducted a 7-year multicenter prospective study (2005 to 2012) of patients with CS or CS-like disease, all of whom underwent comprehensive PTEN mutational analysis. Second malignant neoplasms (SMNs) were ascertained by medical records and confirmed by pathology reports. Standardized incidence ratios (SIRs) for all SMNs combined and for breast, thyroid, endometrial, and renal cancers were calculated. Results Of the 2,912 adult patients included in our analysis, 2,024 had an invasive cancer history. Germline pathogenic PTEN mutations (PTEN mutation positive) were identified in 114 patients (5.6%). Of these 114 patients, 46 (40%) had an SMN. Median age of SMN diagnosis was 50 years (range, 21 to 71 years). Median interval between primary cancer and SMN was 5 years (range, breast cancer, 11 (22%) had a subsequent new primary breast cancer and 10-year second breast cancer cumulative risk of 29% (95% CI, 15.3 to 43.7). Risk of SMNs compared with that of the general population was significantly elevated for all cancers (SIR, 7.74; 95% CI, 5.84 to 10.07), specifically for breast (SIR, 8.92; 95% CI, 5.85 to 13.07), thyroid (SIR, 5.83; 95% CI, 3.01 to 10.18), and endometrial SMNs (SIR, 14.08.07; 95% CI, 7.10 to 27.21). Conclusion Patients with CS with germline PTEN mutations are at higher risk for SMNs compared with the general population. Prophylactic mastectomy should be considered on an individual basis given the significant risk of subsequent breast cancer. PMID:24778394

  5. Identification of Multipotent Stem Cells in Human Brain Tissue Following Stroke.

    Science.gov (United States)

    Tatebayashi, Kotaro; Tanaka, Yasue; Nakano-Doi, Akiko; Sakuma, Rika; Kamachi, Saeko; Shirakawa, Manabu; Uchida, Kazutaka; Kageyama, Hiroto; Takagi, Toshinori; Yoshimura, Shinichi; Matsuyama, Tomohiro; Nakagomi, Takayuki

    2017-06-01

    Perivascular regions of the brain harbor multipotent stem cells. We previously demonstrated that brain pericytes near blood vessels also develop multipotency following experimental ischemia in mice and these ischemia-induced multipotent stem cells (iSCs) can contribute to neurogenesis. However, it is essential to understand the traits of iSCs in the poststroke human brain for possible applications in stem cell-based therapies for stroke patients. In this study, we report for the first time that iSCs can be isolated from the poststroke human brain. Putative iSCs were derived from poststroke brain tissue obtained from elderly stroke patients requiring decompressive craniectomy and partial lobectomy for diffuse cerebral infarction. Immunohistochemistry showed that these iSCs were localized near blood vessels within poststroke areas containing apoptotic/necrotic neurons and expressed both the stem cell marker nestin and several pericytic markers. Isolated iSCs expressed these same markers and demonstrated high proliferative potential without loss of stemness. Furthermore, isolated iSCs expressed other stem cell markers, such as Sox2, c-myc, and Klf4, and differentiated into multiple cells in vitro, including neurons. These results show that iSCs, which are likely brain pericyte derivatives, are present within the poststroke human brain. This study suggests that iSCs can contribute to neural repair in patients with stroke.

  6. 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.

  7. Key Signaling Events for Committing Mouse Pluripotent Stem Cells to the Germline Fate.

    Science.gov (United States)

    Wang, Jian-Qi; Cao, Wen-Guang

    2016-01-01

    The process of germline development carries genetic information and preparatory totipotency across generations. The last decade has witnessed remarkable successes in the generation of germline cells from mouse pluripotent stem cells, especially induced germline cells with the capacity for producing viable offspring, suggesting clinical applications of induced germline cells in humans. However, to date, the culture systems for germline induction with accurate sex-specific meiosis and epigenetic reprogramming have not been well-established. In this study, we primarily focus on the mouse model to discuss key signaling events for germline induction. We review mechanisms of competent regulators on primordial germ cell induction and discuss current achievements and difficulties in inducing sex-specific germline development. Furthermore, we review the developmental identities of mouse embryonic stem cells and epiblast stem cells under certain defined culture conditions as it relates to the differentiation process of becoming germline cells.

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    Lifescience Database Archive (English)

    Full Text Available DNS.Gon.20.AllAg.Germline_stem_cells mm9 DNase-seq Gonad Germline stem cells http:/.../dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Gon.20.AllAg.Germline_stem_cells.bed ...

  9. File list: ALL.Gon.10.AllAg.Germline_stem_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Gon.10.AllAg.Germline_stem_cells mm9 All antigens Gonad Germline stem cells SRX...X1060554,SRX1060555 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Gon.10.AllAg.Germline_stem_cells.bed ...

  10. Differential roles of HOW in male and female Drosophila germline differentiation.

    Directory of Open Access Journals (Sweden)

    Adrian C Monk

    Full Text Available The adult gonads in both male and female Drosophila melanogaster produce gametes that originate from a regenerative pool of germline stem cells (GSCs. The differentiation programme that produces gametes must be co-ordinated with GSC maintenance and proliferation in order to regulate tissue regeneration. The HOW RNA-binding protein has been shown to maintain mitotic progression of male GSCs and their daughters by maintenance of Cyclin B expression as well as suppressing accumulation of the differentiation factor Bam. Loss of HOW function in the male germline results in loss of GSCs due to a delay in G2 and subsequent apoptosis. Here we show that female how mutant GSCs do not have any cell cycle defects although HOW continues to bind bam mRNA and suppress Bam expression. The role of HOW in suppressing germ cell Bam expression appears to be conserved between sexes, leading to different cellular outcomes in how mutants due to the different functions of Bam. In addition the role in maintaining Cyclin B expression has not been conserved so female how GSCs differentiate rather than arrest.

  11. Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

    Directory of Open Access Journals (Sweden)

    Francesca Pacchierotti

    2015-01-01

    Full Text Available The epigenome consists of chemical changes in DNA and chromatin that without modifying the DNA sequence modulate gene expression and cellular phenotype. The epigenome is highly plastic and reacts to changing external conditions with modifications that can be inherited to daughter cells and across generations. Whereas this innate plasticity allows for adaptation to a changing environment, it also implies the potential of epigenetic derailment leading to so-called epimutations. DNA methylation is the most studied epigenetic mark. DNA methylation changes have been associated with cancer, infertility, cardiovascular, respiratory, metabolic, immunologic, and neurodegenerative pathologies. Experiments in rodents demonstrate that exposure to a variety of chemical stressors, occurring during the prenatal or the adult life, may induce DNA methylation changes in germ cells, which may be transmitted across generations with phenotypic consequences. An increasing number of human biomonitoring studies show environmentally related DNA methylation changes mainly in blood leukocytes, whereas very few data have been so far collected on possible epigenetic changes induced in the germline, even by the analysis of easily accessible sperm. In this paper, we review the state of the art on factors impinging on DNA methylation in the germline, highlight gaps of knowledge, and propose priorities for future studies.

  12. 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.

  13. The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

    Science.gov (United States)

    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified.

  14. Common germline polymorphisms associated with breast cancer-specific survival

    NARCIS (Netherlands)

    A. Pirie (Ailith); Q. Guo (Qi); P. Kraft (Peter); S. Canisius (Sander); D. Eccles (Diana); N. Rahman (Nazneen); H. Nevanlinna (Heli); C. Chen (Constance); S. Khan (Sofia); J.P. Tyrer (Jonathan); M.K. Bolla (Manjeet); Q. Wang (Qing); J. Dennis (Joe); K. Michailidou (Kyriaki); M. Lush (Michael); A.M. Dunning (Alison); M. Shah (Mitul); K. Czene (Kamila); H. Darabi (Hatef); M. Eriksson (Mats); D. Lambrechts (Diether); C. Weltens (Caroline); K. Leunen; C. van Ongeval (Chantal); B.G. Nordestgaard (Børge); S.F. Nielsen (Sune); H. Flyger (Henrik); A. Rudolph (Anja); P. Seibold (Petra); D. Flesch-Janys (Dieter); C. Blomqvist (Carl); K. Aittomäki (Kristiina); R. Fagerholm (Rainer); T.A. Muranen (Taru); J.E. Olsen (Janet E.); B. Hallberg (Boubou); C. Vachon (Celine); J.A. Knight (Julia); G. Glendon (Gord); A.M. Mulligan (Anna Marie); A. Broeks (Annegien); S. Cornelissen (Sten); C.A. Haiman (Christopher); B.E. Henderson (Brian); F. Schumacher (Frederick); L. Le Marchand (Loic); J.L. Hopper (John); H. Tsimiklis (Helen); C. Apicella (Carmel); M.C. Southey (Melissa); S.S. Cross (Simon); M.W.R. Reed (Malcolm); G.G. Giles (Graham); R.L. Milne (Roger); C.A. McLean (Catriona Ann); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); J.W.M. Martens (John); A.M.W. van den Ouweland (Ans); F. Marme (Federick); A. Schneeweiss (Andreas); R. Yang (Rongxi); B. Burwinkel (Barbara); J.D. Figueroa (Jonine); S.J. Chanock (Stephen); J. Lissowska (Jolanta); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); H. Brenner (Hermann); K. Butterbach (Katja); B. Holleczek (B.); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); J. Li (Jingmei); J.S. Brand (Judith S.); M.K. Humphreys (Manjeet); P. Devilee (Peter); R.A.E.M. Tollenaar (Rob); C.M. Seynaeve (Caroline); P. Radice (Paolo); P. Peterlongo (Paolo); S. Manoukian (Siranoush); F. Ficarazzi (Filomena); M.W. Beckmann (Matthias); R. Hein (Rebecca); A.B. Ekici (Arif); R. Balleine (Rosemary); K.-A. Phillips (Kelly-Anne); J. Benítez (Javier); M.P. Zamora (Pilar); J.I.A. Perez (Jose Ignacio Arias); P. Menéndez (Primitiva); A. Jakubowska (Anna); J. Lubinski (Jan); J. Gronwald (Jacek); K. Durda (Katarzyna); U. Hamann (Ute); M. Kabisch (Maria); H.U. Ulmer (Hans); T. Rud̈iger (Thomas); S. Margolin (Sara); V. Kristensen (Vessela); S. Nord (Siljie); D.G. Evans (Gareth); J. Abraham (Jean); H. Earl (Helena); C.J. Poole (Christopher J.); L. Hiller (Louise); J.A. Dunn (J.); S. Bowden (Sarah); R. Yang (Rose); D. Campa (Daniele); W.R. Diver (Ryan); S.M. Gapstur (Susan M.); M.M. Gaudet (Mia); S.E. Hankinson (Susan); R.N. Hoover (Robert); A. Hüsing (Anika); R. Kaaks (Rudolf); M.J. Machiela (Mitchell J.); W.C. Willett (Walter C.); M. Barrdahl (Myrto); F. Canzian (Federico); S.-F. Chin (Suet-Feung); C. Caldas (Carlos); D. Hunter (David); S. Lindstrom (Stephen); M. García-Closas (Montserrat); F.J. Couch (Fergus); G. Chenevix-Trench (Georgia); A. Mannermaa (Arto); I.L. Andrulis (Irene); P. Hall (Per); J. Chang-Claude (Jenny); D.F. Easton (Douglas); S.E. Bojesen (Stig); A. Cox (Angela); P.A. Fasching (Peter); P.D.P. Pharoah (Paul); M.K. Schmidt (Marjanka)

    2015-01-01

    textabstractIntroduction: Previous studies have identified common germline variants nominally associated with breast cancer survival. These associations have not been widely replicated in further studies. The purpose of this study was to evaluate the association of previously reported SNPs with brea

  15. Common germline polymorphisms associated with breast cancer-specific survival

    DEFF Research Database (Denmark)

    Pirie, Ailith; Guo, Qi; Kraft, Peter

    2015-01-01

    INTRODUCTION: Previous studies have identified common germline variants nominally associated with breast cancer survival. These associations have not been widely replicated in further studies. The purpose of this study was to evaluate the association of previously reported SNPs with breast cancer...

  16. Risk profile of the RET A883F germline mutation

    DEFF Research Database (Denmark)

    Mathiesen, Jes Sloth; Habra, Mouhammed Amir; Bassett, John Howard Duncan

    2017-01-01

    Context: The A883F germline mutation of the REarranged during Transfection proto-oncogene causes multiple endocrine neoplasia 2B. In the revised American Thyroid Association (ATA) guidelines for the management of medullary thyroid carcinoma (MTC) the A883F mutation has been reclassified from...

  17. Common germline polymorphisms associated with breast cancer-specific survival

    NARCIS (Netherlands)

    A. Pirie (Ailith); Q. Guo (Qi); P. Kraft (Peter); S. Canisius (Sander); D. Eccles (Diana); N. Rahman (Nazneen); H. Nevanlinna (Heli); C. Chen (Constance); S. Khan (Sofia); J.P. Tyrer (Jonathan); M.K. Bolla (Manjeet); Q. Wang (Qing); J. Dennis (Joe); K. Michailidou (Kyriaki); M. Lush (Michael); A.M. Dunning (Alison); M. Shah (Mitul); K. Czene (Kamila); H. Darabi (Hatef); M. Eriksson (Mats); D. Lambrechts (Diether); C. Weltens (Caroline); K. Leunen; C. van Ongeval (Chantal); B.G. Nordestgaard (Børge); S.F. Nielsen (Sune); H. Flyger (Henrik); A. Rudolph (Anja); P. Seibold (Petra); D. Flesch-Janys (Dieter); C. Blomqvist (Carl); K. Aittomäki (Kristiina); R. Fagerholm (Rainer); T.A. Muranen (Taru); J.E. Olsen (Janet E.); B. Hallberg (Boubou); C. Vachon (Celine); J.A. Knight (Julia); G. Glendon (Gord); A.M. Mulligan (Anna Marie); A. Broeks (Annegien); S. Cornelissen (Sten); C.A. Haiman (Christopher); B.E. Henderson (Brian); F. Schumacher (Frederick); L. Le Marchand (Loic); J.L. Hopper (John); H. Tsimiklis (Helen); C. Apicella (Carmel); M.C. Southey (Melissa); S.S. Cross (Simon); M.W.R. Reed (Malcolm); G.G. Giles (Graham); R.L. Milne (Roger); C.A. McLean (Catriona Ann); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); J.W.M. Martens (John); A.M.W. van den Ouweland (Ans); F. Marme (Federick); A. Schneeweiss (Andreas); R. Yang (Rongxi); B. Burwinkel (Barbara); J.D. Figueroa (Jonine); S.J. Chanock (Stephen); J. Lissowska (Jolanta); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); H. Brenner (Hermann); K. Butterbach (Katja); B. Holleczek (B.); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); J. Li (Jingmei); J.S. Brand (Judith S.); M.K. Humphreys (Manjeet); P. Devilee (Peter); R.A.E.M. Tollenaar (Rob); C.M. Seynaeve (Caroline); P. Radice (Paolo); P. Peterlongo (Paolo); S. Manoukian (Siranoush); F. Ficarazzi (Filomena); M.W. Beckmann (Matthias); R. Hein (Rebecca); A.B. Ekici (Arif); R. Balleine (Rosemary); K.-A. Phillips (Kelly-Anne); J. Benítez (Javier); M.P. Zamora (Pilar); J.I.A. Perez (Jose Ignacio Arias); P. Menéndez (Primitiva); A. Jakubowska (Anna); J. Lubinski (Jan); J. Gronwald (Jacek); K. Durda (Katarzyna); U. Hamann (Ute); M. Kabisch (Maria); H.U. Ulmer (Hans); T. Rud̈iger (Thomas); S. Margolin (Sara); V. Kristensen (Vessela); S. Nord (Siljie); D.G. Evans (Gareth); J. Abraham (Jean); H. Earl (Helena); C.J. Poole (Christopher J.); L. Hiller (Louise); J.A. Dunn (J.); S. Bowden (Sarah); R. Yang (Rose); D. Campa (Daniele); W.R. Diver (Ryan); S.M. Gapstur (Susan M.); M.M. Gaudet (Mia); S.E. Hankinson (Susan); R.N. Hoover (Robert); A. Hüsing (Anika); R. Kaaks (Rudolf); M.J. Machiela (Mitchell J.); W.C. Willett (Walter C.); M. Barrdahl (Myrto); F. Canzian (Federico); S.-F. Chin (Suet-Feung); C. Caldas (Carlos); D. Hunter (David); S. Lindstrom (Stephen); M. García-Closas (Montserrat); F.J. Couch (Fergus); G. Chenevix-Trench (Georgia); A. Mannermaa (Arto); I.L. Andrulis (Irene); P. Hall (Per); J. Chang-Claude (Jenny); D.F. Easton (Douglas); S.E. Bojesen (Stig); A. Cox (Angela); P.A. Fasching (Peter); P.D.P. Pharoah (Paul); M.K. Schmidt (Marjanka)

    2015-01-01

    textabstractIntroduction: Previous studies have identified common germline variants nominally associated with breast cancer survival. These associations have not been widely replicated in further studies. The purpose of this study was to evaluate the association of previously reported SNPs with

  18. Germline fumarate hydratase mutations in patients with ovarian mucinous cystadenoma

    DEFF Research Database (Denmark)

    Ylisaukko-oja, Sanna K.; Cybulski, Cezary; Lehtonen, Rainer;

    2006-01-01

    analyzed for somatic FH mutations. Two patients diagnosed with ovarian mucinous cystadenoma (two out of 33, 6%) were found to be FH germline mutation carriers. One of the changes was a novel mutation (Ala231Thr) and the other one (435insAAA) was previously described in FH deficiency families. These results...

  19. Germline promoter hypermethylation of tumor suppressor genes in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Pu-Yuan Wu; Zheng Zhang; Jing-Mei Wang; Wen-Wen Guo; Nong Xiao; Qiong He; Ya-Ping Wang; Yi-Mei Fan

    2012-01-01

    AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1 , CDH1 and P16INK4a in suspected cases of hereditary gastric cancer (GC). METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1 , CDH1 and P16INK4a tumor suppressor genes. Genomic DNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfite DNA sequencing for a specific region of the MLH1 promoter. The methylation status of CDH1 or P16INK4a was assayed using methylation- specific PCR. Clonal bisulfite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions. RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16INK4a genes was detected. CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16INK4a gene is not a frequent event.

  20. Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells

    Directory of Open Access Journals (Sweden)

    Auke B.C. Otten

    2016-07-01

    Full Text Available We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 106 mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC, a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.

  1. The early postnatal nonhuman primate neocortex contains self-renewing multipotent neural progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jihane Homman-Ludiye

    Full Text Available The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs derived from the postnatal day 14 (PD14 marmoset monkey primary visual cortex (V1, striate cortex. While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF and/or fibroblast growth factor 2 (FGF-2, dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine.

  2. Effect of 5-azacytidine: evidence for alteration of the multipotent ability of mesenchymal stem cells.

    Science.gov (United States)

    Rosca, Ana-Maria; Burlacu, Alexandrina

    2011-07-01

    The treatment of cardiac diseases by cell therapy continues to be challenged by a limited supply of appropriate cells. Although stem cells can generate myocytes after local delivery into the heart, this is often accompanied by the generation of several other cell types as a consequence of environment-driven differentiation. One strategy for overcoming dysregulated differentiation is the pretreatment of stem cells with the demethylation agent 5-azacytidine. The effects of 5-azacytidine on various stem cell types vary from cardiomyogenic differentiation to failure of differentiation or from adipogenic and chondrogenic differentiation to uncontrollable expression of a variety of genes. The underlying mechanisms remain poorly understood, and the effect of 5-azacytidine on the multipotent capacity of stem cells has never been addressed. This study was designed to investigate the changes induced by 5-azacytidine in mesenchymal stem cells (MSC), with particular focus on multipotency maintenance and the capacity of 5-azacytidine to boost myogenic differentiation. Our results show that MSCs retained their multipotent capacity after one pulse with 5-azacytidine, whereas additional pulses resulted in a restricted differentiation potential with concomitant increased ability to accomplish chondrogenic commitment. The induction of cardiac differentiation of MSCs was not observed unless the transcriptional activation of several genes was induced by random hypomethylation. Nevertheless, 5-azacytidine treatment promoted cell response to subsequent stimuli and generation of myogenic differentiation under permissive environmental conditions. Therefore, we assume that one pulse with 5-azacytidine might similarly promote the subsequent cardiac differentiation of MSCs, but it is dependent on the finding of adequate conditions for myocardial differentiation.

  3. The early postnatal nonhuman primate neocortex contains self-renewing multipotent neural progenitor cells.

    Science.gov (United States)

    Homman-Ludiye, Jihane; Merson, Tobias D; Bourne, James A

    2012-01-01

    The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine.

  4. Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials.

    Science.gov (United States)

    Palumbo, Carla; Baldini, Andrea; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia; Zaffe, Davide

    2013-04-01

    The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-μm-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model.

  5. Gene expression profiling supports the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as ovarian cancer initiating cells

    Directory of Open Access Journals (Sweden)

    Matyunina Lilya V

    2009-12-01

    Full Text Available Abstract Background Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube. Methods Comparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI isolated by laser capture micro-dissection (LCM from human serous papillary ovarian adenocarcinomas. The results of the gene expression analyses were randomly confirmed in paraffin embedded tissues from ovarian adenocarcinoma of serous subtype and non-neoplastic ovarian tissues using immunohistochemistry. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms. Results Consistent with multipotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenocarcinoma. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development. Conclusions Our results are consistent with the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as the origin of ovarian adenocarcinoma. While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they are inconsistent with claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells.

  6. Methods for derivation of multipotent neural crest cells derived from human pluripotent stem cells

    Science.gov (United States)

    Avery, John; Dalton, Stephen

    2016-01-01

    Summary Multipotent, neural crest cells (NCCs) produce a wide-range of cell types during embryonic development. This includes melanocytes, peripheral neurons, smooth muscle cells, osteocytes, chondrocytes and adipocytes. The protocol described here allows for highly-efficient differentiation of human pluripotent stem cells to a neural crest fate within 15 days. This is accomplished under feeder-free conditions, using chemically defined medium supplemented with two small molecule inhibitors that block glycogen synthase kinase 3 (GSK3) and bone morphogenic protein (BMP) signaling. This technology is well-suited as a platform to understand in greater detail the pathogenesis of human disease associated with impaired neural crest development/migration. PMID:25986498

  7. Berberine: A Potential Multipotent Natural Product to Combat Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Hong-Fang Ji

    2011-08-01

    Full Text Available With the accelerated aging of human society Alzheimer’s disease (AD has become one of the most threatening diseases in the elderly. However, there is no efficient therapeutic agent to combat AD. Berberine is a natural isoquinoline alkaloid that possesses a wide range of pharmacological effects. In the present paper, we review the multiple activities of berberine, including antioxidant, acetylcholinesterase and butyrylcholinesterase inhibitory, monoamine oxidase inhibitory, amyloid-b peptide level-reducing and cholesterol-lowering activities, which suggest that berberine may act as a promising multipotent agent to combat AD.

  8. Bridging long gap peripheral nerve injury using skeletal muscle-derived multipotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Tetsuro Tamaki

    2014-01-01

    Long gap peripheral nerve injuries usually reulting in life-changing problems for patients. Skeletal muscle derived-multipotent stem cells (Sk-MSCs) can differentiate into Schwann and perineurial/endoneurial cells, vascular relating pericytes, and endothelial and smooth muscle cells in the damaged peripheral nerve niche. Application of the Sk-MSCs in the bridging conduit for repairing long nerve gap injury resulted favorable axonal regeneration, which showing supe-rior effects than gold standard therapy--healthy nerve autograft. This means that it does not need to sacriifce of healthy nerves or loss of related functions for repairing peripheral nerve injury.

  9. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells.

    Science.gov (United States)

    Salzmann, Viktoria; Inaba, Mayu; Cheng, Jun; Yamashita, Yukiko M

    2013-12-01

    In the homeostatic state, adult stem cells divide either symmetrically to increase the stem cell number to compensate stem cell loss, or asymmetrically to maintain the population while producing differentiated cells. We have investigated the mode of stem cell division in the testes of Drosophila melanogaster by lineage tracing and confirm the presence of symmetric stem cell division in this system. We found that the rate of symmetric division is limited to 1-2% of total germline stem cell (GSC) divisions, but it increases with expression of a cell adhesion molecule, E-cadherin, or a regulator of the actin cytoskeleton, Moesin, which may modulate adhesiveness of germ cells to the stem cell niche. Our results indicate that the decision regarding asymmetric vs. symmetric division is a dynamically regulated process that contributes to tissue homeostasis, responding to the needs of the tissue.

  10. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective.

    Science.gov (United States)

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ.

  11. Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

    Science.gov (United States)

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

  12. Amniotic fluid as a source of multipotent cells for clinical use.

    Science.gov (United States)

    Young, Bruce K; Chan, Michael K; Liu, Li; Basch, Ross S

    2016-04-01

    Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation.

  13. Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor.

    Science.gov (United States)

    Peng, Tien; Tian, Ying; Boogerd, Cornelis J; Lu, Min Min; Kadzik, Rachel S; Stewart, Kathleen M; Evans, Sylvia M; Morrisey, Edward E

    2013-08-29

    Co-development of the cardiovascular and pulmonary systems is a recent evolutionary adaption to terrestrial life that couples cardiac output with the gas exchange function of the lung. Here we show that the murine pulmonary vasculature develops even in the absence of lung development. We have identified a population of multipotent cardiopulmonary mesoderm progenitors (CPPs) within the posterior pole of the heart that are marked by the expression of Wnt2, Gli1 and Isl1. We show that CPPs arise from cardiac progenitors before lung development. Lineage tracing and clonal analysis demonstrates that CPPs generate the mesoderm lineages within the cardiac inflow tract and lung including cardiomyocytes, pulmonary vascular and airway smooth muscle, proximal vascular endothelium, and pericyte-like cells. CPPs are regulated by hedgehog expression from the foregut endoderm, which is required for connection of the pulmonary vasculature to the heart. Together, these studies identify a novel population of multipotent cardiopulmonary progenitors that coordinates heart and lung co-development that is required for adaptation to terrestrial existence.

  14. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  15. Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow.

    Science.gov (United States)

    Lin, Hsing-Yi; Fujita, Naoki; Endo, Kentaro; Morita, Maresuke; Takeda, Tae; Nakagawa, Takayuki; Nishimura, Ryohei

    2017-03-15

    The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

  16. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors

    Institute of Scientific and Technical Information of China (English)

    Chao Sheng; Ziwei Wang; Changlong Guo; Hua-Jun Wu; Zhonghua Liu; Liu Wang; Shigang He; Xiu-Jie Wang; Zhiguo Chen; Qi Zhou; Qinyuan Zheng; Jianyu Wu; Zhen Xu; Libin Wang; Wei Li; Haijiang Zhang; Xiao-YangZhao; Lei Liu

    2012-01-01

    Multipotent neural stem/progenitor cells hold great promise for cell therapy.The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a muitipotent state without first establishing pluripotency.Here,we demonstrate that sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties.The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers,exhibit a global gene-expression profile similar to normal NSCs,and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons,iNSC-derived neurons stain positive for tyrosine hydroxylase (TH),γ-aminobutyric acid,and choline acetyltransferase.In addition,iNSCs can survive and generate synapses following transplantation into the dentate gyrus.Generation of iNSCs may have important implications for disease modeling and regenerative medicine.

  17. Germline TERT promoter mutations are rare in familial melanoma

    DEFF Research Database (Denmark)

    Harland, Mark; Petljak, Mia; Robles-Espinoza, Carla Daniela;

    2016-01-01

    Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP, POT1), have also been implicated in melanomagenesis. Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.-57...... T>G variant) has been reported in one family. We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls. Germline lymphocyte telomere length was estimated in carriers....... The c.-57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls. One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour. The blood leukocyte...

  18. Prevalence of deleterious ATM germline mutations in gastric cancer patients.

    Science.gov (United States)

    Huang, Dong-Sheng; Tao, Hou-Quan; He, Xu-Jun; Long, Ming; Yu, Sheng; Xia, Ying-Jie; Wei, Zhang; Xiong, Zikai; Jones, Sian; He, Yiping; Yan, Hai; Wang, Xiaoyue

    2015-12-01

    Besides CDH1, few hereditary gastric cancer predisposition genes have been previously reported. In this study, we discovered two germline ATM mutations (p.Y1203fs and p.N1223S) in a Chinese family with a history of gastric cancer by screening 83 cancer susceptibility genes. Using a published exome sequencing dataset, we found deleterious germline mutations of ATM in 2.7% of 335 gastric cancer patients of different ethnic origins. The frequency of deleterious ATM mutations in gastric cancer patients is significantly higher than that in general population (p=0.0000435), suggesting an association of ATM mutations with gastric cancer predisposition. We also observed biallelic inactivation of ATM in tumors of two gastric cancer patients. Further evaluation of ATM mutations in hereditary gastric cancer will facilitate genetic testing and risk assessment.

  19. Human germline antibody gene segments encode polyspecific antibodies.

    Science.gov (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  20. Federal Regulation of Gene Therapy: Who Will Save our Germline?

    OpenAIRE

    2003-01-01

    This paper will attempt to address some of these more complex issues involving human gene therapy and the encompassing regulations. The first section will deal with the science of gene therapy and will briefly touch upon the scientific hurdles that remain for scientists in this field, as this is important to understanding many of the ethical issues. This section will be divided into a basic genetic overview, a description of somatic gene therapy, and a summary of germline gene therapy. The se...

  1. Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer

    OpenAIRE

    Gara, Sudheer Kumar; Jia, Li; Merino, Maria J.; Agarwal, Sunita K.; Zhang, Lisa; Cam, Maggie; Patel, Dhaval; Kebebew, Electron

    2015-01-01

    Familial nonmedullary thyroid cancer accounts for 3 to 9% of all cases of thyroid cancer, but the susceptibility genes are not known. Here, we report a germline variant of HABP2 in seven affected members of a kindred with familial nonmedullary thyroid cancer and in 4.7% of 423 patients with thyroid cancer. This variant was associated with increased HABP2 protein expression in tumor samples from affected family members, as compared with normal adjacent thyroid tissue and samples from sporadic ...

  2. Cell lineage analysis of the mammalian female germline.

    Directory of Open Access Journals (Sweden)

    Yitzhak Reizel

    Full Text Available Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote. We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  3. Cell lineage analysis of the mammalian female germline.

    Science.gov (United States)

    Reizel, Yitzhak; Itzkovitz, Shalev; Adar, Rivka; Elbaz, Judith; Jinich, Adrian; Chapal-Ilani, Noa; Maruvka, Yosef E; Nevo, Nava; Marx, Zipora; Horovitz, Inna; Wasserstrom, Adam; Mayo, Avi; Shur, Irena; Benayahu, Dafna; Skorecki, Karl; Segal, Eran; Dekel, Nava; Shapiro, Ehud

    2012-01-01

    Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  4. Mutation rates and the evolution of germline structure

    Science.gov (United States)

    2016-01-01

    Genome sequencing studies of de novo mutations in humans have revealed surprising incongruities in our understanding of human germline mutation. In particular, the mutation rate observed in modern humans is substantially lower than that estimated from calibration against the fossil record, and the paternal age effect in mutations transmitted to offspring is much weaker than expected from our long-standing model of spermatogenesis. I consider possible explanations for these discrepancies, including evolutionary changes in life-history parameters such as generation time and the age of puberty, a possible contribution from undetected post-zygotic mutations early in embryo development, and changes in cellular mutation processes at different stages of the germline. I suggest a revised model of stem-cell state transitions during spermatogenesis, in which ‘dark’ gonial stem cells play a more active role than hitherto envisaged, with a long cycle time undetected in experimental observations. More generally, I argue that the mutation rate and its evolution depend intimately on the structure of the germline in humans and other primates. This article is part of the themed issue ‘Dating species divergences using rocks and clocks'. PMID:27325834

  5. Age-dependent germline mosaicism of the most common noonan syndrome mutation shows the signature of germline selection.

    Science.gov (United States)

    Yoon, Song-Ro; Choi, Soo-Kung; Eboreime, Jordan; Gelb, Bruce D; Calabrese, Peter; Arnheim, Norman

    2013-06-06

    Noonan syndrome (NS) is among the most common Mendelian genetic diseases (∼1/2,000 live births). Most cases (50%-84%) are sporadic, and new mutations are virtually always paternally derived. More than 47 different sites of NS de novo missense mutations are known in the PTPN11 gene that codes for the protein tyrosine phosphatase SHP-2. Surprisingly, many of these mutations are recurrent with nucleotide substitution rates substantially greater than the genome average; the most common mutation, c.922A>G, is at least 2,400 times greater. We examined the spatial distribution of the c.922A>G mutation in testes from 15 unaffected men and found that the mutations were not uniformly distributed across each testis as would be expected for a mutation hot spot but were highly clustered and showed an age-dependent germline mosaicism. Computational modeling that used different stem cell division schemes confirmed that the data were inconsistent with hypermutation, but consistent with germline selection: mutated spermatogonial stem cells gained an advantage that allowed them to increase in frequency. SHP-2 interacts with the transcriptional activator STAT3. Given STAT3's function in mouse spermatogonial stem cells, we suggest that this interaction might explain the mutant's selective advantage by means of repression of stem cell differentiation signals. Repression of STAT3 activity by cyclin D1 might also play a previously unrecognized role in providing a germline-selective advantage to spermatogonia for the recurrent mutations in the receptor tyrosine kinases that cause Apert syndrome and MEN2B. Looking at recurrent mutations driven by germline selection in different gene families can help highlight common causal signaling pathways.

  6. Analysis of Germline Stem Cell Differentiation Following Loss of GLP-1 Notch Activity in Caenorhabditis elegans.

    Science.gov (United States)

    Fox, Paul M; Schedl, Tim

    2015-09-01

    Stem cells generate the differentiated progeny cells of adult tissues. Stem cells in the Caenorhabditis elegans hermaphrodite germline are maintained within a proliferative zone of ∼230 cells, ∼20 cell diameters in length, through GLP-1 Notch signaling. The distal tip cell caps the germline and supplies GLP-1-activating ligand, and the distal-most germ cells that occupy this niche are likely self-renewing stem cells with active GLP-1 signaling. As germ cells are displaced from the niche, GLP-1 activity likely decreases, yet mitotically cycling germ cells are found throughout the proliferative zone prior to overt meiotic differentiation. Following loss of GLP-1 activity, it remains unclear whether stem cells undergo transit-amplifying (TA) divisions or more directly enter meiosis. To distinguish between these possibilities we employed a temperature-sensitive (ts) glp-1 mutant to manipulate GLP-1 activity. We characterized proliferative zone dynamics in glp-1(ts) mutants at permissive temperature and then analyzed the kinetics of meiotic entry of proliferative zone cells after loss of GLP-1. We found that entry of proliferative zone cells into meiosis following loss of GLP-1 activity is largely synchronous and independent of their distal-proximal position. Furthermore, the majority of cells complete only a single mitotic division before entering meiosis, independent of their distal-proximal position. We conclude that germ cells do not undergo TA divisions following loss of GLP-1 activity. We present a model for the dynamics of the proliferative zone that utilizes cell cycle rate and proliferative zone size and output and incorporates the more direct meiotic differentiation of germ cells following loss of GLP-1 activity.

  7. Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge.

    Science.gov (United States)

    Jung, Jaeyun; Yeom, Chanjoo; Choi, Yeon-Sook; Kim, Sinae; Lee, EunJi; Park, Min Ji; Kang, Sang Wook; Kim, Sung Bae; Chang, Suhwan

    2015-08-21

    The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.

  8. Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population.

    Science.gov (United States)

    Morrison, Jamie I; Lööf, Sara; He, Pingping; Simon, András

    2006-01-30

    In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. Limb regeneration depends on the formation of a blastema, from which the new appendage develops. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. We describe a multipotent Pax7+ satellite cell population located within the skeletal muscle of the salamander limb. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation.

  9. Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest.

    Science.gov (United States)

    Coelho-Aguiar, Juliana M; Le Douarin, Nicole M; Dupin, Elisabeth

    2013-12-01

    The neural crest (NC), an ectoderm-derived structure of the vertebrate embryo, gives rise to the melanocytes, most of the peripheral nervous system and the craniofacial mesenchymal tissues (i.e., connective, bone, cartilage and fat cells). In the trunk of Amniotes, no mesenchymal tissues are derived from the NC. In certain in vitro conditions however, avian and murine trunk NC cells (TNCCs) displayed a limited mesenchymal differentiation capacity. Whether this capacity originates from committed precursors or from multipotent TNCCs was unknown. Here, we further investigated the potential of TNCCs to develop into mesenchymal cell types in vitro. We found that, in fact, quail TNCCs exhibit a high ability to differentiate into myofibroblasts, chondrocytes, lipid-laden adipocytes and mineralizing osteoblasts. In single cell cultures, both mesenchymal and neural cell types coexisted in TNCC clonal progeny: 78% of single cells yielded osteoblasts together with glial cells and neurons; moreover, TNCCs generated heterogenous clones with adipocytes, myofibroblasts, melanocytes and/or glial cells. Therefore, alike cephalic NCCs, early migratory TNCCs comprised multipotent progenitors able to generate both mesenchymal and melanocytic/neural derivatives, suggesting a continuum in NC developmental potentials along the neural axis. The skeletogenic capacity of the TNC, which was present in the exoskeletal armor of the extinct basal forms of Vertebrates and which persisted in the distal fin rays of extant teleost fish, thus did not totally disappear during vertebrate evolution. Mesenchymal potentials of the TNC, although not fulfilled during development, are still present in a dormant state in Amniotes and can be disclosed in in vitro culture. Whether these potentials are not expressed in vivo due to the presence of inhibitory cues or to the lack of permissive factors in the trunk environment remains to be understood. © 2013 Published by Elsevier Inc.

  10. Common germline polymorphisms associated with breast cancer-specific survival.

    Science.gov (United States)

    Pirie, Ailith; Guo, Qi; Kraft, Peter; Canisius, Sander; Eccles, Diana M; Rahman, Nazneen; Nevanlinna, Heli; Chen, Constance; Khan, Sofia; Tyrer, Jonathan; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Michailidou, Kyriaki; Lush, Michael; Dunning, Alison M; Shah, Mitul; Czene, Kamila; Darabi, Hatef; Eriksson, Mikael; Lambrechts, Dieter; Weltens, Caroline; Leunen, Karin; van Ongeval, Chantal; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Blomqvist, Carl; Aittomäki, Kristiina; Fagerholm, Rainer; Muranen, Taru A; Olsen, Janet E; Hallberg, Emily; Vachon, Celine; Knight, Julia A; Glendon, Gord; Mulligan, Anna Marie; Broeks, Annegien; Cornelissen, Sten; Haiman, Christopher A; Henderson, Brian E; Schumacher, Frederick; Le Marchand, Loic; Hopper, John L; Tsimiklis, Helen; Apicella, Carmel; Southey, Melissa C; Cross, Simon S; Reed, Malcolm Wr; Giles, Graham G; Milne, Roger L; McLean, Catriona; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Hooning, Maartje J; Hollestelle, Antoinette; Martens, John Wm; van den Ouweland, Ans Mw; Marme, Federick; Schneeweiss, Andreas; Yang, Rongxi; Burwinkel, Barbara; Figueroa, Jonine; Chanock, Stephen J; Lissowska, Jolanta; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Brenner, Hermann; Butterbach, Katja; Holleczek, Bernd; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Li, Jingmei; Brand, Judith S; Humphreys, Keith; Devilee, Peter; Tollenaar, Robert Aem; Seynaeve, Caroline; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Ficarazzi, Filomena; Beckmann, Matthias W; Hein, Alexander; Ekici, Arif B; Balleine, Rosemary; Phillips, Kelly-Anne; Benitez, Javier; Zamora, M Pilar; Perez, Jose Ignacio Arias; Menéndez, Primitiva; Jakubowska, Anna; Lubinski, Jan; Gronwald, Jacek; Durda, Katarzyna; Hamann, Ute; Kabisch, Maria; Ulmer, Hans Ulrich; Rüdiger, Thomas; Margolin, Sara; Kristensen, Vessela; Nord, Siljie; Evans, D Gareth; Abraham, Jean; Earl, Helena; Poole, Christopher J; Hiller, Louise; Dunn, Janet A; Bowden, Sarah; Yang, Rose; Campa, Daniele; Diver, W Ryan; Gapstur, Susan M; Gaudet, Mia M; Hankinson, Susan; Hoover, Robert N; Hüsing, Anika; Kaaks, Rudolf; Machiela, Mitchell J; Willett, Walter; Barrdahl, Myrto; Canzian, Federico; Chin, Suet-Feung; Caldas, Carlos; Hunter, David J; Lindstrom, Sara; Garcia-Closas, Montserrat; Couch, Fergus J; Chenevix-Trench, Georgia; Mannermaa, Arto; Andrulis, Irene L; Hall, Per; Chang-Claude, Jenny; Easton, Douglas F; Bojesen, Stig E; Cox, Angela; Fasching, Peter A; Pharoah, Paul Dp; Schmidt, Marjanka K

    2015-04-22

    Previous studies have identified common germline variants nominally associated with breast cancer survival. These associations have not been widely replicated in further studies. The purpose of this study was to evaluate the association of previously reported SNPs with breast cancer-specific survival using data from a pooled analysis of eight breast cancer survival genome-wide association studies (GWAS) from the Breast Cancer Association Consortium. A literature review was conducted of all previously published associations between common germline variants and three survival outcomes: breast cancer-specific survival, overall survival and disease-free survival. All associations that reached the nominal significance level of P value pooled analysis of over 37,000 breast cancer cases for association with breast cancer-specific survival. Previous associations were evaluated using a one-sided test based on the reported direction of effect. Fifty-six variants from 45 previous publications were evaluated in the meta-analysis. Fifty-four of these were evaluated in the full set of 37,954 breast cancer cases with 2,900 events and the two additional variants were evaluated in a reduced sample size of 30,000 samples in order to ensure independence from the previously published studies. Five variants reached nominal significance (P pooled GWAS data compared to 2.8 expected under the null hypothesis. Seven additional variants were associated (P <0.05) with ER-positive disease. Although no variants reached genome-wide significance (P <5 x 10(-8)), these results suggest that there is some evidence of association between candidate common germline variants and breast cancer prognosis. Larger studies from multinational collaborations are necessary to increase the power to detect associations, between common variants and prognosis, at more stringent significance levels.

  11. Parallel germline infiltration of a lentivirus in two Malagasy lemurs.

    Directory of Open Access Journals (Sweden)

    Clément Gilbert

    2009-03-01

    Full Text Available Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera -- Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host-virus interactions.

  12. Genetic basis for developmental homeostasis of germline stem cell niche number: a network of Tramtrack-Group nuclear BTB factors.

    Directory of Open Access Journals (Sweden)

    Mathieu Bartoletti

    Full Text Available The potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs, each made of 8-10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches.

  13. Genetic basis for developmental homeostasis of germline stem cell niche number: a network of Tramtrack-Group nuclear BTB factors.

    Science.gov (United States)

    Bartoletti, Mathieu; Rubin, Thomas; Chalvet, Fabienne; Netter, Sophie; Dos Santos, Nicolas; Poisot, Emilie; Paces-Fessy, Mélanie; Cumenal, Delphine; Peronnet, Frédérique; Pret, Anne-Marie; Théodore, Laurent

    2012-01-01

    The potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs), each made of 8-10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches.

  14. Germ-line gene therapy and the medical imperative.

    Science.gov (United States)

    Munson, Ronald; Davis, Lawrence H

    1992-06-01

    Somatic cell gene therapy has yielded promising results. If germ cell gene therapy can be developed, the promise is even greater: hundreds of genetic diseases might be virtually eliminated. But some claim the procedure is morally unacceptable. We thoroughly and sympathetically examine several possible reasons for this claim but find them inadequate. There is no moral reason, then, not to develop and employ germ-line gene therapy. Taking the offensive, we argue next that medicine has a prima facie moral obligation to do so.

  15. CDH1 germline mutations and hereditary lobular breast cancer.

    Science.gov (United States)

    Corso, Giovanni; Intra, Mattia; Trentin, Chiara; Veronesi, Paolo; Galimberti, Viviana

    2016-04-01

    Hereditary diffuse gastric cancer is an autosomal dominant inherited disease associated of CDH1 germline mutations (that encodes for the E-cadherin protein), and lobular breast cancer is the second most frequent type of neoplasia. Recently, novel E-cadherin constitutional alterations have been identified in pedigree clustering only for lobular breast carcinoma without evidence of diffuse gastric tumors and in absence of BRCA1/2 mutations. This first evidence opens novel questions about the inherited correlation between diffuse gastric and lobular breast cancers. In this brief review we revise the literature data about the CDH1 mutation frequency affecting exclusively lobular breast cancer, providing clinical recommendation for asymptomatic mutation carriers.

  16. Human Germline Mutation and the Erratic Evolutionary Clock

    Science.gov (United States)

    Przeworski, Molly

    2016-01-01

    Our understanding of the chronology of human evolution relies on the “molecular clock” provided by the steady accumulation of substitutions on an evolutionary lineage. Recent analyses of human pedigrees have called this understanding into question by revealing unexpectedly low germline mutation rates, which imply that substitutions accrue more slowly than previously believed. Translating mutation rates estimated from pedigrees into substitution rates is not as straightforward as it may seem, however. We dissect the steps involved, emphasizing that dating evolutionary events requires not “a mutation rate” but a precise characterization of how mutations accumulate in development in males and females—knowledge that remains elusive. PMID:27760127

  17. Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer.

    Science.gov (United States)

    Gara, Sudheer Kumar; Jia, Li; Merino, Maria J; Agarwal, Sunita K; Zhang, Lisa; Cam, Maggie; Patel, Dhaval; Kebebew, Electron

    2015-07-30

    Familial nonmedullary thyroid cancer accounts for 3 to 9% of all cases of thyroid cancer, but the susceptibility genes are not known. Here, we report a germline variant of HABP2 in seven affected members of a kindred with familial nonmedullary thyroid cancer and in 4.7% of 423 patients with thyroid cancer. This variant was associated with increased HABP2 protein expression in tumor samples from affected family members, as compared with normal adjacent thyroid tissue and samples from sporadic cancers. Functional studies showed that HABP2 has a tumor-suppressive effect, whereas the G534E variant results in loss of function.

  18. Production of transgenic mice by random recombination of targeted genes in female germline stem cells

    Institute of Scientific and Technical Information of China (English)

    Yong Zhang; Ji Xiong; Jie Xiang; Ji Wu; Zhaojuan Yang; Yunze Yang; Shuzeng Wang; Lingjun Shi; Wenhai Xie; Kejing Sun; Kang Zou; Lei Wang

    2011-01-01

    Oocyte production in most mammalian species is believed to cease before birth. However, this idea has been challenged with the finding that postnatal mouse ovaries possess mitotically active germ cells. A recent study showed that female germline stem cells (FGSCs) from adult mice were isolated, cultured long term and produced oocytes and progeny after transplantation into infertile mice. Here, we demonstrate the successful generation of transgenic or gene knock-down mice using FGSCs. The FGSCs from ovaries of 5-day-old and adult mice were isolated and either infected with recombinant viruses carrying green fluorescent protein, Oocyte-G1 or the mouse dynein axonemal intermediate chain 2 gene, or transfected with the Oocyte-G1 specific shRNA expression vector (pRS shOocyte-G1 vector), and then transplanted into infertile mice. Transplanted cells in the ovaries underwent oogenesis and produced heterozygous offspring after mating with wild-type male mice. The offspring were genetically characterized and the biological functions of the transferred or knock-down genes were investigated. Efficiency of genetransfer or gene knock-down was 29%-37% and it took 2 months to produce transgenic offspring. Gene manipulation of FGSCs is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.

  19. Open-ended question: is immortality exclusively inherent to the germline?--A mini-review.

    Science.gov (United States)

    Lepperdinger, Günter

    2009-01-01

    All somatic cells are subject to aging. Germline links generations, and thus, pluripotent germ cells are considered potentially immortal. The current understanding how the germline escapes this otherwise inevitable phenomenon is outlined in this article. (c) 2008 S. Karger AG, Basel.

  20. Prevalence of low-penetrant germline TP53 D49H mutation in Japanese cancer patients.

    Science.gov (United States)

    Yamaguchi, Ken; Urakami, Kenichi; Nagashima, Takeshi; Shimoda, Yuji; Ohnami, Shumpei; Ohnami, Sumiko; Ohshima, Keiichi; Mochizuki, Tohru; Hatakeyama, Keiichi; Serizawa, Masakuni; Akiyama, Yasuto; Maruyama, Kouji; Katagiri, Hirohisa; Ishida, Yuji; Takahashi, Kaoru; Nishimura, Seiichiro; Terashima, Masanori; Kawamura, Taiichi; Kinugasa, Yusuke; Yamakawa, Yushi; Onitsuka, Tetsuro; Ohde, Yasuhisa; Sugino, Takashi; Ito, Ichiro; Matsubayashi, Hiroyuki; Horiuchi, Yasue; Mizuguchi, Maki; Yamazaki, Mutsumi; Inoue, Kengo; Wakamatsu, Kimiko; Sugiyama, Misato; Uesaka, Katsuhiko; Kusuhara, Masatoshi

    2016-01-01

    Using whole exome sequencing data obtained from 1,685 Japanese cancer patients, we examined genetic variations of germline TP53 and found 10 types of non-synonymous single nucleotide variants. In the present study, we focused on 6 patients with germline D49H mutation located in the transactivation domain 2 of p53 protein, since the mutation seemed to be prevalent in cancer patients and to be pathogenic. According to the initial survey for family history of the proband with the germline TP53 D49H mutation, one osteosarcoma patient and his pedigree fulfill the criteria for Li-Fraumeni-like syndrome and the 2009 Chompret criteria for germline TP53 mutation screening. Since this patient possesses double germline mutations of TP53 D49H and A159D, further studies are required to evaluate contribution of the D49H mutation in this morbidity. The remaining 5 patients had family histories of cancer, but none fulfills the criteria either for the Li-Fraumeni/Li-Fraumeni-like syndromes or the 2009 Chompret criteria for germline TP53 mutation screening. It is possible to postulate that the germline TP53 D49H mutation is likely to be low-penetrant in some pedigrees. The present study also indicates that the survey for the germline TP53 mutation plays an important role in clinical practice as it will prevent mistaking cancer patients with unusual heredities for sporadic cases.

  1. The fate of BRCA1-related germline mutations in triple-negative breast tumors

    Science.gov (United States)

    Kotoula, Vassiliki; Fostira, Florentia; Papadopoulou, Kyriaki; Apostolou, Paraskevi; Tsolaki, Eleftheria; Lazaridis, Georgios; Manoussou, Kyriaki; Zagouri, Flora; Pectasides, Dimitrios; Vlachos, Ioannis; Tikas, Ioannis; Lakis, Sotiris; Konstantopoulou, Irene; Pentheroudakis, George; Gogas, Helen; Papakostas, Pavlos; Christodoulou, Christos; Bafaloukos, Dimitrios; Razis, Evangelia; Karavasilis, Vasilios; Bamias, Christina; Yannoukakos, Drakoulis; Fountzilas, George

    2017-01-01

    The preservation of pathogenic BRCA1/2 germline mutations in tumor tissues is usually not questioned, while it remains unknown whether these interact with somatic genotypes for patient outcome. Herein we compared germline and tumor genotypes in operable triple-negative breast cancer (TNBC) and evaluated their combined effects on prognosis. We analyzed baseline germline and primary tumor genotype data obtained by Sanger and Next Generation Sequencing in 194 TNBC patients. We also performed multiple tests interrogating the preservation of germline mutations in matched tumors and breast tissue from carriers with available material. Patients had been treated within clinical trials with adjuvant anthracyclines-taxanes based chemotherapy. We identified 50 (26%) germline mutation carriers (78% in BRCA1) and 136 (71%) tumors with somatic mutations (83% in TP53). Tumor mutation patterns differed between carriers and non-carriers (P<0.001); PIK3CA mutations were exclusively present in non-carriers (P=0.007). Germline BRCA1/2 mutations were not detected in matched tumors and breast tissues from 14 out of 33 (42%) evaluable carriers. Microsatellite markers revealed tumor loss of the germline mutant allele in one case only. Tumors that had lost the germline mutation demonstrated a higher incidence of somatic TP53 mutations as compared to tumors with preserved germline mutations (P=0.036). Germline mutation status significantly interacted with tumor TP53 mutations for patient disease-free survival (interaction P=0.026): In non-carriers, tumor TP53 mutations did not affect outcome; In carriers, those with mutated TP53 tumors experienced more relapses compared to those with wild-type TP53 tumors (36% vs. 9% relapse rate, respectively). In conclusion, we show that loss of germline BRCA1/2 mutations is not a rare event in TNBC. This finding, the observed differences in tumor genotypes with respect to germline status and the prognostic interaction between germline BRCA1-related and

  2. Recombination, transcription, and diversity of a partially germline-joined VH in a mammal.

    Science.gov (United States)

    Wang, Xinxin; Miller, Robert D

    2012-09-01

    Full or partially germline-joined V genes have been described in a number of different vertebrate lineages where they can contribute to the expressed antibody repertoire through different mechanisms. Here we demonstrate that VH3.1, a partially germline-joined VH gene in the opossum Monodelphis domestica, can undergo V(D)J recombination to generate productive IgH transcripts. VH3.1 is fused to a DH gene segment in the germline DNA and is the only known example of a germline-joined VH in a mammal. B cells that have recombined VH3.1 were not detected until nearly 2 months of age, around the time of weaning, and much later than B cells using the conventional VH. Compared to opossum IgH transcripts using the conventional VH genes, those with VH3.1 have unusually long CDR3 due to the length of the germline-joined DH.

  3. C. elegans germline-deficient mutants respond to pathogen infection using shared and distinct mechanisms.

    Directory of Open Access Journals (Sweden)

    Michael TeKippe

    Full Text Available Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including gram positive and gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16.

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  12. Epigenetic inheritance systems contribute to the evolution of a germline.

    Science.gov (United States)

    Lachmann, Michael; Libby, Eric

    2016-08-19

    Differentiation within multicellular organisms is controlled by epigenetic markers transmitted across cell division. The process of differentiation will modify these epigenetic markers so that information that one cell type possesses can be lost in the transition to another. Many of the systems that encode these markers also exist in unicellular organisms but do not control differentiation. Thus, during the evolution of multicellularity, epigenetic inheritance systems were probably exapted for their current use in differentiation. We show that the simultaneous use of an information carrier for differentiation and transmission across generations can lead to the evolution of cell types that do not directly contribute to the progeny of the organism and ergo a germ-soma distinction. This shows that an intrinsic instability during a transition from unicellularity to multicellularity may contribute to widespread evolution of a germline and its maintenance, a phenomenon also relevant to the evolution of eusociality. The difference in epigenetic information contents between different cell lines in a multicellular organism is also relevant for the full-success cloning of higher animals, as well as for the maintenance of single germlines over evolutionary timescales.This article is part of the themed issue 'The major synthetic evolutionary transitions'.

  13. Sequences sufficient for programming imprinted germline DNA methylation defined.

    Directory of Open Access Journals (Sweden)

    Yoon Jung Park

    Full Text Available Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC; the second carries only the DMD and repeats (DR from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

  14. CDH1 germline mutation in hereditary gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hai-Dan Wang; Jun Ren; Lian Zhang

    2004-01-01

    This paper provides a bird's-eye view both in preclinical and clinical aspects of E-cadherin germline gene (CDH1)in gastric cancer patients and their families. E-cadherin,a product of CDH1 gene, belonging to the functionally related trans-membrane glycoprotein family, is responsible for the Ca2+-dependent cell-cell adhesion mechanism and contributes to dissociation followed by acquisition of cell motility, which usually occurs in the first step of cancer invasion and metastasis. CDH1 gene germline mutation is common in many types of carcinoma,and occurs very frequent in hereditary gastric carcinoma (HGC) patients and their families. Recently, more and more researches support that E-cadherin plays an important role in the differentiation, growth and invasion of HGC. So it is of great value to clarify its mechanisms both for understanding HGC pathogenesis and for clinical therapy, especially in China, where there are a high risk population of gastric cancer and a high HGC incidence rate. In this paper, recent researches on CDH1 gene mutation, especially its role in tumor genesis and progress of HGC, are reviewed, and advances in evaluation of its mutation status for HGC diagnosis, therapy and prognosis,are also discussed briefly.

  15. Epigenetic inheritance systems contribute to the evolution of a germline

    Science.gov (United States)

    Libby, Eric

    2016-01-01

    Differentiation within multicellular organisms is controlled by epigenetic markers transmitted across cell division. The process of differentiation will modify these epigenetic markers so that information that one cell type possesses can be lost in the transition to another. Many of the systems that encode these markers also exist in unicellular organisms but do not control differentiation. Thus, during the evolution of multicellularity, epigenetic inheritance systems were probably exapted for their current use in differentiation. We show that the simultaneous use of an information carrier for differentiation and transmission across generations can lead to the evolution of cell types that do not directly contribute to the progeny of the organism and ergo a germ–soma distinction. This shows that an intrinsic instability during a transition from unicellularity to multicellularity may contribute to widespread evolution of a germline and its maintenance, a phenomenon also relevant to the evolution of eusociality. The difference in epigenetic information contents between different cell lines in a multicellular organism is also relevant for the full-success cloning of higher animals, as well as for the maintenance of single germlines over evolutionary timescales. This article is part of the themed issue ‘The major synthetic evolutionary transitions’. PMID:27431523

  16. Germ cell transplantation using sexually competent fish: an approach for rapid propagation of endangered and valuable germlines.

    Directory of Open Access Journals (Sweden)

    Sullip K Majhi

    Full Text Available The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae, were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg and high water temperature (25 degrees C treatments. The observation of the donor cells' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2-13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.

  17. Giant Panda (Ailuropoda melanoleuca Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Hilary M A Prescott

    Full Text Available Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca; red panda (Ailurus fulgens; and Asiatic lion (Panthera leo persica. m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of

  18. Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells.

    Science.gov (United States)

    Prescott, Hilary M A; Manning, Craig; Gardner, Aaron; Ritchie, William A; Pizzi, Romain; Girling, Simon; Valentine, Iain; Wang, Chengdong; Jahoda, Colin A B

    2015-01-01

    Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers

  19. Exclusive Multipotency and Preferential Asymmetric Divisions in Post-Embryonic Neural Stem Cells of the Fish Retina

    OpenAIRE

    Centanin, L.; Ander, J.-J.; Hoeckendorf, B.; Lust, K.; T. Kellner; Kraemer, I.; Urbany, C.; Hasel, E.; Harris, W.A.; Simons, B. D.; Wittbrodt, J

    2014-01-01

    This is the final version of the article. It has been published by The Company of Biologists Ltd in Development here: http://dev.biologists.org/content/early/2014/08/19/dev.109892.long. The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic or...

  20. Roles of chromatin remodelers in maintenance mechanisms of multipotency of mouse trunk neural crest cells in the formation of neural crest-derived stem cells.

    Science.gov (United States)

    Fujita, Kyohei; Ogawa, Ryuhei; Kawawaki, Syunsaku; Ito, Kazuo

    2014-08-01

    We analyzed roles of two chromatin remodelers, Chromodomain Helicase DNA-binding protein 7 (CHD7) and SWItch/Sucrose NonFermentable-B (SWI/SNF-B), and Bone Morphogenetic Protein (BMP)/Wnt signaling in the maintenance of the multipotency of mouse trunk neural crest cells, leading to the formation of mouse neural crest-derived stem cells (mouse NCSCs). CHD7 was expressed in the undifferentiated neural crest cells and in the dorsal root ganglia (DRG) and sciatic nerve, typical tissues containing NCSCs. BMP/Wnt signaling stimulated the expression of CHD7 and participated in maintaining the multipotency of neural crest cells. Furthermore, the promotion of CHD7 expression maintained the multipotency of these cells. The inhibition of CHD7 and SWI/SNF-B expression significantly suppressed the maintenance of the multipotency of these cells. In addition, BMP/Wnt treatment promoted CHD7 expression and caused the increase of the percentage of multipotent cells in DRG. Thus, the present data suggest that the chromatin remodelers as well as BMP/Wnt signaling play essential roles in the maintenance of the multipotency of mouse trunk neural crest cells and in the formation of mouse NCSCs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies

    Directory of Open Access Journals (Sweden)

    Burt Austin

    2009-07-01

    Full Text Available Abstract Background Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae. Results We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae. Conclusion We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

  2. Zebrafish Müller glia-derived progenitors are multipotent, exhibit proliferative biases and regenerate excess neurons.

    Science.gov (United States)

    Powell, Curtis; Cornblath, Eli; Elsaeidi, Fairouz; Wan, Jin; Goldman, Daniel

    2016-04-20

    Unlike mammals, zebrafish can regenerate a damaged retina. Key to this regenerative response are Müller glia (MG) that respond to injury by reprogramming and adopting retinal stem cell properties. These reprogrammed MG divide to produce a proliferating population of retinal progenitors that migrate to areas of retinal damage and regenerate lost neurons. Previous studies have suggested that MG-derived progenitors may be biased to produce that are lost with injury. Here we investigated MG multipotency using injury paradigms that target different retinal nuclear layers for cell ablation. Our data indicate that regardless of which nuclear layer was damaged, MG respond by generating multipotent progenitors that migrate to all nuclear layers and differentiate into layer-specific cell types, suggesting that MG-derived progenitors in the injured retina are intrinsically multipotent. However, our analysis of progenitor proliferation reveals a proliferative advantage in nuclear layers where neurons were ablated. This suggests that feedback inhibition from surviving neurons may skew neuronal regeneration towards ablated cell types.

  3. Ultrastructural features of human adipose-derived multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Manea, Claudiu Marius; Rusu, Mugurel Constantin; Constantin, Daniel; Mănoiu, Valentina Mariana; Moldovan, Lucia; Jianu, Adelina Maria

    2014-01-01

    Multipotent mesenchymal stromal cells (MMSCs) are plastic-adherent cells with a well-established phenotype. Equine, but not human, adipose MMSCs have been characterized ultrastructurally. The purpose of our study was to evaluate ultrastructurally the adipose-derived human MMSCs. Cell cultures were prepared from human lipoaspirate. The flow cytometry evaluation of surface markers of cultured cells confirmed the expected profile of MMSCs, that were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. We examined these human adipose-derived MMSCs in transmission electron microscopy (TEM) by Epon en-face embedding the fixed MMSCs. The main ultrastructural features of MMSCs were the extremely rich content of endosomal/vesicular elements, long mitochondria, dilated RER (rough endoplasmic reticulum) cisternae, and abundant intermediate filaments and microtubules. We found two types of MMSCS prolongations: (a) thick processes, with opposite, vesicular and filaments-rich, sides and (b) slender processes (pseudopodes and filopodes), with occasional proximal dilated segments housing mitochondria, vesicles and secretory granules. These TEM features of MMSCs characterized an in vitro cell population and could use to distinguish between different cell types in culture.

  4. Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke.

    Science.gov (United States)

    Sakuma, Rika; Kawahara, Maiko; Nakano-Doi, Akiko; Takahashi, Ai; Tanaka, Yasue; Narita, Aya; Kuwahara-Otani, Sachi; Hayakawa, Tetsu; Yagi, Hideshi; Matsuyama, Tomohiro; Nakagomi, Takayuki

    2016-03-07

    Microglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear. In this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia. We demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-β (PDGFRβ), a hallmark of vascular PCs. PDGFRβ(+) PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity. The study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.

  5. Umbilical cord blood: a trustworthy source of multipotent stem cells for regenerative medicine.

    Science.gov (United States)

    Jaing, Tang-Her

    2014-01-01

    It is conservatively estimated that one in three individuals in the US might benefit from regenerative medicine therapy. However, the relation of embryonic stem cells (ESCs) to human blastocysts always stirs ethical, political, moral, and emotional debate over their use in research. Thus, for the reasonably foreseeable future, the march of regenerative medicine to the clinic will depend upon the development of non-ESC therapies. Current sources of non-ESCs easily available in large numbers can be found in the bone marrow, adipose tissue, and umbilical cord blood (UCB). UCB provides an immune-compatible source of stem cells for regenerative medicine. Owing to inconsistent results, it is certainly an important and clinically relevant question whether UCB will prove to be therapeutically effective. This review will show that UCB contains multiple populations of multipotent stem cells, capable of giving rise to hematopoietic, epithelial, endothelial, and neural tissues both in vitro and in vivo. Here we raise the possibility that due to unique immunological properties of both the stem cell and non-stem cell components of cord blood, it may be possible to utilize allogeneic cells for regenerative applications without needing to influence or compromise the recipient immune system.

  6. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells.

    Science.gov (United States)

    Rawlins, Emma L; Clark, Cheryl P; Xue, Yan; Hogan, Brigid L M

    2009-11-01

    The conducting airways (bronchi and bronchioles) and peripheral gas exchange (alveolar) regions of the mammalian lung are generated by a process of branching morphogenesis. Evidence suggests that during embryonic development, the undifferentiated epithelial progenitors are located at the distal tips of the branching epithelium. To test this hypothesis, we used an Id2-CreER(T2) knock-in mouse strain to lineage trace the distal epithelial tip cells during either the pseudoglandular or canalicular phases of development. During the pseudoglandular stage, the tip cells both self-renew and contribute descendents to all epithelial cell lineages, including neuroendocrine cells. In addition, individual Id2(+) tip cells can self-renew and contribute descendents to both the bronchiolar and alveolar compartments. By contrast, during the later canalicular stage, the distal epithelial tip cells only contribute descendents to the alveoli. Taken together, this evidence supports a model in which the distal tip of the developing lung contains a multipotent epithelial population, the fate of which changes during development.

  7. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.

    Science.gov (United States)

    Oguro, Hideyuki; Ding, Lei; Morrison, Sean J

    2013-07-03

    Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitors (MPPs) are routinely isolated using various markers but remain heterogeneous. Here we show that four SLAM family markers, CD150, CD48, CD229, and CD244, can distinguish HSCs and MPPs from restricted progenitors and subdivide them into a hierarchy of functionally distinct subpopulations with stepwise changes in cell-cycle status, self-renewal, and reconstituting potential. CD229 expression largely distinguished lymphoid-biased HSCs from rarely dividing myeloid-biased HSCs, enabling prospective enrichment of these HSC subsets. Differences in CD229 and CD244 expression resolved CD150(-)CD48(-/low)Lineage(-/low)Sca-1(+)c-Kit(+) cells into a hierarchy of highly purified MPPs that retained erythroid and platelet potential but exhibited progressive changes in mitotic activity and reconstituting potential. Use of these markers, and reconstitution assays, showed that conditional deletion of Scf from endothelial cells and perivascular stromal cells eliminated the vast majority of bone marrow HSCs, including nearly all CD229(-/low) HSCs, demonstrating that quiescent HSCs are maintained by a perivascular niche. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Multipotent neuroblasts generate a biochemical neuroarchitecture in the central complex of the grasshopper Schistocerca gregaria.

    Science.gov (United States)

    Boyan, George; Williams, Leslie; Herbert, Zsofia

    2010-04-01

    We have examined the developmental expression of the neuromodulators locustatachykinin, leucokinin-1, allatostatin and serotonin in a subset of lineages (Y, Z) of the central complex in the brain of the grasshopper Schistocerca gregaria. First, we show that all these neuromodulators are expressed in the same lineages during embryogenesis. The neuroblasts generating these lineages are therefore biochemically multipotent. Second, the neurons expressing the different neuromodulators are found clustered at stereotypic locations in their respective lineages. Locustatachykinin and leucokinin-1 map to the apical region of the lineage, allatostatin medially and serotonin to the base of the lineage. Since the location in these lineages translates into their birth order, we have been able ontogenetically to analyse their biochemical expression patterns. The age-profile within a lineage reveals that locustatachykinin- and leucokinin-1-expressing neurons are born first, then allatostatin neurons and finally serotoninergic neurons. Co-expression has been tested for serotonin with locustatachykin, leucokinin-1 or allatostatin and is negative but is positive for locustatachykinin and leucokinin-1, consistent with the stereotypic location of cells in the lineages. The delay between the birth of a neuron and the expression of its neuromodulator is stereotypic for each substance. Combined with a known birth date, this delay translates into a developmental expression pattern for the central complex itself.

  9. Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells

    Science.gov (United States)

    Di Liddo, Rosa; Aguiari, Paola; Barbon, Silvia; Bertalot, Thomas; Mandoli, Amit; Tasso, Alessia; Schrenk, Sandra; Iop, Laura; Gandaglia, Alessandro; Parnigotto, Pier Paolo; Conconi, Maria Teresa; Gerosa, Gino

    2016-01-01

    Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality. PMID:27789941

  10. Characterization and Comparison of Canine Multipotent Stromal Cells Derived from Liver and Bone Marrow

    Science.gov (United States)

    Malagola, Ermanno; Teunissen, Michelle; van der Laan, Luc J.W.; Verstegen, Monique M.A.; Schotanus, Baukje A.; van Steenbeek, Frank G.; Penning, Louis C.; van Wolferen, Monique E.; Tryfonidou, Marianna A.

    2016-01-01

    Liver-derived multipotent stromal cells (L-MSCs) may prove preferable for treatment strategies of liver diseases, in comparison to the widely studied bone marrow-derived MSCs (BM-MSCs). Canines are a large animal model, in which the pathologies of liver diseases are similar to man. This study further promotes the implementation of canine models in MSC-based treatments of liver diseases. L-MSCs were characterized and compared to BM-MSCs from the same individual. Both cell types demonstrated a spindle-shaped fibroblast-like morphology, possessed the same growth potential, and demonstrated similar immunomodulation gene expression of CD274, PTGS-1, and PTGS-2. Marked differences in cell surface markers, CD105 and CD146, distinguished these two cell populations, and L-MSCs retained a liver-specific imprinting, observed by expression of CK18 and CK19. Finally, both populations differentiated toward the osteogenic and adipogenic lineage; however, L-MSCs failed to differentiate into the chondrogenic lineage. In conclusion, characterization of canine L-MSCs and BM-MSCs demonstrated that the two cell type populations are highly comparable. Although it is still unclear which cell source is preferred for clinical application in liver treatment strategies, this study provides a foundation for future controlled studies with MSC therapy in various liver diseases in dogs before their application in man. PMID:26462417

  11. FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors

    Directory of Open Access Journals (Sweden)

    Adam J. Mead

    2013-06-01

    Full Text Available Whether signals mediated via growth factor receptors (GFRs might influence lineage fate in multipotent progenitors (MPPs is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.

  12. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  13. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes.

    Science.gov (United States)

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  14. Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency

    Science.gov (United States)

    Chuah, Yon Jin; Koh, Yi Ting; Lim, Kaiyang; Menon, Nishanth V.; Wu, Yingnan; Kang, Yuejun

    2015-01-01

    Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies. PMID:26647719

  15. Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Naoya; Yamada, Shigeru [Division of Pharmacology, National Institute of Health Sciences (Japan); Asanagi, Miki [Division of Pharmacology, National Institute of Health Sciences (Japan); Faculty of Engineering, Department of Materials Science and Engineering, Yokohama National University (Japan); Sekino, Yuko [Division of Pharmacology, National Institute of Health Sciences (Japan); Kanda, Yasunari, E-mail: kanda@nihs.go.jp [Division of Pharmacology, National Institute of Health Sciences (Japan)

    2016-02-05

    Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

  16. Elicitation of broadly neutralizing antibodies against HIV-1 – the germline/maturation hypothesis

    Directory of Open Access Journals (Sweden)

    Prabakaran ePonraj

    2014-08-01

    Full Text Available We have previously observed that all known broadly neutralizing antibodies (bnAbs against HIV-1 are highly divergent from their putative germline predecessors in contrast to bnAbs against henipaviruses and SARS coronavirus, which are much less divergent from their germline counterparts. We have hypothesized that because the germline antibodies are so different compared to the highly somatically mutated HIV-1 bnAbs they may not bind to the Env. This led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the HIV-1 envelope glycoproteins (Envs is reduced or eliminated by their very weak or absent interactions with germline antibodies. Thus immune responses leading to elicitation of bnAbs may not be initiated and/or sustained; even if they are, the maturation pathways are so complex that prolonged periods of time may be required for elicitation of such bnAbs. In support of the hypothesis, our initial experiments showed that germline-like precursors of several bnAbs do not bind to their epitopes. Recently, a number of research groups working in the HIV vaccine field have obtained data supporting and further expanding the germline/maturation hypothesis. Vaccine immunogens that could bind putative germline antibody predecessors of known bnAbs were successfully generated. However, guiding the immune system through the exceptionally complex antibody maturation pathways in order to elicit those bnAbs remains a major challenge. Here, we summarize developments in the HIV-1 vaccine field based on the germline/maturation hypothesis including our recent data demonstrating germline-like VRC01 antibodies in a human cord blood IgM library.

  17. Lynch Syndrome Caused by Germline PMS2 Mutations

    DEFF Research Database (Denmark)

    Ten Broeke, Sanne W; Brohet, Richard M; Tops, Carli M

    2015-01-01

    . Standardized incidence ratios (SIRs) were calculated to estimate risks for other Lynch syndrome-associated cancers. RESULTS: The cumulative risk (CR) of CRC for male mutation carriers by age 70 years was 19%. The CR among female carriers was 11% for CRC and 12% for EC. The mean age of CRC development was 52......PURPOSE: The clinical consequences of PMS2 germline mutations are poorly understood compared with other Lynch-associated mismatch repair gene (MMR) mutations. The aim of this European cohort study was to define the cancer risk faced by PMS2 mutation carriers. METHODS: Data were collected from 98...... years, and there was a significant difference in mean age of CRC between the probands (mean, 47 years; range, 26 to 68 years) and other family members with a PMS2 mutation (mean, 58 years; range, 31 to 86 years; P

  18. Germline transcription: a key regulator of accessibility and recombination.

    Science.gov (United States)

    Abarrategui, Iratxe; Krangel, Michael S

    2009-01-01

    The developmental control of V(D)J recombination is imposed at the level of chromatin accessibility of recombination signal sequences (RSSs) to the recombinase machinery. Cis-acting transcriptional regulatory elements such as promoters and enhancers play a central role in the control of accessibility in vivo. However, the molecular mechanisms by which these elements influence accessibility are still under investigation. Although accessibility for V(D)J recombination is usually accompanied by germline transcription at antigen receptor loci, the functional significance of this transcription in directing RSS accessibility has been elusive. In this chapter, we review past studies outlining the complex relationship between V(D)J recombination and transcription as well as our current understanding on how chromatin structure is regulated during gene expression. We then summarize recent work that directly addresses the functional role of transcription in V(D)J recombination.

  19. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal.

    Science.gov (United States)

    Kubota, Hiroshi; Brinster, Ralph L

    2008-01-01

    Spermatogonial stem cells (SSCs), postnatal male germline stem cells, are the foundation of spermatogenesis, during which an enormous number of spermatozoa is produced daily by the testis throughout life of the male. SSCs are unique among stem cells in the adult body because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. In addition, SSCs provide an excellent and powerful model to study stem cell biology because of the availability of a functional assay that unequivocally identifies the stem cell. Development of an in vitro culture system that allows an unlimited supply of SSCs is a crucial technique to manipulate genes of the SSC to generate valuable transgenic animals, to study the self-renewal mechanism, and to develop new therapeutic strategies for infertility. In this chapter, we describe a detailed protocol for the culture of mouse and rat SSCs. A key factor for successful development of the SSC culture system was identification of in vitro growth factor requirements for the stem cell using a defined serum-free medium. Because transplantation assays using immunodeficient mice demonstrated that extrinsic factors for self-renewal of SSCs appear to be conserved among many mammalian species, culture techniques for SSCs of other species, including farm animals and humans, are likely to be developed in the coming 5-10 years.

  20. Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem Cell Lineage in Drosophila melanogaster.

    Science.gov (United States)

    Matsuoka, Shinya; Armstrong, Alissa R; Sampson, Leesa L; Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2017-06-01

    Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems. Copyright © 2017 by the Genetics Society of America.

  1. Understanding what determines the frequency and pattern of human germline mutations.

    Science.gov (United States)

    Arnheim, Norman; Calabrese, Peter

    2009-07-01

    Surprising findings about human germline mutation have come from applying new technologies to detect rare mutations in germline DNA, from analysing DNA sequence divergence between humans and closely related species, and from investigating human polymorphic variation. In this Review we discuss how these approaches affect our current understanding of the roles of sex, age, mutation hot spots, germline selection and genomic factors in determining human nucleotide substitution mutation patterns and frequencies. To enhance our understanding of mutation and disease, more extensive molecular data on the human germ line with regard to mutation origin, DNA repair, epigenetic status and the effect of newly arisen mutations on gamete development are needed.

  2. Temporal and spatial localization of three germline-specific proteins in medaka.

    Science.gov (United States)

    Aoki, Yumiko; Nagao, Issei; Saito, Daisuke; Ebe, Youko; Kinjo, Masataka; Tanaka, Minoru

    2008-03-01

    We show that germline-specific proteins, olvas (vasa), nanos, and tdrd1 (tudor), alter their localization in the cytoplasm during germline development in the medaka (Oryzias latipes). By immunohistochemical analysis, these three germline-specific proteins were detectable on granule-like structures in the cytoplasm of migrating primordial germ cells. In the germ cells of the gonadal primordia, these granules formed a hollow area lacking these three protein components. During the sexual differentiation of the female gonads, the granules were found to be reduced in size in the germ cells undergoing cystic division and they showed a perinuclear localization in the oocytes. However, the germ cells in the male gonads retained their hollow granules during this early sex differentiation stage. We further demonstrate the differential localization of olvas, nanos, and tdrd1 on these granules during medaka germline development.

  3. A germline RET proto-oncogene mutation in multiple members of an ...

    African Journals Online (AJOL)

    Makia Marafie

    2016-09-17

    Sep 17, 2016 ... multiple members of an Arab family with variable onset of MEN type ... fashion and caused by germline mutation in RET proto- oncogene. The main .... ing sudden severe high blood pressure crises that required immediate ...

  4. Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

    Science.gov (United States)

    Hamilton, Eileen P; Kapusta, Aurélie; Huvos, Piroska E; Bidwell, Shelby L; Zafar, Nikhat; Tang, Haibao; Hadjithomas, Michalis; Krishnakumar, Vivek; Badger, Jonathan H; Caler, Elisabet V; Russ, Carsten; Zeng, Qiandong; Fan, Lin; Levin, Joshua Z; Shea, Terrance; Young, Sarah K; Hegarty, Ryan; Daza, Riza; Gujja, Sharvari; Wortman, Jennifer R; Birren, Bruce W; Nusbaum, Chad; Thomas, Jainy; Carey, Clayton M; Pritham, Ellen J; Feschotte, Cédric; Noto, Tomoko; Mochizuki, Kazufumi; Papazyan, Romeo; Taverna, Sean D; Dear, Paul H; Cassidy-Hanley, Donna M; Xiong, Jie; Miao, Wei; Orias, Eduardo; Coyne, Robert S

    2016-11-28

    The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

  5. Identification of eight novel SDHB, SDHC, SDHD germline variants in Danish pheochromocytoma/paraganglioma patients

    DEFF Research Database (Denmark)

    Bennedbæk, Marc; Rossing, Maria; Rasmussen, Åse K

    2016-01-01

    BACKGROUND: Germline mutations in the succinate dehydrogenase complex genes SDHB, SDHC, and SDHD predispose to pheochromocytomas and paragangliomas. Here, we examine the SDHB, SDHC, and SDHD mutation spectrum in the Danish population by screening of 143 Danish pheochromocytoma and paraganglioma...

  6. Defending the genome from the enemy within: mechanisms of retrotransposon suppression in the mouse germline.

    Science.gov (United States)

    Crichton, James H; Dunican, Donncha S; Maclennan, Marie; Meehan, Richard R; Adams, Ian R

    2014-05-01

    The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline.

  7. Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells

    Directory of Open Access Journals (Sweden)

    Karen M. Chapman

    2015-03-01

    Full Text Available Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate “pure,” non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.

  8. ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells

    Directory of Open Access Journals (Sweden)

    Tompkins Kathleen

    2010-10-01

    Full Text Available Abstract Background Unrepaired DNA double-stranded breaks (DSBs cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms. Results Here we investigate roles for the NHEJ factor ARTEMIS in multipotent mesenchymal stem/progenitor cells (MSCs, as putative sarcomagenic cells of origin. We demonstrate a key role for ARTEMIS in sarcoma suppression in a sensitized mouse tumor model. In this context, we found that ARTEMIS deficiency led to chromosomal damage but, paradoxically, enhanced resistance and proliferative potential in primary MSCs subjected to various stresses. Gene expression analysis revealed abnormally regulated stress response, cell proliferation, and signal transduction pathways in ARTEMIS-defective MSCs. Finally, we identified candidate regulatory genes that may, in part, mediate a stress-resistant, hyperproliferative phenotype in preneoplastic ARTEMIS-deficient MSCs. Conclusions Our discoveries suggest that Art prevents genome damage and restrains proliferation in MSCs exposed to various stress stimuli. We propose that deficiency leads to a preneoplastic state in primary MSCs and is associated with aberrant proliferative control and cellular stress

  9. A Fate Map of the Murine Pancreas Buds Reveals a Multipotent Ventral Foregut Organ Progenitor

    Science.gov (United States)

    Angelo, Jesse R.; Guerrero-Zayas, Mara-Isel; Tremblay, Kimberly D.

    2012-01-01

    The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2–4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification. PMID:22815796

  10. A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor.

    Directory of Open Access Journals (Sweden)

    Jesse R Angelo

    Full Text Available The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2-4 between the 2 and 11 somite stage (SS. The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL. Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.

  11. Multipotent mesenchymal stem cells from human subacromial bursa: potential for cell based tendon tissue engineering.

    Science.gov (United States)

    Song, Na; Armstrong, April D; Li, Feng; Ouyang, Hongsheng; Niyibizi, Christopher

    2014-01-01

    Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a

  12. Generation of multipotent early lymphoid progenitors from human embryonic stem cells.

    Science.gov (United States)

    Larbi, Aniya; Mitjavila-Garcia, Maria Teresa; Flamant, Stéphane; Valogne, Yannick; Clay, Denis; Usunier, Benoît; l'Homme, Bruno; Féraud, Olivier; Casal, Ibrahim; Gobbo, Emilie; Divers, Dominique; Chapel, Alain; Turhan, Ali G; Bennaceur-Griscelli, Annelise; Haddad, Rima

    2014-12-15

    During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34(+)CD45RA(+)CD7(-) HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34(+)CD45RA(+)CD7(+) HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs express genes of the lymphoid specification and that CD34(+)CD45RA(+)CD7(-) cells express B-cell-associated genes, while CD34(+)CD45RA(+)CD7(+) HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies.

  13. Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling.

    Science.gov (United States)

    Chen, Cheng-Yi; Tsai, Chin-Han; Chen, Chia-Yu; Wu, Yi-Hsin; Chen, Chie-Pein

    2016-03-03

    The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.

  14. Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation

    Directory of Open Access Journals (Sweden)

    Vidane AS

    2014-08-01

    Full Text Available Atanasio S Vidane,1 Aline F Souza,1 Rafael V Sampaio,1 Fabiana F Bressan,2 Naira C Pieri,1 Daniele S Martins,2 Flavio V Meirelles,2 Maria A Miglino,1 Carlos E Ambrósio2 1Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil; 2Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, São Paulo, Brazil Abstract: Amnion-derived mesenchymal stem cells (AMSCs are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases. Keywords: amnion, cats, cell differentiation, fetal membranes, mesenchymal cells

  15. Angiogenesis in differentiated placental multipotent mesenchymal stromal cells is dependent on integrin alpha5beta1.

    Directory of Open Access Journals (Sweden)

    Ming-Yi Lee

    Full Text Available Human placental multipotent mesenchymal stromal cells (hPMSCs can be isolated from term placenta, but their angiogenic ability and the regulatory pathways involved are not known. hPMSCs were shown to express integrins alpha(v, alpha(4, alpha(5, beta(1, beta(3, and beta(5 and could be induced to differentiate into cells expressing endothelial markers. Increases in cell surface integrins alpha(5 and beta(1, but not alpha(4, alpha(vbeta(3, or alpha(vbeta(5, accompanied endothelial differentiation. Vascular endothelial growth factor-A augmented the effect of fibronectin in enhancing adhesion and migration of differentiated hPMSC through integrin alpha(5beta(1, but not alpha(vbeta(3 or alpha(vbeta(5. Formation of capillary-like structures in vitro from differentiated cells was inhibited by pre-treatment with function-blocking antibodies to integrins alpha(5 and beta(1. When hPMSCs were seeded onto chick chorioallantoic membranes (CAM, human von Willebrand factor-positive cells were observed to engraft in the chick endothelium. CAMs transplanted with differentiated hPMSCs had a greater number of vessels containing human cells and more incorporated cells per vessel compared to CAMs transplanted with undifferentiated hPMSCs, and overall angiogenesis was enhanced more by the differentiated cells. Function-blocking antibodies to integrins alpha(5 and beta(1 inhibited angiogenesis in the CAM assay. These results suggest that differentiated hPMSCs may contribute to blood vessel formation, and this activity depends on integrin alpha(5beta(1.

  16. Gene markers of cellular aging in human multipotent stromal cells in culture.

    Science.gov (United States)

    Bellayr, Ian H; Catalano, Jennifer G; Lababidi, Samir; Yang, Amy X; Lo Surdo, Jessica L; Bauer, Steven R; Puri, Raj K

    2014-04-28

    Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs

  17. Investigating the role of tbx4 in the female germline in mice.

    Science.gov (United States)

    Douglas, Nataki C; Arora, Ripla; Chen, Cayla Yiyu; Sauer, Mark V; Papaioannou, Virginia E

    2013-12-01

    Normal development of germ cells is essential for fertility and mammalian reproduction. Although abnormal development of oocytes or follicles may lead to primary ovarian insufficiency (POI), a disorder that causes infertility in 1% of women less than 40 yr of age, the genes and signaling pathways activated in POI are not as yet fully elucidated. Tbx4, a member of the T-box family of transcription factors, is expressed in embryonic germ cells and postnatal oocytes at all stages of folliculogenesis. To investigate the requirement for Tbx4 in the germline, we analyzed germ cell development in the absence of Tbx4. We show that primordial germ cells (PGCs) are reduced in Tbx4 homozygous null (Tbx4(-/-)) embryos at Embryonic Day (E) 10.0. Tbx4(-/-) embryos die by E10.5; to study later time points in vitro, a tamoxifen-inducible estrogen receptor Cre recombinase was used to delete Tbx4 conditional mutant alleles. In addition, Gdf9cre and Zp3cre, two oocyte-specific Cre recombinases, were used to delete Tbx4 from postnatal primordial and primary follicles, respectively. We show that in vitro differentiation of the gonad into morphologically distinct testes and ovaries occurs normally starting at E11.5 when Tbx4 is deleted. In Gdf9cre; Tbx4(fl/-) and Zp3cre; Tbx4(fl/-) adult females, primordial, primary, secondary, and antral follicles form, ovulation occurs, corpus luteum formation is normal, and the mice are fertile without any evidence of diminished ovarian reserve. Although postnatal deletion of Tbx4 in oocytes does not obviously impair fertility, it is possible that the reduction in PGCs observed in Tbx4 homozygous null mutant embryos could affect long-term fertility in adults.

  18. Familial Myelodysplastic/Acute Leukemia Syndromes—Myeloid Neoplasms with Germline Predisposition

    Science.gov (United States)

    Baptista, Renata Lyrio Rafael; dos Santos, Anna Cláudia Evangelista; Gutiyama, Luciana Mayumi; Solza, Cristiana; Zalcberg, Ilana Renault

    2017-01-01

    Although most cases of myeloid neoplasms are sporadic, a small subset has been associated with germline mutations. The 2016 revision of the World Health Organization classification included these cases in a myeloid neoplasm group with a predisposing germline mutational background. These patients must have a different management and their families should get genetic counseling. Cases identification and outline of the major known syndromes characteristics will be discussed in this text. PMID:28955657

  19. Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.

    Science.gov (United States)

    Flanagan, Sarah E; Haapaniemi, Emma; Russell, Mark A; Caswell, Richard; Lango Allen, Hana; De Franco, Elisa; McDonald, Timothy J; Rajala, Hanna; Ramelius, Anita; Barton, John; Heiskanen, Kaarina; Heiskanen-Kosma, Tarja; Kajosaari, Merja; Murphy, Nuala P; Milenkovic, Tatjana; Seppänen, Mikko; Lernmark, Åke; Mustjoki, Satu; Otonkoski, Timo; Kere, Juha; Morgan, Noel G; Ellard, Sian; Hattersley, Andrew T

    2014-08-01

    Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.

  20. Breast cancer risk and clinical implications for germline PTEN mutation carriers.

    Science.gov (United States)

    Ngeow, Joanne; Sesock, Kaitlin; Eng, Charis

    2017-08-01

    PTEN Hamartoma Tumor syndrome (PHTS) encompasses a clinical spectrum of heritable disorders including Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and Proteus and Proteus-like syndrome that are associated with germline mutations in the PTEN tumor suppressor gene. Breast cancer risk estimates (67-85 %) for women with germline PTEN mutations are similar to those quoted for patients with germline mutations in the BRCA1/2 genes. With PTEN on several germline gene testing panels, finding PTEN mutations and variants have increased exponentially. PHTS can be differentiated from other hereditary cancer syndromes including Hereditary Breast Ovarian Cancer syndrome, Lynch syndrome, and hamartomatous polyposis syndromes based on personal as well as family history. However, many of the benign features of CS are common in the general population, making the diagnosis of CS challenging. Breast cancer patients with an identified germline PTEN mutation are at increased risk of endometrial, thyroid, renal, and colorectal cancers as well as a second breast cancer. Increased screening for the various component cancers as well as predictive testing in first-degree relatives is recommended. Prophylactic mastectomy may be considered especially if breast tissue is dense or if repeated breast biopsies have been necessary. Management of women with breast cancer suspected of CS who test negative for germline PTEN mutations should be managed as per a mutation carrier if she meets CS diagnostic criteria, and should be offered enrollment in research to identify other predisposition genes.

  1. Germline Mutations in Predisposition Genes in Pediatric Cancer

    Science.gov (United States)

    Edmonson, Michael N.; Gruber, Tanja A.; Easton, John; Hedges, Dale; Ma, Xiaotu; Zhou, Xin; Yergeau, Donald A.; Wilkinson, Mark R.; Vadodaria, Bhavin; Chen, Xiang; McGee, Rose B.; Hines-Dowell, Stacy; Nuccio, Regina; Quinn, Emily; Shurtleff, Sheila A.; Rusch, Michael; Patel, Aman; Becksfort, Jared B.; Wang, Shuoguo; Weaver, Meaghann S.; Ding, Li; Mardis, Elaine R.; Wilson, Richard K.; Gajjar, Amar; Ellison, David W.; Pappo, Alberto S.; Pui, Ching-Hon; Downing, James R.

    2016-01-01

    BACKGROUND The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. METHODS In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism). RESULTS Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer. CONCLUSIONS Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American

  2. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

    Science.gov (United States)

    Chandrakanthan, Vashe; Yeola, Avani; Kwan, Jair C.; Oliver, Rema A.; Qiao, Qiao; Kang, Young Chan; Zarzour, Peter; Beck, Dominik; Boelen, Lies; Unnikrishnan, Ashwin; Villanueva, Jeanette E.; Nunez, Andrea C.; Knezevic, Kathy; Palu, Cintia; Nasrallah, Rabab; Carnell, Michael; Macmillan, Alex; Whan, Renee; Yu, Yan; Hardy, Philip; Grey, Shane T.; Gladbach, Amadeus; Delerue, Fabien; Ittner, Lars; Mobbs, Ralph; Walkley, Carl R.; Purton, Louise E.; Ward, Robyn L.; Wong, Jason W. H.; Hesson, Luke B.; Walsh, William; Pimanda, John E.

    2016-01-01

    Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration. PMID:27044077

  3. Synergistic Effects of a Mixture of Glycosaminoglycans to Inhibit Adipogenesis and Enhance Chondrocyte Features in Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Petar D. Petrov

    2015-11-01

    Full Text Available Background/Aims: Multipotent mesenchymal stem cells affect homeostasis of adipose and joint tissues. Factors influencing their differentiation fate are of interest for both obesity and joint problems. We studied the impact of a mixture of glycosaminoglycans (GAGs (hyaluronic acid: dermatan sulfate 1:0.25, w/w used in an oral supplement for joint discomfort (Oralvisc™ on the differentiation fate of multipotent cells. Methods: Primary mouse embryo fibroblasts (MEFs were used as a model system. Post-confluent monolayer MEF cultures non-stimulated or hormonally stimulated to adipogenesis were chronically exposed to the GAGs mixture, its individual components or vehicle. The appearance of lipid laden cells, lipid accumulation and expression of selected genes at the mRNA and protein level was assessed. Results: Exposure to the GAGs mixture synergistically suppressed spontaneous adipogenesis and induced the expression of cartilage extracellular matrix proteins, aggrecan core protein, decorin and cartilage oligomeric matrix protein. Hormonally-induced adipogenesis in the presence of the GAGs mixture resulted in decreased adipogenic differentiation, down-regulation of adipogenic/lipogenic factors and genes for insulin resistance-related adipokines (resistin and retinol binding protein 4, and up-regulation of oxidative metabolism-related genes. Adipogenesis in the presence of dermatan sulfate, the minor component of the mixture, was not impaired but resulted in smaller lipid droplets and the induction of a more complete brown adipocyte-related transcriptional program in the cells in the adipose state. Conclusions: The Oralvisc™ GAGs mixture can tip the adipogenic/chondrogenic fate balance of multipotent cells away from adipogenesis while favoring chondrocyte related gene expression. The mixture and its dermatan sulfate component also have modulatory effects of interest on hormonally-induced adipogenesis and on metabolic and secretory capabilities of

  4. Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells

    OpenAIRE

    2015-01-01

    Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSC) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass ...

  5. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis.

    Science.gov (United States)

    Iohara, Koichiro; Zheng, Li; Ito, Masataka; Tomokiyo, Atsushi; Matsushita, Kenji; Nakashima, Misako

    2006-11-01

    Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor gamma and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

  6. Germline Chd8 haploinsufficiency alters brain development in mouse.

    Science.gov (United States)

    Gompers, Andrea L; Su-Feher, Linda; Ellegood, Jacob; Copping, Nycole A; Riyadh, M Asrafuzzaman; Stradleigh, Tyler W; Pride, Michael C; Schaffler, Melanie D; Wade, A Ayanna; Catta-Preta, Rinaldo; Zdilar, Iva; Louis, Shreya; Kaushik, Gaurav; Mannion, Brandon J; Plajzer-Frick, Ingrid; Afzal, Veena; Visel, Axel; Pennacchio, Len A; Dickel, Diane E; Lerch, Jason P; Crawley, Jacqueline N; Zarbalis, Konstantinos S; Silverman, Jill L; Nord, Alex S

    2017-08-01

    The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8(+/del5) mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8(+/del5) mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8(+/del5) mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8(+/del5) mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.

  7. Interspecific germline transmission of cultured primordial germ cells.

    Directory of Open Access Journals (Sweden)

    Marie-Cecile van de Lavoir

    Full Text Available In birds, the primordial germ cell (PGC lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus and the recipient species is guinea fowl (Numida meleagris, a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund.

  8. Germline transmission of exogenous genes in the chicken.

    Science.gov (United States)

    Bosselman, R A; Hsu, R Y; Boggs, T; Hu, S; Bruszewski, J; Ou, S; Kozar, L; Martin, F; Green, C; Jacobsen, F

    1989-01-27

    Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.

  9. Thyroid Follicle Formation and Thyroglobulin Expression in Multipotent Endodermal Stem Cells

    Science.gov (United States)

    Latif, Rauf; Davies, Terry F.

    2013-01-01

    Objective The aim of this study was to assess the impact of transcriptional induction on thyroid follicular cell (TFC) differentiation from endodermally matured embryonic stem (ES) cells. The thyroid transcription factors—NKx2 homeobox 1 (NKx2-1, formerly called TTF-1) and Paired box gene 8 (Pax8)—are known to associate biochemically and synergistically in the activation of thyroid functional genes including the sodium/iodide symporter (NIS), thyrotropin (TSH) receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO) genes. In this study, we investigated the ability of ectopically expressed Pax8 and NKx2-1 to further the induction and differentiation of murine ES cells into potential TFCs. Methods ES cells were stably transfected with either the Pax8 gene, the NKx2-1 gene, or both genes to study the induction of NIS, TSHR, Tg, and TPO genes as assessed using both quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and protein expression. The derived cells were cultured with or without the presence of activin A to allow their differentiation into multipotent endodermal cells. Results The four thyroid-specific genes NIS, TSHR, Tg, and TPO were all significantly activated by expressing both transcription factors within the same ES cell. In contrast, significant but much lower transcriptional activity of the TSHR, Tg, and TPO genes was detected in cells expressing just NKx2-1, and only the NIS and TSHR genes responded to Pax8 alone. No Tg protein expression could be detected prior to their development into endodermal derivatives. However, after further differentiation of postembryoid body ES cells with activin A and TSH into endodermal cell lines, those cells with dual transfection of Pax8 and NKx2-1 demonstrated greatly enhanced expression of the NIS, TSHR, Tg, and TPO genes to such a degree that it was similar to that found in control thyroid cells. Furthermore, these same cells formed three-dimensional neofollicles in vitro and

  10. Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

    Energy Technology Data Exchange (ETDEWEB)

    Curtis, Brandon M.; Leix, Kyle Alexander [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States); Ji, Yajing [Department of Biomedical Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States); Glaves, Richard Samuel Elliot [Department of Biology, Central Michigan University, Mount Pleasant, MI 48859 (United States); Ash, David E. [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States); Mohanty, Dillip K., E-mail: Mohan1dk@cmich.edu [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States)

    2014-07-18

    Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.

  11. Chronic activation of pattern recognition receptors suppresses brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes.

    Science.gov (United States)

    Bae, Jiyoung; Chen, Jiangang; Zhao, Ling

    2015-06-01

    Brown adipose tissue (BAT) holds promise to combat obesity through energy-spending, non-shivering thermogenesis. Understanding of regulation of BAT development can lead to novel strategies to increase BAT mass and function for obesity treatment and prevention. Here, we report the effects of chronic activation of PRR on brown adipogenesis of multipotent mesodermal stem C3H10T1/2 cells and immortalized brown pre-adipocytes from the classical interscapular BAT of mice. Activation of NOD1, TLR4, or TLR2 by their respective synthetic ligand suppressed brown marker gene expression and lipid accumulation during differentiation of brown-like adipocytes of C3H10T1/2. Activation of the PRR only during the commitment was sufficient to suppress the differentiation. PRR activation suppressed PGC-1α mRNA, but induced PRDM16 mRNA at the commitment. Consistently, PRR activation suppressed the differentiation of immortalized brown pre-adipocytes. Activation of PRR induced NF-κB activation in both cells, which correlated with their abilities to suppress PPARγ transactivation, a critical event for brown adipogenesis. Taken together, our results demonstrate that chronic PRR activation suppressed brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes, possibly through suppression of PPARγ transactivation. The results suggest that anti- inflammatory therapies targeting PRRs may be beneficial for the BAT development.

  12. Induced multipotency in adult keratinocytes through down-regulation of ΔNp63 or DGCR8

    OpenAIRE

    Chakravarti, Deepavali; Su, Xiaohua; Cho, Min Soon; Bui, Ngoc Hoang Bao; Coarfa, Cristian; Venkatanarayan, Avinashnarayan; Ashley L Benham; Flores González, Ramón E.; Alana, Jennifer; Xiao, Weimin; Leung, Marco L.; Vin, Harina; Chan, Io Long; Aquino, Arianexys; Müller, Nicole

    2014-01-01

    The p53 family member deltaNp63 (ΔNp63) is required for transcriptional activation of the microprocessor complex subunit DGCR8 in epidermal cells, leading to terminal differentiation of tissues such as the epidermis. We show here that loss of ΔNp63 leads to the generation of cells with self-renewing but limited differentiation capacity. When DGCR8 is reexpressed in cells deficient for ΔNp63, these cells can terminally differentiate into all three germ layers. We dubbed these cells induced mul...

  13. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality.

    Science.gov (United States)

    Buckley, Bethany A; Burkhart, Kirk B; Gu, Sam Guoping; Spracklin, George; Kershner, Aaron; Fritz, Heidi; Kimble, Judith; Fire, Andrew; Kennedy, Scott

    2012-09-20

    Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

  14. Characterisation and germline transmission of cultured avian primordial germ cells.

    Directory of Open Access Journals (Sweden)

    Joni Macdonald

    Full Text Available BACKGROUND: Avian primordial germ cells (PGCs have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. PRINCIPAL FINDINGS: We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. CONCLUSIONS: The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

  15. Germline copy number variation and ovarian cancer survival

    Directory of Open Access Journals (Sweden)

    Brooke L Fridley

    2012-08-01

    Full Text Available Copy number variants (CNVs have been implicated in many complex diseases. We examined whether inherited CNVs were associated with overall survival among women with invasive epithelial ovarian cancer. Germline DNA from 1,056 cases (494 deceased, average of 3.7 years follow-up was interrogated with the Illumina 610quad genome-wide array containing, after quality control exclusions, 581,903 single nucleotide polymorphisms (SNPs and 17,917 CNV probes. Comprehensive analysis capitalized upon the strengths of three complementary approaches to CNV classification. First, to identify small CNVs, single markers were evaluated and, where associated with survival, consecutive markers were combined. Two chromosomal regions were associated with survival using this approach (14q31.3 rs2274736 p=1.59x10-6, p=0.001; 22q13.31 rs2285164 p=4.01x10-5, p=0.009, but were not significant after multiple testing correction. Second, to identify large CNVs, genome-wide segmentation was conducted to characterize chromosomal gains and losses, and association with survival was evaluated by segment. Four regions were associated with survival (1q21.3 loss p=0.005, 5p14.1 loss p=0.004, 9p23 loss p=0.002, and 15q22.31 gain p=0.002; however, again, after correcting for multiple testing, no regions were statistically significant, and none were in common with the single-marker approach. Finally, to evaluate associations with general amounts of copy number changes across the genome, we estimated CNV burden based on genome-wide numbers of gains and losses; no associations with survival were observed (p>0.40. Although CNVs that were not well-covered by the Illumina 610quad array merit investigation, these data suggest no association between inherited CNVs and survival after ovarian cancer.

  16. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    Science.gov (United States)

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  17. Transgenerational epigenetic imprinting of the male germline by endocrine disruptor exposure during gonadal sex determination.

    Science.gov (United States)

    Chang, Hung-Shu; Anway, Matthew D; Rekow, Stephen S; Skinner, Michael K

    2006-12-01

    Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.

  18. Pan-cancer analysis reveals technical artifacts in TCGA germline variant calls.

    Science.gov (United States)

    Buckley, Alexandra R; Standish, Kristopher A; Bhutani, Kunal; Ideker, Trey; Lasken, Roger S; Carter, Hannah; Harismendy, Olivier; Schork, Nicholas J

    2017-06-12

    Cancer research to date has largely focused on somatically acquired genetic aberrations. In contrast, the degree to which germline, or inherited, variation contributes to tumorigenesis remains unclear, possibly due to a lack of accessible germline variant data. Here we called germline variants on 9618 cases from The Cancer Genome Atlas (TCGA) database representing 31 cancer types. We identified batch effects affecting loss of function (LOF) variant calls that can be traced back to differences in the way the sequence data were generated both within and across cancer types. Overall, LOF indel calls were more sensitive to technical artifacts than LOF Single Nucleotide Variant (SNV) calls. In particular, whole genome amplification of DNA prior to sequencing led to an artificially increased burden of LOF indel calls, which confounded association analyses relating germline variants to tumor type despite stringent indel filtering strategies. The samples affected by these technical artifacts include all acute myeloid leukemia and practically all ovarian cancer samples. We demonstrate how technical artifacts induced by whole genome amplification of DNA can lead to false positive germline-tumor type associations and suggest TCGA whole genome amplified samples be used with caution. This study draws attention to the need to be sensitive to problems associated with a lack of uniformity in data generation in TCGA data.

  19. Transposition and gene disruption in the male germline of the mouse.

    Science.gov (United States)

    Dupuy, A J; Fritz, S; Largaespada, D A

    2001-06-01

    We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation-site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)-trap transposon vectors, were outcrossed to wild-type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics.

  20. Transgenerational effects of proton beam irradiation on Caenorhabditis elegans germline apoptosis.

    Science.gov (United States)

    Min, Hyemin; Sung, Minhee; Son, Miseol; Kawasaki, Ichiro; Shim, Yhong-Hee

    2017-08-26

    When treating cancer using radiation therapy, it is critical to increase patient survival rates and to reduce side effects. In this respect, proton beam radiation treatment performs better than other radiation treatments because of its high target specificity. However, complications still remain after proton beam radiation treatment. Among them, the risk to progeny after irradiation of their parents is a major concern. In this study, we analyzed the transgenerational effects of proton beam irradiation using the model organism Caenorhabditis. elegans. We found that germline apoptosis increased after proton beam irradiation and its effects were sustained transgenerationally. Moreover, we identified that a germline-specific histone methyltransferase component, SET-2, has a critical role in transmitting the transgenerational effect on germline apoptosis to the next generation after proton beam irradiation. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Germline mosaicism in osteopathia striata with cranial sclerosis--recurrence in siblings.

    Science.gov (United States)

    O'Byrne, James J; Phelan, Ethna; Steenackers, Ellen; van Hul, Wim; Reardon, William

    2016-04-01

    We report recurrence of osteopathia striata with cranial sclerosis (OSCS) in two full siblings conceived by unaffected parents. Molecular confirmation of OSCS in both siblings was achieved by identification of a novel heterozygous mutation in the WTX gene. Neither parent had clinical features of OSCS nor was the pathogenic mutation demonstrable in DNA extracted from both peripheral blood leucocytes and buccal cells. This case demonstrates germline mosaicism in OSCS and represents the third report of mosaicism affecting the germline in families with OSCS. Previous reports were of parental gonadosomal mosaicism, with one showing recurrence in multiple children. Our observation adds to a body of evidence that suggests that germline mosaicism in OSCS may occur more frequently than believed previously and may have implications for counselling families with OSCS.

  2. PUF-8, a Pumilio homolog, inhibits the proliferative fate in the Caenorhabditis elegans germline.

    Science.gov (United States)

    Racher, Hilary; Hansen, Dave

    2012-10-01

    Stem cell populations are maintained by keeping a balance between self-renewal (proliferation) and differentiation of dividing stem cells. Within the Caenorhabditis elegans germline, the key regulator maintaining this balance is the canonical Notch signaling pathway, with GLP-1/Notch activity promoting the proliferative fate. We identified the Pumilio homolog, PUF-8, as an inhibitor of the proliferative fate of stem cells in the C. elegans germline. puf-8(0) strongly enhances overproliferation of glp-1(gf) mutants and partially suppresses underproliferation of a weak glp-1(lf) mutant. The germline tumor that is formed in a puf-8(0); glp-1(gf) double mutant is due to a failure of germ cells to enter meiotic prophase. puf-8 likely inhibits the proliferative fate through negatively regulating GLP-1/Notch signaling or by functioning parallel to it.

  3. Ascl3 marks adult progenitor cells of the mouse salivary gland.

    Science.gov (United States)

    Rugel-Stahl, Anastasia; Elliott, Marilyn E; Ovitt, Catherine E

    2012-05-01

    The Ascl3 transcription factor marks a subset of salivary gland duct cells present in the three major salivary glands of the mouse. In vivo, these cells generate both duct and secretory acinar cell descendants. Here, we have analyzed whether Ascl3-expressing cells retain this multipotent lineage potential in adult glands. Cells isolated from mouse salivary glands were cultured in vitro as non-adherent spheres. Lineage tracing of the Ascl3-expressing cells within the spheres demonstrates that Ascl3+ cells isolated from adult glands remain multipotent, generating both duct and acinar cell types in vitro. Furthermore, we demonstrate that the progenitor cells characterized by Keratin 5 expression are an independent population from Ascl3+ progenitor cells. We conclude that the Ascl3+ cells are intermediate lineage-restricted progenitor cells of the adult salivary glands.

  4. NF1 germline mutation differentially dictates optic glioma formation and growth in neurofibromatosis-1.

    Science.gov (United States)

    Toonen, Joseph A; Anastasaki, Corina; Smithson, Laura J; Gianino, Scott M; Li, Kairong; Kesterson, Robert A; Gutmann, David H

    2016-05-01

    Neurofibromatosis type 1 (NF1) is a common neurogenetic condition characterized by significant clinical heterogeneity. A major barrier to developing precision medicine approaches for NF1 is an incomplete understanding of the factors that underlie its inherent variability. To determine the impact of the germline NF1 gene mutation on the optic gliomas frequently encountered in children with NF1, we developed genetically engineered mice harboring two representative NF1-patient-derived Nf1 gene mutations (c.2542G>C;p.G848R and c.2041C>T;p.R681X). We found that each germline Nf1 gene mutation resulted in different levels of neurofibromin expression. Importantly, only R681X(CKO) but not G848R(CKO), mice develop optic gliomas with increased optic nerve volumes, glial fibrillary acid protein immunoreactivity, proliferation and retinal ganglion cell death, similar to Nf1 conditional knockout mice harboring a neomycin insertion (neo) as the germline Nf1 gene mutation. These differences in optic glioma phenotypes reflect both cell-autonomous and stromal effects of the germline Nf1 gene mutation. In this regard, primary astrocytes harboring the R681X germline Nf1 gene mutation exhibit increased basal astrocyte proliferation (BrdU incorporation) indistinguishable from neo(CKO) astrocytes, whereas astrocytes with the G848R mutation have lower levels of proliferation. Evidence for paracrine effects from the tumor microenvironment were revealed when R681X(CKO) mice were compared with conventional neo(CKO) mice. Relative to neo(CKO) mice, the optic gliomas from R681X(CKO) mice had more microglia infiltration and JNK(Thr183/Tyr185) activation, microglia-produced Ccl5, and glial AKT(Thr308) activation. Collectively, these studies establish that the germline Nf1 gene mutation is a major determinant of optic glioma development and growth through by both tumor cell-intrinsic and stromal effects.

  5. Germline mutation analysis of MLH1 and MSH2 in Malaysian Lynch syndrome patients

    Institute of Scientific and Technical Information of China (English)

    Mohd Nizam Zahary; Gurjeet Kaur; Muhammad Radzi Abu Hassan; Harjinder Singh; Venkatesh R Naik; Ravindran Ankathil

    2012-01-01

    AIM:To investigate the protein expression profile of mismatch repair (MMR) genes in suspected cases of Lynch syndrome and to characterize the associated germline mutations.METHODS:Immunohistochemical analysis of tumor samples was performed to determine the protein expression profile of MMR protein.Germline mutation screening was carried out on peripheral blood samples.The entire exon regions of MLH1 and MSH2 genes were amplified by polymerase chain reaction,screened by denaturing high performance liquid chromatography (dHPLC) and analyzed by DNA sequencing to characterize the germline mutations.RESULTS:Three out of 34 tissue samples (8.8%) and four out of 34 tissue samples (11.8%) showed loss of nuclear staining by immunohistochemistry,indicating the absence of MLH1 and MSH2 protein expression in carcinoma cells,respectively.dHPLC analysis followed by DNA sequencing showed these samples to have germline mutations of MSH2 gene.However,no deleterious mutations were identified in any of the 19 exons or coding regions of MLH1 gene,but we were able to identify MLH1 promoter polymorphism,-93G >A (rs1800734),in 21 out of 34 patients (61.8%).We identified one novel mutation,transversion mutation c.2005G > C,which resulted in a missense mutation (Gly669Arg),a transversion mutation in exon 1,c.142G > T,which resulted in a nonsense mutation (Glu48Stop)and splice-site mutation,c.2006-6T > C,which was adjacent to exon 13 of MSH2 gene.CONCLUSION:Germline mutations were identified in four Malaysian Lynch syndrome patients.Immunohistochemical analysis of tumor tissue proved to be a good pre-screening test before proceeding to germline mutation analysis of DNA MMR genes.

  6. Simultaneous Analysis of Germline Crips and Snips in High Risk Prostate Cancer Families

    Science.gov (United States)

    2007-10-01

    AUTHOR(S) Jianfeng Xu, M.D., Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: jxu@wfubmc.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION...support that we can use the panel to accurately identify germline CNPs. Identifying germline CNPs among 48 HapMap subjects using Affymetrix 500K...dataset, which contains allele intensity data for 48 individuals (including 5 HapMap CEPH trios, 5 Yoruban trios, three other non- HapMap trios, and 9

  7. Identification of Germline Genetic Mutations in Pancreatic Cancer Patients

    Science.gov (United States)

    Salo-Mullen, Erin E.; O’Reilly, Eileen; Kelsen, David; Ashraf, Asad M.; Lowery, Maeve; Yu, Kenneth; Reidy, Diane; Epstein, Andrew S.; Lincoln, Anne; Saldia, Amethyst; Jacobs, Lauren M.; Rau-Murthy, Rohini; Zhang, Liying; Kurtz, Robert; Saltz, Leonard; Offit, Kenneth; Robson, Mark; Stadler, Zsofia K.

    2016-01-01

    Background Pancreatic adenocarcinoma (PAC) is part of several cancer predisposition syndromes; however, indications for genetic counseling/testing are not well-defined. We sought to determine mutation prevalence and characteristics that predict for inherited predisposition to PAC. Methods We identified 175 consecutive PAC patients who underwent clinical genetics assessment at Memorial Sloan Kettering between 2011–2014. Clinical data, family history, and germline results were evaluated. Results Among 159 PAC patients who pursued genetic testing, 24 pathogenic mutations were identified (15.1%; 95%CI, 9.5%–20.7%), including BRCA2(n=13), BRCA1(n=4), p16(n=2), PALB2(n=1), and Lynch syndrome(n=4). BRCA1/BRCA2 prevalence was 13.7% in Ashkenazi Jewish(AJ) (n=95) and 7.1% in non-AJ(n=56) patients. In AJ patients with strong, weak, or absent family history of BRCA-associated cancers, mutation prevalence was 16.7%, 15.8%, and 7.4%, respectively. Mean age at diagnosis in all mutation carriers was 58.5y(range 45–75y) compared to 64y(range 27–87y) in non-mutation carriers(P=0.02). Although BRCA2 was the most common mutation identified, no patients with early-onset PAC(≤50y) harbored a BRCA2 mutation and the mean age at diagnosis in BRCA2 carriers was equivalent to non-mutation carriers(P=0.34). Mutation prevalence in early-onset patients(n=21) was 28.6%, including BRCA1(n=2), p16(n=2), MSH2(n=1) and MLH1(n=1). Conclusion Mutations in BRCA2 account for over 50% of PAC patients with an identified susceptibility syndrome. AJ patients had high BRCA1/BRCA2 prevalence regardless of personal/family history, suggesting that ancestry alone indicates a need for genetic evaluation. With the exception of BRCA2-associated PAC, inherited predisposition to PAC is associated with earlier age at PAC diagnosis suggesting that this subset of patients may also represent a population warranting further evaluation. PMID:26440929

  8. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity.

    Science.gov (United States)

    Attaf, Meriem; Holland, Stephan J; Bartok, Istvan; Dyson, Julian

    2016-10-24

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface.

  9. Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status

    Directory of Open Access Journals (Sweden)

    de Carvalho Marcelo

    2011-03-01

    Full Text Available Abstract Background In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions. Results Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2 versus hypoxia (5% O2 for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1 were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold involved in extracellular matrix assembly (SMOC2, neural and muscle development (NOG, GPR56, SNTG2, LAMA and epithelial development (DMKN. This group described herein for the first time was assigned by the Gene Ontology program to "plasticity". Conclusion The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.

  10. Adult neural stem cells in the mammalian central nervous system

    Institute of Scientific and Technical Information of China (English)

    Dengke K Ma; Michael A Bonaguidi; Guo-li Ming; Hongjun Song

    2009-01-01

    Neural stem cells (NSCs) are present not only during the embryonic development but also in the adult brain of all mammalian species, including humans. Stem cell niche architecture in vivo enables adult NSCs to continuously generate functional neurons in specific brain regions throughout life. The adult neurogenesis process is subject to dynamic regulation by various physiological, pathological and pharmacological stimuli. Multipotent adult NSCs also appear to be intrinsically plastic, amenable to genetic programing during normal differentiation, and to epigenetic reprograming during de-differentiation into pluripotency. Increasing evidence suggests that adult NSCs significantly contribute to specialized neural functions under physiological and pathological conditions. Fully understanding the biology of adult NSCs will provide crucial insights into both the etiology and potential therapeutic interventions of major brain disorders. Here, we review recent progress on adult NSCs of the mammalian central nervous system, in-cluding topics on their identity, niche, function, plasticity, and emerging roles in cancer and regenerative medicine.

  11. Protection of germline gene expression by the C. elegans Argonaute CSR-1.

    Science.gov (United States)

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2013-12-23

    In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

  12. A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs.

    Directory of Open Access Journals (Sweden)

    Allison C Billi

    Full Text Available Piwi-interacting RNAs (piRNAs fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.

  13. Functional analysis of Tudor-domain-containing proteins in the zebrafish germline

    NARCIS (Netherlands)

    Huang, H.Y.

    2012-01-01

    The Argonaute protein family consists of two distinct sub-clades: AGO and PIWI proteins. AGO proteins can form RNA induced silencing complex (RISC) and are responsible for RNAi and miRNA pathways. PIWI proteins are mostly expressed in the germline and together with specific small RNA groups, named

  14. Prophylactic Laparoscopic Total Gastrectomy with Jejunal Pouch Reconstruction in Patients Carrying a CDH1 Germline Mutation

    NARCIS (Netherlands)

    Haverkamp, L.; Sluis, P.C. van der; Ausems, M.G.; Horst, S. van der; Siersema, P.D.; Ruurda, J.P.; Offerhaus, G.J.; Hillegersberg, R. van

    2015-01-01

    BACKGROUND: For patients with an identified germline E-cadherin-1 (CDH1) mutation, prophylactic gastrectomy is the treatment of choice to eliminate the high risk of developing diffuse gastric cancer. Laparoscopic total gastrectomy with jejunal pouch reconstruction is a novel approach that may be esp

  15. Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers

    NARCIS (Netherlands)

    Post, R.S. van der; Vogelaar, I.P.; Carneiro, F.; Guilford, P.; Huntsman, D.; Hoogerbrugge, N.; Caldas, C.; Schreiber, K.E.; Hardwick, R.H.; Ausems, M.G.; Bardram, L.; Benusiglio, P.R.; Bisseling, T.M.; Blair, V.; Bleiker, E.; Boussioutas, A.; Cats, A.; Coit, D.; DeGregorio, L.; Figueiredo, J.; Ford, J.M.; Heijkoop, E.; Hermens, R.; Humar, B.; Kaurah, P.; Keller, G.; Lai, J.; Ligtenberg, M.J.; O'Donovan, M.; Oliveira, C.; Pinheiro, H.; Ragunath, K.; Rasenberg, E.; Richardson, S.; Roviello, F.; Schackert, H.; Seruca, R.; Taylor, A.; Huurne, A. Ter; Tischkowitz, M.; Joe, S.T.; Dijck, B. van; Grieken, N.C. van; Hillegersberg, R. van; Sandick, J.W. van; Vehof, R.; Krieken, J.H.J.M. van; Fitzgerald, R.C.

    2015-01-01

    Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, inc

  16. Morphological predictors of BRCA1 germline mutations in young women with breast cancer

    NARCIS (Netherlands)

    Southey, M. C.; Ramus, S. J.; Dowty, J. G.; Smith, L. D.; Tesoriero, A. A.; Wong, E. E. M.; Dite, G. S.; Jenkins, M. A.; Byrnes, G. B.; Winship, I.; Phillips, K-A; Giles, G. G.; Hopper, J. L.

    2011-01-01

    BACKGROUND: Knowing a young woman with newly diagnosed breast cancer has a germline BRCA1 mutation informs her clinical management and that of her relatives. We sought an optimal strategy for identifying carriers using family history, breast cancer morphology and hormone receptor status data. METHOD

  17. Neurofibromatosis type 1 in two siblings due to maternal germline mosaicism.

    Science.gov (United States)

    Trevisson, E; Forzan, M; Salviati, L; Clementi, M

    2014-04-01

    Neurofibromatosis type 1 (NF1) is caused by loss of function mutations of the NF1 gene, which are de novo in 50% of cases. Although this gene shows one of the highest mutation rates in the human genome, germline mosaicism is very rare in this condition. We describe the molecular analysis of a family in which neurofibromatosis type 1 occurred in two out of four siblings born to unaffected parents. Molecular analysis of the NF1 gene identified in both patients the same splicing mutation c.1392+1G>A, which was absent in parental lymphocytes. Microsatellite analysis showed that the two affected siblings shared the same maternal allele, however a specific PCR-RFLP assay excluded the presence of the NF1 splicing mutation in multiple maternal tissues. Our molecular and clinical findings are consistent with a germline mosaicism for the NF1 splicing mutation. This is the first case of maternal germline mosaicism for a NF1 mutation characterized so far at the molecular level. Our data confirm that germline mosaicism is rare in neurofibromatosis 1, but it has important implications for genetic counseling.

  18. Elucidating the impact of neurofibromatosis-1 germline mutations on neurofibromin function and dopamine-based learning.

    Science.gov (United States)

    Anastasaki, Corina; Woo, Albert S; Messiaen, Ludwine M; Gutmann, David H

    2015-06-15

    Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate neurofibromin function in the brain. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. TP53 germline mutations in Portugal and genetic modifiers of age at cancer onset

    NARCIS (Netherlands)

    Pinto, Carla; Veiga, Isabel; Pinheiro, Manuela; Peixoto, Ana; Pinto, Armando; Lopes, Jose M.; Reis, Rui M.; Oliveira, Carla; Baptista, Manuela; Roque, Lucia; Regateiro, Fernando; Cirnes, Luis; Hofstra, Robert M. W.; Seruca, Raquel; Castedo, Sergio; Teixeira, Manuel R.

    2009-01-01

    The Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant disease caused by TP53 germline mutations. This study aimed to characterize the TP53 mutational spectrum in patients suspected to have LFS in Portugal and to evaluate the influence of the MDM2-SNP309 and TP53-72Arg variants and of telomere

  20. First report of a de novo germline mutation in the MLH1 gene

    NARCIS (Netherlands)

    Stulp, Rein P; Vos, Yvonne J; Mol, Bart; Karrenbeld, Arend; de Raad, Monique; van der Mijle, Huub J C; Sijmons, Rolf H

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC) is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast m

  1. PARENTAL ORIGIN AND GERMLINE MOSAICISM OF DELETIONS AND DUPLICATIONS OF THE DYSTROPHIN GENE - A EUROPEAN STUDY

    NARCIS (Netherlands)

    VANESSEN, AJ; ABBS, S; BAIGET, M; BAKKER, E; BOILEAU, C; VANBROECKHOVEN, C; BUSHBY, K; CLARKE, A; CLAUSTRES, M; COVONE, AE; FERRARI, M; FERLINI, A; GALLUZZI, G; GRIMM, T; GRUBBEN, C; JEANPIERRE, M; KAARIAINEN, H; LIECHTIGALLATI, S; MELIS, MA; VANOMMEN, GJB; PONCIN, JE; SCHEFFER, H; SCHWARTZ, M; SPEER, A; STUHRMANN, M; VERELLENDUMOULIN, C; WILCOX, DE; TENKATE, LP

    1992-01-01

    Knowledge about the parental origin of new mutations and the occurrence of germline mosaicism is important for estimating recurrence risks in Duchenne (DMD) and Becker muscular dystrophy (BMD). However, there are problems in resolving these issues partly because not all mutations can as yet be direc

  2. Hereditary diffuse gastric cancer : updated clinical guidelines with an emphasis on germline CDH1 mutation carriers

    NARCIS (Netherlands)

    van der Post, Rachel S.; Vogelaar, Ingrid P.; Carneiro, Fatima; Guilford, Parry; Huntsman, David; Hoogerbrugge, Nicoline; Caldas, Carlos; Schreiber, Karen E. Chelcun; Hardwick, Richard H.; Ausems, Margreet G. E. M.; Bardram, Linda; Benusiglio, Patrick R.; Bisseling, Tanya M.; Blair, Vanessa; Bleiker, Eveline; Boussioutas, Alex; Cats, Annemieke; Coit, Daniel; DeGregorio, Lynn; Figueiredo, Joana; Ford, James M.; Heijkoop, Esther; Hermens, Rosella; Humar, Bostjan; Kaurah, Pardeep; Keller, Gisella; Lai, Jennifer; Ligtenberg, Marjolijn J. L.; O'Donovan, Maria; Oliveira, Carla; Pinheiro, Hugo; Ragunath, Krish; Rasenberg, Esther; Richardson, Susan; Roviello, Franco; Schackert, Hans; Seruca, Raquel; Taylor, Amy; ter Huurne, Anouk; Tischkowitz, Marc; Joe, Sheena Tjon A.; van Dijck, Benjamin; van Grieken, Nicole C. T.; van Hillegersberg, Richard; van Sandick, Johanna W.; Vehof, Rianne; van Krieken, J. Han; Fitzgerald, Rebecca C.

    2015-01-01

    Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, inc

  3. A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Shah, S.; Schrader, K.A.; Waanders, E.; Timms, A.E.; Vijai, J.; Miething, C.; Wechsler, J.; Yang, J.; Hayes, J.; Klein, R.J.; Zhang, Jinghui; Wei, L.; Wu, G.; Rusch, M.; Nagahawatte, P.; Ma, J; Chen, S.C.; Song, G.; Cheng, J.; Meyers, P.; Bhojwani, D.; Jhanwar, S.; Maslak, P.; Fleisher, M.; Littman, J.; Offit, L.; Rau-Murthy, R.; Fleischut, M.H.; Corines, M.; Murali, R.; Gao, X.; Manschreck, C.; Kitzing, T.; Murty, V.V.; Raimondi, S.C.; Kuiper, R.P.; Simons, A.; Schiffman, J.D.; Onel, K.; Plon, S.E.; Wheeler, D.A.; Ritter, D.; Ziegler, D.S.; Tucker, K.; Sutton, R.; Chenevix-Trench, G.; Li, J.; Huntsman, D.G.; Hansford, S.; Senz, J.; Walsh, T.; Lee, M. van der; Hahn, C.N.; Roberts, K.G.; King, M.C.; Lo, S.M.; Levine, R.L.; Viale, A.; Socci, N.D.; Nathanson, K.L.; Scott, H.S.; Daly, M.; Lipkin, S.M.; Lowe, S.W.; Downing, J.R.; Altshuler, D.; Sandlund, J.T.; Horwitz, M.S.; Mullighan, C.G.; Offit, K.

    2013-01-01

    Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly1

  4. Germline FAS gene mutation in a case of ALPS and NLP Hodgkin lymphoma

    NARCIS (Netherlands)

    van den Berg, Anke; Maggio, Ewerton; Diepstra, A; de Jong, Doetje; van Krieken, J; Poppema, S

    2002-01-01

    FAS germline mutations have been associated with the development of autoimmune lymphoproliferative syndrome (ALPS). Occurrence of Hodgkin lymphoma (HL) has been reported in 2 families with ALPS. In both families an uncle of the index patient developed HL. A 15-year-old boy with autoommune thrombopen

  5. No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

    Science.gov (United States)

    Hagström, Erik; Freyer, Christoph; Battersby, Brendan J.; Stewart, James B.; Larsson, Nils-Göran

    2014-01-01

    Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals. PMID:24163253

  6. In Genes We Trust: Germline Engineering, Eugenics, and the Future of the Human Genome.

    Science.gov (United States)

    Powell, Russell

    2015-12-01

    Liberal proponents of genetic engineering maintain that developing human germline modification technologies is morally desirable because it will result in a net improvement in human health and well-being. Skeptics of germline modification, in contrast, fear evolutionary harms that could flow from intervening in the human germline, and worry that such programs, even if well intentioned, could lead to a recapitulation of the scientifically and morally discredited projects of the old eugenics. Some bioconservatives have appealed as well to the value of retaining our "given" human biological nature as a reason for restraining the development and use of human genetic modification technologies even where they would tend to increase well-being. In this article, I argue that germline intervention will be necessary merely to sustain the levels of genetic health that we presently enjoy for future generations-a goal that should appeal to bioliberals and bioconservatives alike. This is due to the population-genetic consequences of relaxed selection pressures in human populations caused by the increasing efficacy and availability of conventional medicine. This heterodox conclusion, which I present as a problem of intergenerational justice, has been overlooked in medicine and bioethics due to certain misconceptions about human evolution, which I attempt to rectify, as well as the sordid history of Darwinian approaches to medicine and social policy, which I distinguish from the present argument.

  7. Meiotic double-strand breaks uncover and protect against mitotic errors in the C. elegans germline.

    Science.gov (United States)

    Stevens, Deanna; Oegema, Karen; Desai, Arshad

    2013-12-01

    In sexually reproducing multicellular organisms, genetic information is propagated via the germline, the specialized tissue that generates haploid gametes. The C. elegans germline generates gametes in an assembly line-like process-mitotic divisions under the control of the stem cell niche produce nuclei that, upon leaving the niche, enter into meiosis and progress through meiotic prophase [1]. Here, we characterize the effects of perturbing cell division in the mitotic region of the C. elegans germline. We show that mitotic errors result in a spindle checkpoint-dependent cell-cycle delay, but defective nuclei are eventually formed and enter meiosis. These defective nuclei are eliminated by programmed cell death during meiotic prophase. The cell death-based removal of defective nuclei does not require the spindle checkpoint but instead depends on the DNA damage checkpoint. Removal of nuclei resulting from errors in mitosis also requires Spo11, the enzyme that creates double-strand breaks to initiate meiotic recombination. Consistent with this, double-strand breaks are increased in number and persist longer in germlines with mitotic defects. These findings reveal that the process of initiating meiotic recombination inherently selects against nuclei with abnormal chromosomal content generated by mitotic errors, thereby ensuring the genomic integrity of gametes.

  8. Plant germline formation: common concepts and developmental flexibility in sexual and asexual reproduction

    OpenAIRE

    Schmidt, Anja; Schmid, Marc W.; Grossniklaus, Ueli

    2015-01-01

    The life cycle of flowering plants alternates between two heteromorphic generations: a diploid sporophytic generation and a haploid gametophytic generation. During the development of the plant reproductive lineages - the germlines - typically, single sporophytic (somatic) cells in the flower become committed to undergo meiosis. The resulting spores subsequently develop into highly polarized and differentiated haploid gametophytes that harbour the gametes. Recent studies have provided insights...

  9. Chk2 and p53 regulate the transmission of healed chromosomes in the Drosophila male germline.

    Science.gov (United States)

    Titen, Simon W A; Lin, Ho-Chen; Bhandari, Jayaram; Golic, Kent G

    2014-02-01

    When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

  10. Chk2 and p53 regulate the transmission of healed chromosomes in the Drosophila male germline.

    Directory of Open Access Journals (Sweden)

    Simon W A Titen

    2014-02-01

    Full Text Available When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.

  11. First report of a de novo germline mutation in the MLH1 gene

    Institute of Scientific and Technical Information of China (English)

    Rein P Stulp; Yvonne J Vos; Bart Mol; Arend Karrenbeld; Monique de Raad; Huub JC van der Mijle; Rolf H Sijmons

    2006-01-01

    Hereditary non-polyposis colorectal carcinoma (HNPCC)is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven,or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient's parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  12. A population-based analysis of germline BAP1 mutations in melanoma

    Science.gov (United States)

    O’Shea, Sally J.; Robles-Espinoza, Carla Daniela; Harrigan, Jeanine; Jacq, Xavier; Hewinson, James; Iyer, Vivek; Merchant, Will; Elliott, Faye; Harland, Mark; Bishop, D. Timothy; Newton-Bishop, Julia A.

    2017-01-01

    Abstract Germline mutation of the BRCA1 associated protein-1 (BAP1) gene has been linked to uveal melanoma, mesothelioma, meningioma, renal cell carcinoma and basal cell carcinoma. Germline variants have also been found in familial cutaneous melanoma pedigrees, but their contribution to sporadic melanoma has not been fully assessed. We sequenced BAP1 in 1,977 melanoma cases and 754 controls and used deubiquitinase assays, a pedigree analysis, and a histopathological review to assess the consequences of the mutations found. Sequencing revealed 30 BAP1 variants in total, of which 27 were rare (ExAc allele frequency <0.002). Of the 27 rare variants, 22 were present in cases (18 missense, one splice acceptor, one frameshift and two near splice regions) and five in controls (all missense). A missense change (S98R) in a case that completely abolished BAP1 deubiquitinase activity was identified. Analysis of cancers in the pedigree of the proband carrying the S98R variant and in two other pedigrees carrying clear loss-of-function alleles showed the presence of BAP1-associated cancers such as renal cell carcinoma, mesothelioma and meningioma, but not uveal melanoma. Two of these three probands carrying BAP1 loss-of-function variants also had melanomas with histopathological features suggestive of a germline BAP1 mutation. The remaining cases with germline mutations, which were predominantly missense mutations, were associated with less typical pedigrees and tumours lacking a characteristic BAP1-associated histopathological appearances, but may still represent less penetrant variants. Germline BAP1 alleles defined as loss-of-function or predicted to be deleterious/damaging are rare in cutaneous melanoma. PMID:28062663

  13. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells

    Directory of Open Access Journals (Sweden)

    Kanatsu-Shinohara Mito

    2006-07-01

    Full Text Available Abstract Background We previously identified a set of genes called ECATs (ES cell-associated transcripts that are expressed at high levels in mouse ES cells. Here, we examine the expression and DNA methylation of ECATs in somatic cells and germ cells. Results In all ECATs examined, the promoter region had low methylation levels in ES cells, but higher levels in somatic cells. In contrast, in spite of their lack of pluripotency, male germline stem (GS cells expressed most ECATs and exhibited hypomethylation of ECAT promoter regions. We observed a similar hypomethylation of ECAT loci in adult testis and isolated sperm. Some ECATs were even less methylated in male germ cells than in ES cells. However, a few ECATs were not expressed in GS cells, and most of them targets of Oct3/4 and Sox2. The Octamer/Sox regulatory elements were hypermethylated in these genes. In addition, we found that GS cells express little Sox2 protein and low Oct3/4 protein despite abundant expression of their transcripts. Conclusion Our results suggest that DNA hypermethylation and transcriptional repression of a small set of ECATs, together with post-transcriptional repression of Oct3/4 and Sox2, contribute to the loss of pluripotency in male germ cells.

  14. Translational activation maintains germline tissue homeostasis during adulthood

    OpenAIRE

    Nousch, Marco; Eckmann, Christian R.

    2015-01-01

    Adult tissue maintenance is achieved through a tightly controlled equilibrium of 2 opposing cell fates: stem cell proliferation and differentiation. In recent years, the germ line emerged as a powerful in vivo model tissue to investigate the underlying gene expression mechanisms regulating this balance. Studies in numerous organisms highlighted the prevalence of post-transcriptional mRNA regulation, which relies on RNA-targeting factors that influence mRNA fates (e.g. decay or translational e...

  15. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    Science.gov (United States)

    McKeithan, Wesley; Colas, Alexandre; Bushway, Paul J.; Ray, Saugata; Mercola, Mark

    2013-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. The oligonucleotide transfection protocol used to functionally screen siRNA and microRNA libraries is a powerful tool to reveal networks of genes, signaling proteins and microRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. The discussion addresses technical limitations, troubleshooting and potential applications. PMID:23154934

  16. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    OpenAIRE

    2012-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent step...

  17. Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

    Energy Technology Data Exchange (ETDEWEB)

    Sotoca, Ana M., E-mail: a.sotoca@science.ru.nl [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Roelofs-Hendriks, Jose [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Boeren, Sjef [Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen (Netherlands); Kraan, Peter M. van der [Department of Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Vervoort, Jacques [Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen (Netherlands); Zoelen, Everardus J.J. van; Piek, Ester [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands)

    2013-10-15

    This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function. - Highlights: • PDGFs (C and D) significantly increased the number of multipotent undifferentiated hMSCs. • Enhanced proliferation did not impair the ability to undergo lineage-specific differentiation. • Proteomic analysis confirmed the overall signatures of the ‘intact’ cells.

  18. Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers.

    Science.gov (United States)

    Sliepen, Kwinten; Medina-Ramírez, Max; Yasmeen, Anila; Moore, John P; Klasse, Per Johan; Sanders, Rogier W

    2015-12-01

    HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs.

  19. Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche.

    Science.gov (United States)

    Dorn, David C; Dorn, August

    2011-03-01

    The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst

  20. Germline single nucleotide polymorphisms associated with response of urothelial carcinoma to platinum-based therapy: the role of the host.

    LENUS (Irish Health Repository)

    Gallagher, D J

    2013-09-01

    Variations in urothelial carcinoma (UC) response to platinum chemotherapy are common and frequently attributed to genetic and epigenetic variations of somatic DNA. We hypothesized that variations in germline DNA may contribute to UC chemosensitivity.

  1. Refractory ceramic fibers (RCFs) induce germline aneuploidy in Drosophila oocytes.

    Science.gov (United States)

    Osgood, C J

    1994-06-01

    Mineral fibers are ubiquitous in the natural environment and are widely used in industry in such applications as insulators. We have previously shown that asbestos fibers induce aneuploidy in oocytes of Drosophila melanogaster; here we extend those studies by testing refractory ceramic fibers (RCFs) for their mutagenicity to germ cells. The results establish that the tested RCFs are inducers of aneuploidy following feeding of adult females. A subset of the RCFs were also effective following larval feeding. Our results suggest that RCFs, like certain asbestos fibers, may pose a hazard to germ cells.

  2. Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development.

    Science.gov (United States)

    Chang, Yi-Jie; Pi, Haiwei; Hsieh, Chang-Che; Fuller, Margaret T

    2013-11-01

    Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.

  3. Hair follicle: a novel source of multipotent stem cells for tissue engineering and regenerative medicine.

    Science.gov (United States)

    Mistriotis, Panagiotis; Andreadis, Stelios T

    2013-08-01

    The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle (HF) is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that were shown to have surprisingly broad differentiation potential. In this review, we discuss the biology of the HF with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative HF stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of HF stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility makes the HF an ideal source of stem cells for gene and cell therapies.

  4. Hair Follicle: A Novel Source of Multipotent Stem Cells for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Mistriotis, Panagiotis

    2013-01-01

    The adult body harbors powerful reservoirs of stem cells that enable tissue regeneration under homeostatic conditions or in response to disease or injury. The hair follicle (HF) is a readily accessible mini organ within the skin and contains stem cells from diverse developmental origins that were shown to have surprisingly broad differentiation potential. In this review, we discuss the biology of the HF with particular emphasis on the various stem cell populations residing within the tissue. We summarize the existing knowledge on putative HF stem cell markers, the differentiation potential, and technologies to isolate and expand distinct stem cell populations. We also discuss the potential of HF stem cells for drug and gene delivery, tissue engineering, and regenerative medicine. We propose that the abundance of stem cells with broad differentiation potential and the ease of accessibility makes the HF an ideal source of stem cells for gene and cell therapies. PMID:23157470

  5. Novel Regenerative Therapies Based on Regionally Induced Multipotent Stem Cells in Post-Stroke Brains: Their Origin, Characterization, and Perspective.

    Science.gov (United States)

    Takagi, Toshinori; Yoshimura, Shinichi; Sakuma, Rika; Nakano-Doi, Akiko; Matsuyama, Tomohiro; Nakagomi, Takayuki

    2017-07-25

    Brain injuries such as ischemic stroke cause severe neural loss. Until recently, it was believed that post-ischemic areas mainly contain necrotic tissue and inflammatory cells. However, using a mouse model of cerebral infarction, we demonstrated that stem cells develop within ischemic areas. Ischemia-induced stem cells can function as neural progenitors; thus, we initially named them injury/ischemia-induced neural stem/progenitor cells (iNSPCs). However, because they differentiate into more than neural lineages, we now refer to them as ischemia-induced multipotent stem cells (iSCs). Very recently, we showed that putative iNSPCs/iSCs are present within post-stroke areas in human brains. Because iNSPCs/iSCs isolated from mouse and human ischemic tissues can differentiate into neuronal lineages in vitro, it is possible that a clearer understanding of iNSPC/iSC profiles and the molecules that regulate iNSPC/iSC fate (e.g., proliferation, differentiation, and survival) would make it possible to perform neural regeneration/repair in patients following stroke. In this article, we introduce the origin and traits of iNSPCs/iSCs based on our reports and recent viewpoints. We also discuss their possible contribution to neurogenesis through endogenous and exogenous iNSPC/iSC therapies following ischemic stroke.

  6. Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures.

    Science.gov (United States)

    Gryshkov, Oleksandr; Hofmann, Nicola; Lauterboeck, Lothar; Pogozhykh, Denys; Mueller, Thomas; Glasmacher, Birgit

    2015-08-01

    Multipotent stromal cells derived from the common marmoset monkey Callithrix jacchus (cjMSCs) possess high phylogenetic similarity to humans, with a great potential for preclinical studies in the field of regenerative medicine. Safe and effective long-term storage of cells is of great significance to clinical and research applications. Encapsulation of such cell types within alginate beads that can mimic an extra-cellular matrix and provide a supportive environment for cells during cryopreservation, has several advantages over freezing of cells in suspension. In this study we have analysed the effect of dimethyl sulfoxide (Me2SO, 2.5-10%, v/v) and pre-freeze loading time of alginate encapsulated cjMSCs in Me2SO (0-45 min) on the viability and metabolic activity of the cells after freezing using a slow cooling rate (-1°C/min). It was found that these parameters affect the stability and homogeneity of alginate beads after thawing. Moreover, the cjMSCs can be frozen in alginate beads with lower Me2SO concentration of 7.5% after 30 min of loading, while retaining high cryopreservation outcome. We demonstrated the maximum viability, membrane integrity and metabolic activity of the cells under optimized, less cytotoxic conditions. The results of this study are another step forward towards the application of cryopreservation for the long-term storage and subsequent applications of transplants in cell-based therapies.

  7. Spontaneous formation of tumorigenic hybrids between breast cancer and multipotent stromal cells is a source of tumor heterogeneity.

    Science.gov (United States)

    Rappa, Germana; Mercapide, Javier; Lorico, Aurelio

    2012-06-01

    Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

  8. Impact of various modifications of biodegradable membranous scaffolds surface on multipotent mesenchimal stromal cells adhesion and viability

    Directory of Open Access Journals (Sweden)

    L. V. Antonova

    2012-01-01

    Full Text Available Designing a hybrid vascular graft using biocompatible and biodegradable polymers is one of the ways to fill the market with vascular grafts of a small diameter necessary for coronary artery bypass surgery. Good adhesive properties of cultivated cells and low cytotoxicity of biopolymers can improve biocompatibility of polymer-based vascular grafts. The impact of protein modification of membranous polyhydroxyalkanoate and polycaprolactone scaffolds surface on their adhesive properties and cytotoxicity to multipotent mesenchimal stromal cells was evaluated. We used the following compositions of membranous scaffolds: № 1 polyhydroxybutyrate valerate ММ 2 307 kDa with polycaprolactone ММ 80 000 kDa, № 2 had the same composition but was made using a magnetic stirrer, №3 polyhydroxybutyrate ММ 541 kDa with polycaprolactone ММ 80 000 kDa and № 4 polycaprolactone ММ 80 000 kDa only. Premodification of scaffolds surfaces with 75, 50 and 25% fetal calf serum was found to significantly reduce cytotoxicity of scaffold № 4 and improve the adhesive properties of scaffolds № 1, 2 and 3. Premodification with fibronectin (10 µg/ml mostly improved the adhesive properties of scaffolds № 1—4.

  9. C-MYC transcriptionally amplifies SOX2 target genes to regulate self-renewal in multipotent otic progenitor cells.

    Science.gov (United States)

    Kwan, Kelvin Y; Shen, Jun; Corey, David P

    2015-01-13

    Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santamaria-Martinez, Albert [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat de Barcelona, Barcelona (Spain); Barquinero, Jordi [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat Autonoma de Barcelona, Barcelona (Spain); Banc de Sang i Teixits, Barcelona (Spain); Barbosa-Desongles, Anna; Hurtado, Antoni; Pinos, Tomas [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat Autonoma de Barcelona, Barcelona (Spain); Seoane, Joan [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat Autonoma de Barcelona, Barcelona (Spain); Medical Oncology program, Vall d' Hebron Institute of Oncology, Barcelona (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona (Spain); Poupon, Marie-France [Institut Curie, Paris (France); Morote, Joan [Universitat Autonoma de Barcelona, Barcelona (Spain); Servei d' Urologia. Hospital Vall d' Hebron, Barcelona (Spain); Reventos, Jaume [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat Autonoma de Barcelona, Barcelona (Spain); Munell, Francina, E-mail: fmunell@ir.vhebron.net [Institut de Recerca Hospital Vall d' Hebron, Barcelona (Spain); Universitat Autonoma de Barcelona, Barcelona (Spain)

    2009-10-15

    Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

  11. Bone Marrow-Derived Multipotent Stromal Cells Attenuate Inflammation in Obliterative Airway Disease in Mouse Tracheal Allografts

    Directory of Open Access Journals (Sweden)

    Alicia Casey

    2014-01-01

    Full Text Available Obliterative bronchiolitis (OB remains the most significant cause of death in long-term survival of lung transplantation. Using an established murine heterotopic tracheal allograft model, the effects of different routes of administration of bone marrow-derived multipotent stromal cells (MSCs on the development of OB were evaluated. Tracheas from BALB/c mice were implanted into the subcutaneous tissue of major histocompatibility complex- (MHC- disparate C57BL/6 mice. At the time of transplant, bone marrow-derived MSCs were administered either systemically or locally or via a combination of the two routes. The allografts were explanted at various time points after transplantation and were evaluated for epithelial integrity, inflammatory cell infiltration, fibrosis, and luminal obliteration. We found that the most effective route of bone marrow-derived MSC administration is the combination of systemic and local delivery. Treatment of recipient mice with MSCs suppressed neutrophil, macrophage, and T-cell infiltration and reduced fibrosis. These beneficial effects were observed despite lack of significant MSC epithelial engraftment or new epithelial cell generation. Our study suggests that optimal combination of systemic and local delivery of MSCs may ameliorate the development of obliterative airway disease through modulation of immune response.

  12. Adult stem cells: hopes and hypes of regenerative medicine.

    Science.gov (United States)

    Dulak, Józef; Szade, Krzysztof; Szade, Agata; Nowak, Witold; Józkowicz, Alicja

    2015-01-01

    Stem cells are self-renewing cells that can differentiate into specialized cell type(s). Pluripotent stem cells, i.e. embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) differentiate into cells of all three embryonic lineages. Multipotent stem cells, like hematopoietic stem cells (HSC), can develop into multiple specialized cells in a specific tissue. Unipotent cells differentiate only into one cell type, like e.g. satellite cells of skeletal muscle. There are many examples of successful clinical applications of stem cells. Over million patients worldwide have benefited from bone marrow transplantations performed for treatment of leukemias, anemias or immunodeficiencies. Skin stem cells are used to heal severe burns, while limbal stem cells can regenerate the damaged cornea. Pluripotent stem cells, especially the patient-specific iPSC, have a tremendous therapeutic potential, but their clinical application will require overcoming numerous drawbacks. Therefore, the use of adult stem cells, which are multipotent or unipotent, can be at present a more achievable strategy. Noteworthy, some studies ascribed particular adult stem cells as pluripotent. However, despite efforts, the postulated pluripotency of such events like "spore-like cells", "very small embryonic-like stem cells" or "multipotent adult progenitor cells" have not been confirmed in stringent independent studies. Also plasticity of the bone marrow-derived cells which were suggested to differentiate e.g. into cardiomyocytes, has not been positively verified, and their therapeutic effect, if observed, results rather from the paracrine activity. Here we discuss the examples of recent studies on adult stem cells in the light of current understanding of stem cell biology.

  13. Absence of germline mutations in BAP1 in sporadic cases of malignant mesothelioma.

    Science.gov (United States)

    Sneddon, Sophie; Leon, Justine S; Dick, Ian M; Cadby, Gemma; Olsen, Nola; Brims, Fraser; Allcock, Richard J N; Moses, Eric K; Melton, Phillip E; de Klerk, Nicholas; Musk, A W Bill; Robinson, Bruce W S; Creaney, Jenette

    2015-05-25

    Malignant mesothelioma (MM) is a uniformly fatal tumour caused predominantly by exposure to asbestos. It is not known why some exposed individuals get mesothelioma and others do not. There is some epidemiological evidence of host susceptibility. BAP1 gene somatic mutations and allelic loss are common in mesothelioma and recently a BAP1 cancer syndrome was described in which affected individuals and families had an increased risk of cancer of multiple types, including MM. To determine if BAP1 mutations could underlie any of the sporadic mesothelioma cases in our cohort of patients, we performed targeted deep sequencing of the BAP1 exome on the IonTorrent Proton sequencer in 115 unrelated MM cases. No exonic germline BAP1 mutations of known functional significance were observed, further supporting the notion that sporadic germline BAP1 mutations are not relevant to the genetic susceptibility of MM.

  14. A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory.

    Science.gov (United States)

    Katz, David J; Edwards, T Matthew; Reinke, Valerie; Kelly, William G

    2009-04-17

    Epigenetic information undergoes extensive reprogramming in the germline between generations. This reprogramming may be essential to establish a developmental ground state in the zygote. We show that mutants in spr-5, the Caenorhabditis elegans ortholog of the H3K4me2 demethylase LSD1/KDM1, exhibit progressive sterility over many generations. This sterility correlates with the misregulation of spermatogenesis-expressed genes and transgenerational accumulation of the histone modification dimethylation of histone H3 on lysine 4 (H3K4me2). This suggests that H3K4me2 can serve as a stable epigenetic memory, and that erasure of H3K4me2 by LSD/KDM1 in the germline prevents the inappropriate transmission of this epigenetic memory from one generation to the next. Thus, our results provide direct mechanistic insights into the processes that are required for epigenetic reprogramming between generations.

  15. Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano.

    Science.gov (United States)

    Grudniewska, Magda; Mouton, Stijn; Simanov, Daniil; Beltman, Frank; Grelling, Margriet; de Mulder, Katrien; Arindrarto, Wibowo; Weissert, Philipp M; van der Elst, Stefan; Berezikov, Eugene

    2016-12-20

    The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

  16. Paternal Age Explains a Major Portion of De Novo Germline Mutation Rate Variability in Healthy Individuals

    Science.gov (United States)

    Bourassa, Cynthia V.; Lemieux Perreault, Louis-Philippe; Legault, Marc-André; Barhdadi, Amina; Ambalavanan, Amirthagowri; Brendgen, Mara; Vitaro, Frank; Noreau, Anne; Dionne, Ginette; Tremblay, Richard E.; Dion, Patrick A.; Boivin, Michel; Dubé, Marie-Pierre; Rouleau, Guy A.

    2016-01-01

    De novo mutations (DNM) are an important source of rare variants and are increasingly being linked to the development of many diseases. Recently, the paternal age effect has been the focus of a number of studies that attempt to explain the observation that increasing paternal age increases the risk for a number of diseases. Using disease-free familial quartets we show that there is a strong positive correlation between paternal age and germline DNM in healthy subjects. We also observed that germline CNVs do not follow the same trend, suggesting a different mechanism. Finally, we observed that DNM were not evenly distributed across the genome, which adds support to the existence of DNM hotspots. PMID:27723766

  17. Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

    Science.gov (United States)

    Khattak, Shahryar; Schuez, Maritta; Richter, Tobias; Knapp, Dunja; Haigo, Saori L.; Sandoval-Guzmán, Tatiana; Hradlikova, Kristyna; Duemmler, Annett; Kerney, Ryan; Tanaka, Elly M.

    2013-01-01

    The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible. PMID:24052945

  18. Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function.

    Science.gov (United States)

    Chen, Yichang; Shu, Le; Qiu, Zhiqun; Lee, Dong Yeon; Settle, Sara J; Que Hee, Shane; Telesca, Donatello; Yang, Xia; Allard, Patrick

    2016-07-01

    Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.

  19. Production of fat-1 transgenic rats using a post-natal female germline stem cell line.

    Science.gov (United States)

    Zhou, Li; Wang, Lei; Kang, Jing X; Xie, Wenhai; Li, Xiaoyong; Wu, Changqing; Xu, Bo; Wu, Ji

    2014-03-01

    Germline stem cell lines possess the abilities of self-renewal and differentiation, and have been established from both mouse and human ovaries. Here, we established a new female germline stem cell (FGSC) line from post-natal rats by immunomagnetic sorting for Fragilis, which showed a normal karyotype, high telomerase activity, and a consistent gene expression pattern of primordial germ cells after 1 year of culture. Using an in vitro differentiation system, the FGSC line could differentiate into oocytes. After liposome-based transfection with green fluorescent protein (GFP) or fat-1 vectors, the FGSCs were transplanted into the ovaries of infertile rats. The transplanted FGSCs underwent oogenesis, and the rats produced offspring carrying the GFP or fat-1 transgene after mating with wild-type male rats. The efficiency of gene transfer was 27.86-28.00%, and 2 months was needed to produce transgenic rats. These findings have implications in biomedical research and potential applications in biotechnology.

  20. Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

    Directory of Open Access Journals (Sweden)

    Joachim Forner

    Full Text Available Transcription activator-like effector nucleases (TALENs are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.

  1. Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

    Science.gov (United States)

    Forner, Joachim; Pfeiffer, Anne; Langenecker, Tobias; Manavella, Pablo A; Manavella, Pablo; Lohmann, Jan U

    2015-01-01

    Transcription activator-like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM) did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.

  2. Induction of Atherosclerosis in Mice and Hamsters Without Germline Genetic Engineering

    DEFF Research Database (Denmark)

    Bjørklund, Martin Mæng; Hollensen, Anne K; Hagensen, Mette K

    2014-01-01

    RATIONALE: Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation. OBJECTIVE......: To develop a method for induction of atherosclerosis without germline genetic engineering. METHODS AND RESULTS: Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector...... are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models....

  3. Effective gene silencing in Drosophila ovarian germline by artificial microRNAs

    Institute of Scientific and Technical Information of China (English)

    Hailong Wang; YanJun Mu; Dahua Chen

    2011-01-01

    @@ Dear Editor, Drosophila oogenesis is of great interest because it represents an excellent model system to study a number of fascinating biological processes, such as stem cell regulation, germ cell meiosis and oocyte determination, as well as signal interactions between germline and soma.A typical Drosophila ovary is composed of 16-20 ovarioles, each consisting of an anterior functional unit called a germarium and a linear string of differentiated egg chambers posterior to the germarium [1] (Figure 1A and 1B).Drosophila oogenesis initiates at the tip of the germarium, when a germline stem cell (GSC) divides asymmetrically to generate a daughter GSC and a cystoblast that eventually develops into a mature egg [2] (Figure 1C and 1E).

  4. Maternal Metabolic Syndrome Programs Mitochondrial Dysfunction via Germline Changes across Three Generations.

    Science.gov (United States)

    Saben, Jessica L; Boudoures, Anna L; Asghar, Zeenat; Thompson, Alysha; Drury, Andrea; Zhang, Wendy; Chi, Maggie; Cusumano, Andrew; Scheaffer, Suzanne; Moley, Kelle H

    2016-06-28

    Maternal obesity impairs offspring health, but the responsible mechanisms are not fully established. To address this question, we fed female mice a high-fat/high-sugar diet from before conception until weaning and then followed the outcomes in the next three generations of offspring, all fed a control diet. We observed that female offspring born to obese mothers had impaired peripheral insulin signaling that was associated with mitochondrial dysfunction and altered mitochondrial dynamic and complex proteins in skeletal muscle. This mitochondrial phenotype persisted through the female germline and was passed down to the second and third generations. Our results indicate that maternal programming of metabolic disease can be passed through the female germline and that the transfer of aberrant oocyte mitochondria to subsequent generations may contribute to the increased risk for developing insulin resistance.

  5. Maternal Metabolic Syndrome Programs Mitochondrial Dysfunction via Germline Changes across Three Generations

    Directory of Open Access Journals (Sweden)

    Jessica L. Saben

    2016-06-01

    Full Text Available Maternal obesity impairs offspring health, but the responsible mechanisms are not fully established. To address this question, we fed female mice a high-fat/high-sugar diet from before conception until weaning and then followed the outcomes in the next three generations of offspring, all fed a control diet. We observed that female offspring born to obese mothers had impaired peripheral insulin signaling that was associated with mitochondrial dysfunction and altered mitochondrial dynamic and complex proteins in skeletal muscle. This mitochondrial phenotype persisted through the female germline and was passed down to the second and third generations. Our results indicate that maternal programming of metabolic disease can be passed through the female germline and that the transfer of aberrant oocyte mitochondria to subsequent generations may contribute to the increased risk for developing insulin resistance.

  6. Synchronous Onset of Breast and Pancreatic Cancers: Results of Germline and Somatic Genetic Analysis

    Directory of Open Access Journals (Sweden)

    Michael Castro

    2016-07-01

    Full Text Available Background: Synchronous cancers have occasionally been detected at initial diagnosis among patients with breast and ovarian cancer. However, simultaneous coexistence and diagnosis of breast and pancreas cancer has not previously been reported. Case Report: Paternal transmission of a germline BRCA2 mutation to a patient who was diagnosed at age 40 with locally advanced breast and pancreas cancer is presented. Somatic genomic analysis of both cancers with next-generation DNA sequencing confirmed the germline result and reported a variety of variants of unknown significance alterations, of which two were present in both the breast and pancreas cancers. Discussion: The possibility that genomic alterations could have been responsible for modulating the phenotypic or clinical expression of this rare presentation is considered. The authors call attention to the practice of privatizing the clinicogenetic information gained from genetic testing and call for health policy that will facilitate sharing in order to advance the outcomes of patients diagnosed with hereditary cancers.

  7. High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells

    Science.gov (United States)

    Pech, Matthew F.; Garbuzov, Alina; Hasegawa, Kazuteru; Sukhwani, Meena; Zhang, Ruixuan J.; Benayoun, Bérénice A.; Brockman, Stephanie A.; Lin, Shengda; Brunet, Anne; Orwig, Kyle E.; Artandi, Steven E.

    2015-01-01

    Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of TertHigh undifferentiated spermatogonia and TertLow differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the TertHigh cKit− population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans. PMID:26584619

  8. Germ-line Gene Editing: What are the Social and Moral Risks?

    OpenAIRE

    Sutton, Agneta

    2016-01-01

    Should we welcome all developments in gene editing? Somatic cell gene editing would be on a par with conventional therapies aimed at treating particular conditions or alleviating symptoms. It would solely affect the individual patient treated. It could thus serve as a welcome new kind of treatment for cancers and blood diseases such as ß-thalassaemia. Germ-line gene editing, on the other hand, would have hereditary effects. This raises special concerns about medical mishaps. Medical risks are...

  9. Male and female Drosophila germline stem cells: two versions of immortality.

    Science.gov (United States)

    Fuller, Margaret T; Spradling, Allan C

    2007-04-20

    Drosophila male and female germline stem cells (GSCs) are sustained by niches and regulatory pathways whose common principles serve as models for understanding mammalian stem cells. Despite striking cellular and genetic similarities that suggest a common evolutionary origin, however, male and female GSCs also display important differences. Comparing these two stem cells and their niches in detail is likely to reveal how a common heritage has been adapted to the differing requirements of male and female gamete production.

  10. A germline-centric view of cell fate commitment, reprogramming and immortality.

    Science.gov (United States)

    Torres-Padilla, Maria-Elena; Ciosk, Rafal

    2013-02-01

    To ensure species continuity, the tantalising developmental plasticity of early embryonic cells, also called totipotency, must be transmitted to the offspring. This responsibility rests within the reproductive cell lineage: the germ line. At the recent EMBO/EMBL symposium 'Germline - Immortality through Totipotency', researchers discussed the mechanisms that establish and control totipotency, with an eye towards the mechanisms that may endow germ cells with the ability to propagate totipotency across generations.

  11. Report of 16 kindreds and one kindred with hMLH1 germline mutation

    Institute of Scientific and Technical Information of China (English)

    Bo Zhao; Zhen-Jun Wang; Yu-Feng Xu; Yuan-Lian Wan; Peng Li; Yan-Ting Huang

    2002-01-01

    AIM: To analyze the diagnosis and treatment of 16 hereditarynonpolyposis colorectal cancer (HNPCC) kindreds, and toreport the first kindred with hMLH1 germline mutation inMainland China.METHODS: The diagnosis, treatment and follow-up study of16 HNPCC kindreds were retrospectively reviewed. Dataconceming site of the malignant tumor, age st the diagnosis,history of synchronous and/ or metachronous cancer, andhistopathology of tumors were recorded. All treatments hadwon formal consent. PCR and SSCP were used to screen thecoding region of hMLH1 and hMSH2 genes. Variant bandswere sequenced by a 377 DNA sequencer.RESULTS: Among sixteen kindreds, sixty-eight patients hada mean age of 50.8 years, including twenty-one multiplecancer patients and forty-six colorectal cancer patients(metachronous colorectal cancers in sixteen). A total of onehundred and one malignant neoplasms were found in thesesixty-eight patients, including 50 colonic, 17 rectal, 11gastric, 7 endometrial, and 4 esophageal cancers. 39.5%colorectal patients had metachronous cancers within tenyears who needed reopeertions. A germline G265T nonsensemutation was found in the third exon of hMLH1, resulting in astop codon and truncated protein. Three phenotypically normalfamily members were also found to carry the mutated gene.ONCLUSION: HNPCC is a typical auto-dominant hereditaryisease, the main characteristics include early onset andfrequency of cancers; predominance of colorectal,especially right-sided colon cancers; frequency of multipleprimary cancers (especially colorectal cancers). Segmentalresection for colorectal cancers is not eligible for colorectalcancer patient in HNPCC kindreds. Intensive follow-up isessential for all patients and possible gene carriers. The firstHNPCC kindred with hMUH1 gene germline mutation wasidentified in Mainland China, and three phenotypicallynormal family members were found to be carriers of themutated gene. The G265T germline (nonsense) mutation inthe third exon of hMLH1

  12. Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch.

    Science.gov (United States)

    Schoenmakers, Sam; Wassenaar, Evelyne; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2010-06-01

    During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).

  13. Risk of Cancer in Cases of Suspected Lynch Syndrome Without Germline Mutation

    OpenAIRE

    Rodríguez-Soler, María; Pérez-Carbonell, Lucía; Guarinos, Carla; Zapater, Pedro; Castillejo, Adela; Barberá, Víctor Manuel; Juárez, Miriam; Bessa, Xavier; Xicola, Rosa M; Clofent, Juan; Bujanda, Luis; Balaguer, Francesc; Reñé, Josep-Maria; de Castro, Luisa; Marín-Gabriel, José C.

    2013-01-01

    Background & Aims: Colorectal cancers (CRCs) with microsatellite instability (MSI) and a mismatch repair (MMR) immunohistochemical deficit without hypermethylation of the MLH1 promoter are likely to be caused by Lynch syndrome. Some patients with these cancers have not been found to have pathogenic germline mutations and are considered to have Lynch-like syndrome (LLS). The aim of this study was to determine the risk of cancer in families of patients with LLS. Methods: We studied a population...

  14. High Throughput Sequencing of Germline and Tumor from Men With Early-Onset Metastatic Prostate Cancer

    Science.gov (United States)

    2014-10-01

    challenge, Dr. Tomlins has continued to develop state of the art technologies to use formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens...men with early-onset, metastatic prostate cancer PRINCIPAL INVESTIGATOR: Kathleen A. Cooney, M.D. CONTRACTING ORGANIZATION...High-Throughput Sequencing of Germline and Tumor From Men with Early-Onset Metastatic Prostate Cancer 5b. GRANT NUMBER W81XWH-13-1-0371 5c

  15. HABP2 germline variants are uncommon in familial nonmedullary thyroid cancer

    OpenAIRE

    Weeks, Alexia L.; Scott G Wilson; Ward, Lynley; Goldblatt, Jack; Hui, Jennie; Walsh, John P.

    2016-01-01

    Background The genetic basis of nonsyndromic familial nonmedullary thyroid cancer (FNMTC) is poorly understood. A recent study identified HABP2 as a tumor suppressor gene and identified a germline variant (G534E) in an extended FNMTC kindred. The relevance of this to other FNMTC kindreds is uncertain. Methods Sanger sequencing was performed on peripheral blood DNA from probands from 37 Australian FNMTC kindreds to detect the G534E variant. Whole exome data from 59 participants from 20 kindred...

  16. The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential.

    Science.gov (United States)

    Fiorelli, Roberto; Cebrian-Silla, Arantxa; Garcia-Verdugo, Jose-Manuel; Raineteau, Olivier

    2013-12-01

    Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP(+) cells that share the morphology and the antigenic properties of SVZ-NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non-proliferative in the intact spinal cord, but incorporate the S-phase marker EdU following spinal cord injury. Multipotent, clonal YFP-expressing neurospheres (i.e., deriving from recombined GFAP-expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self-renewal properties when compared with SVZ-neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non-neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP(+) cells co-exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self-renewal properties.

  17. Protein phosphatase 1ß limits ring canal constriction during Drosophila germline cyst formation.

    Science.gov (United States)

    Yamamoto, Shinya; Bayat, Vafa; Bellen, Hugo J; Tan, Change

    2013-01-01

    Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

  18. Protein phosphatase 1ß limits ring canal constriction during Drosophila germline cyst formation.

    Directory of Open Access Journals (Sweden)

    Shinya Yamamoto

    Full Text Available Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw, a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT. This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

  19. Familial gastrointestinal stromal tumors associated with dysphagia and novel type germline mutation of KIT gene.

    Science.gov (United States)

    Hirota, Seiichi; Nishida, Toshirou; Isozaki, Koji; Taniguchi, Masahiko; Nishikawa, Kazuhiro; Ohashi, Akiko; Takabayashi, Arimichi; Obayashi, Tadashi; Okuno, Tomoko; Kinoshita, Kazuo; Chen, Hui; Shinomura, Yasuhisa; Kitamura, Yukihiko

    2002-05-01

    A family with multiple gastrointestinal stromal tumors (GISTs), a new type of germline mutation of KIT gene, and dysphagia is reported. The mutation was observed at Asp-820 in tyrosine kinase (TK) II domain. Mutations in TK II domain have been found in mast cell and germ cell tumors but not in GISTs, and the present family members are the first reported cases of GISTs with TK II domain mutations, including sporadic GISTs. Because interleukin 3-dependent Ba/F3 murine lymphoid cells transfected with the mutant KIT complementary DNA grew autonomously without any growth factors and formed tumors in nude mice, the mutation was considered to be gain-of-function type. Family members with the germline KIT mutation reported dysphagia, but those without the mutation did not. The mechanism of dysphagia was examined with gastrointestinal fiberscopy, endoscopic ultrasonography, and esophageal manometry. No mechanical obstruction was found, and the esophagus was not remarkably dilated. In the family members with dysphagia, endoscopic ultrasonography at the esophagocardiac junction showed a thickened hyperechoic layer between the circular and longitudinal muscle layers, suggesting hyperplasia of interstitial cells of Cajal at the myenteric plexus layer. Manometry showed low resting lower esophageal sphincter pressure and abnormal simultaneous contractions of the esophagus without normal peristalsis. These findings indicate that the dysphagia of the present family is different from typical achalasia. This is the first report of familial dysphagia caused by germline gain-of-function mutation of the KIT gene at the TK II domain.

  20. MORC-1 Integrates Nuclear RNAi and Transgenerational Chromatin Architecture to Promote Germline Immortality.

    Science.gov (United States)

    Weiser, Natasha E; Yang, Danny X; Feng, Suhua; Kalinava, Natallia; Brown, Kristen C; Khanikar, Jayshree; Freeberg, Mallory A; Snyder, Martha J; Csankovszki, Györgyi; Chan, Raymond C; Gu, Sam G; Montgomery, Taiowa A; Jacobsen, Steven E; Kim, John K

    2017-05-22

    Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1, which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(-) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(-) mutants revealed a progressive, met-1-dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Comprehensive assessment of germline chemical toxicity using the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Parodi, Daniela A; Damoiseaux, Robert; Allard, Patrick

    2015-02-22

    Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals. We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

  2. Fitness loss and germline mutations in barn swallows breeding in Chernobyl

    Energy Technology Data Exchange (ETDEWEB)

    Ellegren, Hans; Lindgren, Gabriella; Primmer, C.R. [Swedish Univ. of Agricultural Sciences, Animal Breeding and Genetics Dept., Uppsala (Sweden); Moeller, A.P. [Universite Pierre et Marie Curie. Lab. d`Ecologie, Paris, 75 (France)

    1997-10-09

    The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer, infant leukaemia, and congenital malformations in newborns. Two recent studies have reported, although with some controversy, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heretability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy. (author).

  3. A germline polymorphism of DNA polymerase beta induces genomic instability and cellular transformation.

    Directory of Open Access Journals (Sweden)

    Jennifer Yamtich

    Full Text Available Several germline single nucleotide polymorphisms (SNPs have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β, encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER, a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797 in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.

  4. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.

    Science.gov (United States)

    Snow, Andrew L; Xiao, Wenming; Stinson, Jeffrey R; Lu, Wei; Chaigne-Delalande, Benjamin; Zheng, Lixin; Pittaluga, Stefania; Matthews, Helen F; Schmitz, Roland; Jhavar, Sameer; Kuchen, Stefan; Kardava, Lela; Wang, Wei; Lamborn, Ian T; Jing, Huie; Raffeld, Mark; Moir, Susan; Fleisher, Thomas A; Staudt, Louis M; Su, Helen C; Lenardo, Michael J

    2012-11-19

    Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.

  5. Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Quan-Xiang Wei

    Full Text Available We previously reported a rare germline variant (c.1-6531 that resulted in allele-specific expression (ASE of death-associated protein kinase 1 (DAPK1 and predisposition to chronic lymphocytic leukemia (CLL. We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2% CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

  6. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  7. Aspirin increases metabolism through germline signalling to extend the lifespan of Caenorhabditis elegans.

    Science.gov (United States)

    Huang, Xiao-Bing; Mu, Xiao-Hui; Wan, Qin-Li; He, Xiao-Ming; Wu, Gui-Sheng; Luo, Huai-Rong

    2017-01-01

    Aspirin is a prototypic cyclooxygenase inhibitor with a variety of beneficial effects on human health. It prevents age-related diseases and delays the aging process. Previous research has shown that aspirin might act through a dietary restriction-like mechanism to extend lifespan. To explore the mechanism of action of aspirin on aging, we determined the whole-genome expression profile of Caenorhabditis elegans treated with aspirin. Transcriptome analysis revealed the RNA levels of genes involved in metabolism were primarily increased. Reproduction has been reported to be associated with metabolism. We found that aspirin did not extend the lifespan or improve the heat stress resistance of germline mutants of glp-1. Furthermore, Oil Red O staining showed that aspirin treatment decreased lipid deposition and increased expression of lipid hydrolysis and fatty acid β-oxidation-related genes. The effect of germline ablation on lifespan was mainly mediated by DAF-12 and DAF-16. Next, we performed genetic analysis with a series of worm mutants and found that aspirin did not further extend the lifespans of daf-12 and daf-16 single mutants, glp-1;daf-12 and glp-1;daf-16 double mutants, or glp-1;daf-12;daf-16 triple mutants. The results suggest that aspirin increase metabolism and regulate germline signalling to activate downstream DAF-12 and DAF-16 to extend lifespan.

  8. The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer

    Science.gov (United States)

    Esteban-Jurado, Clara; Franch-Expósito, Sebastià; Muñoz, Jenifer; Ocaña, Teresa; Carballal, Sabela; López-Cerón, Maria; Cuatrecasas, Miriam; Vila-Casadesús, Maria; Lozano, Juan José; Serra, Enric; Beltran, Sergi; Brea-Fernández, Alejandro; Ruiz-Ponte, Clara; Castells, Antoni; Bujanda, Luis; Garre, Pilar; Caldés, Trinidad; Cubiella, Joaquín; Balaguer, Francesc; Castellví-Bel, Sergi

    2016-01-01

    Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC. PMID:27165003

  9. Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

    Science.gov (United States)

    Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Aspesi, Anna; Morleo, Giulia; Biasi, Alessandra; Sculco, Marika; Mancuso, Giuseppe; Guarrera, Simonetta; Righi, Luisella; Grosso, Federica; Libener, Roberta; Pavesi, Mansueto; Mariani, Narciso; Casadio, Caterina; Boldorini, Renzo; Mirabelli, Dario; Pasini, Barbara; Magnani, Corrado; Matullo, Giuseppe; Dianzani, Irma

    2017-10-01

    Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing

    Science.gov (United States)

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T.; Scarpa, Aldo

    2016-01-01

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  11. Proven germline mosaicism in a father of two children with CHARGE syndrome.

    Science.gov (United States)

    Pauli, S; Pieper, L; Häberle, J; Grzmil, P; Burfeind, P; Steckel, M; Lenz, U; Michelmann, H W

    2009-05-01

    CHARGE syndrome is an autosomal dominant malformation syndrome caused by mutations in the CHD7 gene. The majority of cases are sporadic and only few familial cases have been reported. In these families, mosaicism in one parent, as well as parent- to-child transmission of a CHD7 mutation, has been described. In some further cases, germline mosaicism has been suggested. Here, we report the first case in which germline mosaicism could be demonstrated in a father of two affected children with CHARGE syndrome. The truncating mutation c.7302dupA in exon 34 of the CHD7 gene was found in both affected children but was not detected in parental lymphocytes. However, in DNA extracted from the father's spermatozoa, the c.7302dupA mutation could be identified. Furthermore, mutation analysis of DNA isolated from 59 single spermatozoa revealed that the c.7302dupA mutation occurs in 16 spermatozoa, confirming germline mosaicism in the father of the affected children. This result has a high impact for genetic counselling of the family and for their recurrence risk in further pregnancies.

  12. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing.

    Science.gov (United States)

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T; Scarpa, Aldo

    2016-01-12

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.

  13. Low prevalence of germline hMSH6 mutations in colorectal cancer families from Spain

    Institute of Scientific and Technical Information of China (English)

    Ana Sánchez de Abajo; Trinidad Caldes; Miguel de la Hoya; Alicia Tosar; Javier Godino; Juan Manuel Fernández; Jose Lopez Asenjo; Beatriz Perez Villamil; Pedro Perez Segura; Eduardo Diaz-Rubio

    2005-01-01

    AIM: To investigate the prevalence and penetrance of hMSH6 mutations in Spanish HNPCC families that was negative for mutation in hMLH1 or hMSH2.METHODS: We used PCR-based DGGE assay and direct Sequencing to screen for hMSH6 gene in 91 HNPCC families.RESULTS: we have identified 10 families with germ-line mutations in the DNA sequence. These mutations included two intronic variation, three missense mutation, one nonsense mutation, and four silent mutations. Among the 10 germ-line mutations identified in the Spanish cohort,8 were novel, perhaps, suggesting different mutational spectra in the Spanish population. Detailed pedigrees were constructed for the three families with a possible pathogenic hMSH6 mutation. The two silent mutations H388H and L758L, detected in a person affected of colorectal cancer at age 29, produce loss of the wild-type allele in the tumor sample. Immunohistochemical analysis showed that expression of MSH6 protein was lost only in the tumors from the carriers of V878A and Q263X mutations.CONCLUSION: Altogether, our results indicate that disease-causing germ-line mutations of hMSH6 are very less frequent in Spanish HNPCC families.

  14. Bridging the generation gap: flowering plant gametophytes and animal germlines reveal unexpected similarities.

    Science.gov (United States)

    Dickinson, Hugh G; Grant-Downton, Robert

    2009-11-01

    Alternation of generations underpins all plant life histories and is held to possess important adaptive features. A wide range of data have accumulated over the past century which suggest that alternation from sporophyte to gametophyte in angiosperms includes a significant phase of 'informational reprogramming', leaving the founder cells of the gametophyte developmentally uncommitted. This review attempts to bring together results from these historic studies with more recent data on molecular and epigenetic events which accompany alternation, gametophyte development and gametogenesis in angiosperms. It is striking that most members of the other principal group of multicellular eukaryotes--the animals--have a completely different a life history: animals generate their gametes directly from diploid germlines, often set aside early in development. Nevertheless, a comparison between animal germlines and angiosperm gametophyte development reveals a number of surprising similarities at the cytological and molecular levels. This difference in life history but similarity in developmental process is reviewed in the context of the very different life strategies adopted by plants and animals, and particularly the fact that plants do not set aside diploid germlines early in development.

  15. POLD1 Germline Mutations in Patients Initially Diagnosed with Werner Syndrome.

    Science.gov (United States)

    Lessel, Davor; Hisama, Fuki M; Szakszon, Katalin; Saha, Bidisha; Sanjuanelo, Alexander Barrios; Salbert, Bonnie A; Steele, Pamela D; Baldwin, Jennifer; Brown, W Ted; Piussan, Charles; Plauchu, Henri; Szilvássy, Judit; Horkay, Edit; Högel, Josef; Martin, George M; Herr, Alan J; Oshima, Junko; Kubisch, Christian

    2015-11-01

    Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%-15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome.

  16. HOXB4 Increases Runx1 Expression to Promote the de novo Formation of Multipotent Hematopoietic Cells.

    Science.gov (United States)

    Teichweyde, Nadine; Horn, Peter A; Klump, Hannes

    2017-06-01

    The de novo generation of patient-specific hematopoietic stem and progenitor cells from induced pluripotent stem cells (iPSCs) has become a promising approach for cell replacement therapies in the future. However, efficient differentiation protocols for producing fully functional human hematopoietic stem cells are still missing. In the mouse model, ectopic expression of the human homeotic selector protein HOXB4 has been shown to enforce the development of hematopoietic stem cells (HSCs) in differentiating pluripotent stem cell cultures. However, the mechanism how HOXB4 mediates the formation of HSCs capable of long-term, multilineage repopulation after transplantation is not well understood yet. Using a mouse embryonic stem (ES) cell-based differentiation model, we asked whether retrovirally expressed HOXB4 induces the expression of Runx1/AML1, a gene whose expression is absolutely necessary for the formation of definitive, adult HSCs during embryonic development. During ES cell differentiation, basal expression of Runx1 was observed in all cultures, irrespective of ectopic HOXB4 expression. However, only in those cultures ectopically expressing HOXB4, substantial amounts of hematopoietic progenitors were generated which exclusively displayed increased Runx1 expression. Our results strongly suggest that HOXB4 does not induce basal Runx1 expression but, instead, mediates an increase of Runx1 expression which appears to be a prerequisite for the formation of hematopoietic stem and progenitor cells.

  17. 1,25-dihydroxyvitamin D3 treatment delays cellular aging in human mesenchymal stem cells while maintaining their multipotent capacity.

    Directory of Open Access Journals (Sweden)

    Barbara Klotz

    Full Text Available 1,25-dihydroxyvitamin D3 (1,25D3 was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23 and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC. Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105 and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.

  18. Preferential and comprehensive reconstitution of severely damaged sciatic nerve using murine skeletal muscle-derived multipotent stem cells.

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    Tetsuro Tamaki

    Full Text Available Loss of vital functions in the somatic motor and sensory nervous systems can be induced by severe peripheral nerve transection with a long gap following trauma. In such cases, autologous nerve grafts have been used as the gold standard, with the expectation of activation and proliferation of graft-concomitant Schwann cells associated with their paracrine effects. However, there are a limited number of suitable sites available for harvesting of nerve autografts due to the unavoidable sacrifice of other healthy functions. To overcome this problem, the potential of skeletal muscle-derived multipotent stem cells (Sk-MSCs was examined as a novel alternative cell source for peripheral nerve regeneration. Cultured/expanded Sk-MSCs were injected into severely crushed sciatic nerve corresponding to serious neurotmesis. After 4 weeks, engrafted Sk-MSCs preferentially differentiated into not only Schwann cells, but also perineurial/endoneurial cells, and formed myelin sheath and perineurium/endoneurium, encircling the regenerated axons. Increased vascular formation was also observed, leading to a favorable blood supply and waste product excretion. In addition, engrafted cells expressed key neurotrophic and nerve/vascular growth factor mRNAs; thus, endocrine/paracrine effects for the donor/recipient cells were also expected. Interestingly, skeletal myogenic capacity of expanded Sk-MSCs was clearly diminished in peripheral nerve niche. The same differentiation and tissue reconstitution capacity of Sk-MSCs was sufficiently exerted in the long nerve gap bridging the acellular conduit, which facilitated nerve regeneration/reconnection. These effects represent favorable functional recovery in Sk-MSC-treated mice, as demonstrated by good corduroy walking. We also demonstrated that these differentiation characteristics of the Sk-MSCs were comparable to native peripheral nerve-derived cells, whereas the therapeutic capacities were largely superior in Sk

  19. Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

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    Kosuke Saito

    Full Text Available Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL. We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs, which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST. Culture medium was transplanted as a control (NT. In the mouse experiment, facial-nerve-palsy (FNP scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

  20. The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells

    Science.gov (United States)

    Coffelt, Seth B.; Marini, Frank C.; Watson, Keri; Zwezdaryk, Kevin J.; Dembinski, Jennifer L.; LaMarca, Heather L.; Tomchuck, Suzanne L.; zu Bentrup, Kerstin Honer; Danka, Elizabeth S.; Henkle, Sarah L.; Scandurro, Aline B.

    2009-01-01

    Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells. PMID:19234121

  1. Inflammatory pre-conditioning of mesenchymal multipotent stromal cells improves their immunomodulatory potency in acute pyelonephritis in rats.

    Science.gov (United States)

    Plotnikov, Egor Y; Pulkova, Natalya V; Pevzner, Irina B; Zorova, Ljubava D; Silachev, Denis N; Morosanova, Maria A; Sukhikh, Gennady T; Zorov, Dmitry B

    2013-06-01

    Acute pyelonephritis is one of the most frequent infectious diseases of the urinary tract and a leading cause of kidney failure worldwide. One strategy for modulating excessive inflammatory responses in pyelonephritis is administration of mesenchymal multipotent stromal cells (MMSCs). The putative protective effect of injection of MMSCs against experimental acute pyelonephritis was examined. We used in vivo experimental model of APN where bacteria are introduced in the bladder of rat. Three days after, intravenous injection of MMSCs was done. On the 7th day blood samples and kidneys were taken for further analysis. We found obvious signs of oxidative stress and inflammation in the kidney in acute pyelonephritis in rats. Particularly, pro-inflammatory cytokine tumor necrosis factor-α levels, malondialdehyde, nitrite and myeloperoxidase activity were significantly increased. Histologic evaluation revealed numerous attributes of inflammation and tissue damage in the kidney. Treatment with MMSCs caused a remarkable decrease of all of these pathologic signs in renal tissue. Also, activated leukocytes induced pre-conditioning-like signaling in MMSCs. We showed alterations of expression or activity of inducible nitric oxide synthase, transforming growth factor-β, matrix metalloproteinase-2 and glycogen synthase kinase-3β, which could mediate immunomodulation and protective effects of MMSCs. This signaling could be characterized as inflammatory pre-conditioning. The beneficial capacity of MMSCs to alleviate renal inflammation was more pronounced when pre-conditioned MMSCs were used. This approach could be used to prime MMSCs with different inflammatory modulators to enhance their engraftment and function in an immunoprotected fashion. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs.

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    Sam Hoot

    2013-01-01

    Full Text Available Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env. To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56 of recombinant Envs (from clades A, B and C for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.

  3. Recombinant HIV envelope proteins fail to engage germline versions of anti-CD4bs bNAbs.

    Science.gov (United States)

    Hoot, Sam; McGuire, Andrew T; Cohen, Kristen W; Strong, Roland K; Hangartner, Lars; Klein, Florian; Diskin, Ron; Scheid, Johannes F; Sather, D Noah; Burton, Dennis R; Stamatatos, Leonidas

    2013-01-01

    Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.

  4. Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas.

    Science.gov (United States)

    Pennington, Kathryn P; Walsh, Tom; Harrell, Maria I; Lee, Ming K; Pennil, Christopher C; Rendi, Mara H; Thornton, Anne; Norquist, Barbara M; Casadei, Silvia; Nord, Alexander S; Agnew, Kathy J; Pritchard, Colin C; Scroggins, Sheena; Garcia, Rochelle L; King, Mary-Claire; Swisher, Elizabeth M

    2014-02-01

    Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain. Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway. Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation. Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials. ©2013 AACR.

  5. Characterization of age-related changes of tendon stem cells from adult human tendons.

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    Ruzzini, Laura; Abbruzzese, Franca; Rainer, Alberto; Longo, Umile Giuseppe; Trombetta, Marcella; Maffulli, Nicola; Denaro, Vincenzo

    2014-11-01

    The present study evaluated the presence of stem cells in hamstring tendons from adult subjects of different ages. The aim was to isolate, characterize and expand these cells in vitro, and to evaluate whether cell activities are influenced by age. Tendon stem cells (TSCs) were isolated through magnetic sorting from the hamstring tendons of six patients. TSC percentage, morphology and clonogenic potential were evaluated, as well as the expression of specific surface markers. TSC multi-potency was also investigated as a function of age, and quantitative polimerase chain reaction was used to evaluate gene expression of TSCs cultured in suitable differentiating media. The presence of easily harvestable stem cell population within adult human hamstring tendons was demonstrated. These cells exhibit features such as clonogenicity, multi-potency and mesenchymal stem cells markers expression. The age-related variations in human TSCs affect the number of isolated cells and their self-renewal potential, while multi-potency assays are not influenced by tendon ageing, even though cells from younger individuals expressed higher levels of osteogenic and adipogenic genes, while chondrogenic genes were highly expressed in cells from older individuals. These results may open new opportunities to study TSCs to better understand tendon physiology, healing and pathological processes such as tendinopathy and degenerative age-related changes opening new frontiers in the management of tendinopathy and tendon ruptures.

  6. Real-time imaging of bHLH transcriptional factors reveals their dynamic control in the multipotency and fate choice of neural stem cells

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    Itaru eImayoshi

    2015-08-01

    Full Text Available The basic-helix-loop-helix (bHLH transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate the fate choice of neurons, astrocytes, and oligodendrocytes, respectively; however, these factors are coexpressed in self-renewing multipotent neural stem cells (NSCs even before cell fate determination. This fact raises the possibility these fate determination factors are differentially expressed between self-renewing and differentiating NSCs with unique expression dynamics. Real-time imaging analysis utilizing fluorescent proteins is a powerful strategy for monitoring expression dynamics. Fusion with fluorescent reporters makes it possible to analyze the dynamic behavior of specific proteins in living cells. However, it is technically challenging to conduct long-term imaging of proteins, particularly those with low expression levels, because a high-sensitivity and low-noise imaging system is required, and very often bleaching of fluorescent proteins and cell toxicity by prolonged laser exposure are problematic. Furthermore, to analyze the functional roles of the dynamic expression of cellular proteins, it is essential to image reporter fusion proteins that are expressed at comparable levels to their endogenous expression. In this review, we introduce our recent reports about the dynamic control of bHLH transcription factors in multipotency and fate choice of NSCs, focusing on real-time imaging of fluorescent reporters fused with bHLH transcription factors. Our imaging results indicate that bHLH transcription factors are expressed in an oscillatory manner by NSCs, and that one of them becomes dominant during fate choice. We propose that the multipotent state of NSCs correlates with the oscillatory expression of several bHLH transcription factors, whereas the differentiated state correlates with the sustained expression of a single bHLH transcription factor.

  7. Real-time imaging of bHLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells.

    Science.gov (United States)

    Imayoshi, Itaru; Ishidate, Fumiyoshi; Kageyama, Ryoichiro

    2015-01-01

    The basic-helix-loop-helix (bHLH) transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate the fate choice of neurons, astrocytes, and oligodendrocytes, respectively; however, these factors are coexpressed in self-renewing multipotent neural stem cells (NSCs) even before cell fate determination. This fact raises the possibility that these fate determination factors are differentially expressed between self-renewing and differentiating NSCs with unique expression dynamics. Real-time imaging analysis utilizing fluorescent proteins is a powerful strategy for monitoring expression dynamics. Fusion with fluorescent reporters makes it possible to analyze the dynamic behavior of specific proteins in living cells. However, it is technically challenging to conduct long-term imaging of proteins, particularly those with low expression levels, because a high-sensitivity and low-noise imaging system is required, and very often bleaching of fluorescent proteins and cell toxicity by prolonged laser exposure are problematic. Furthermore, to analyze the functional roles of the dynamic expression of cellular proteins, it is essential to image reporter fusion proteins that are expressed at comparable levels to their endogenous expression. In this review, we introduce our recent reports about the dynamic control of bHLH transcription factors in multipotency and fate choice of NSCs, focusing on real-time imaging of fluorescent reporters fused with bHLH transcription factors. Our imaging results indicate that bHLH transcription factors are expressed in an oscillatory manner by NSCs, and that one of them becomes dominant during fate choice. We propose that the multipotent state of NSCs correlates with the oscillatory expression of several bHLH transcription factors, whereas the differentiated state correlates with the sustained expression of a single bHLH transcription factor.

  8. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.

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    Lin, H; Spradling, A C

    1997-06-01

    Germline stem cells play a pivotal role in gametogenesis; yet little is known about how they are formed, how they divide to self-renew, and how these processes are genetically controlled. Here we describe the self-renewing asymmetric division of germline stem cells in the Drosophila ovarian germline, as marked by the spectrosome, a cytoplasmic structure rich in membrane skeletal proteins. The ontogeny of the spectrosome marks the lineage of germline stem cells. We identified two new groups of mutations in which the divisional asymmetry is disrupted. The first, which we refer to as ovarette (ovt) mutations, was shown to correspond to a novel class of mutations in the pumilio locus. Since pumilio is known to posttranscriptionally repress the expression of target genes at earlier stages of germ cell development, our results suggest that a similar activity is needed to maintain germ line stem cells. We have also identified a second and novel gene, piwi, whose mutations abolish germline stem cell division.

  9. The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

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    Dunnick, Wesley A; Shi, Jian; Holden, Victoria; Fontaine, Clinton; Collins, John T

    2011-01-01

    Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is type level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

  10. JNK signaling regulates E-cadherin junctions in germline cysts and determines primordial follicle formation in mice.

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    Niu, Wanbao; Wang, Ye; Wang, Zhengpin; Xin, Qiliang; Wang, Yijing; Feng, Lizhao; Zhao, Lihua; Wen, Jia; Zhang, Hua; Wang, Chao; Xia, Guoliang

    2016-05-15

    Physiologically, the size of the primordial follicle pool determines the reproductive lifespan of female mammals, while its establishment largely depends on a process of germline cyst breakdown during the perinatal period. The mechanisms regulating this process are poorly understood. Here we demonstrate that c-Jun amino-terminal kinase (JNK) signaling is crucial for germline cyst breakdown and primordial follicle formation. JNK was specifically localized in oocytes and its activity increased as germline cyst breakdown progressed. Importantly, disruption of JNK signaling with a specific inhibitor (SP600125) or knockdown technology (Lenti-JNK-shRNAs) resulted in significantly suppressed cyst breakdown and primordial follicle formation in cultured mouse ovaries. Our results show that E-cadherin is intensely expressed in germline cysts, and that its decline is necessary for oocyte release from the cyst. However, inhibition of JNK signaling leads to aberrantly enhanced localization of E-cadherin at oocyte-oocyte contact sites. WNT4 expression is upregulated after SP600125 treatment. Additionally, similar to the effect of SP600125 treatment, WNT4 overexpression delays cyst breakdown and is accompanied by abnormal E-cadherin expression patterns. In conclusion, our results suggest that JNK signaling, which is inversely correlated with WNT4, plays an important role in perinatal germline cyst breakdown and primordial follicle formation by regulating E-cadherin junctions between oocytes in mouse ovaries. © 2016. Published by The Company of Biologists Ltd.

  11. Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.

    Science.gov (United States)

    Naito, Mitsuru; Harumi, Takashi; Kuwana, Takashi

    2015-02-01

    Production of germline chimaeric chickens by the transfer of cultured primordial germ cells (PGC) is a useful system for germline manipulation. A novel culture system was developed for chicken PGC isolated from embryonic blood. The isolated PGC were cultured on feeder cells derived from chicken embryonic fibroblast. The cultured PGC formed colonies and they proliferated about 300-times during the first 30 days. The cultured PGC retained the ability to migrate to recipient gonads and were also chicken VASA homologue (CVH)-positive. Female PGC were present in the mixed-sex PGC populations cultured for more than 90 days and gave rise to viable offspring efficiently via germline chimaeric chickens. Male cultured PGC were transferred to recipient embryos and produced putative chimaeric chickens. The DNA derived from the cultured PGC was detected in the sperm samples of male putative chimaeric chickens, but no donor derived offspring were obtained. Donor-derived offspring were also obtained from germline chimaeric chickens by the transfer of frozen-thawed cultured PGC. The culture method for PGC developed in the present study is useful for manipulation of the germline in chickens, such as preservation of genetic resources and gene transfer.

  12. The function of Msx1 gene in promoting meiosis of dairy goat male germline stem cells (mGSCs).

    Science.gov (United States)

    Mu, Hailong; Wu, Jiang; Zhu, Haijing; Li, Na; Tang, Furong; Yao, Xi; Yang, Churong; Peng, Sha; Li, Guangpeng; Hua, Jinlian

    2013-12-01

    During sequential stages of meiosis, numerous cytoplasmic and nuclear events take place in which many germline and non-germline genes involved. It is demonstrated that the germline gene Stra8 and synaptonemal complex protein 3 (Scp3) play an important role in the meiosis. Recently, studies showed Msx1, a DNA-binding protein taking part in the skeletal development, also having a functional attractive factor to Stra8 and Scp3 in the meiosis. In this study, we cloned the gene Msx1 then transfected the Msx1 constructed recombination plasmid, pMsx1-Ires2-AcGFP, into the dairy goat germline stem cells (male germline stem cells) and analysed the effects of Msx1 on the expression of Stra8 and Scp3. The results showed that Msx1 could enhance the expression of Stra8 and Scp3 and promote the meiosis in goat testicular cells. Bmp4 activated the expression of Msx1 and Stra8. This study suggests that Msx1 plays an important role in spermatogenesis and meiosis. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Src64 controls a novel actin network required for proper ring canal formation in the Drosophila male germline.

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    Eikenes, Åsmund Husabø; Malerød, Lene; Lie-Jensen, Anette; Sem Wegner, Catherine; Brech, Andreas; Liestøl, Knut; Stenmark, Harald; Haglund, Kaisa

    2015-12-01

    In many organisms, germ cells develop as cysts in which cells are interconnected via ring canals (RCs) as a result of incomplete cytokinesis. However, the molecular mechanisms of incomplete cytokinesis remain poorly understood. Here, we address the role of tyrosine phosphorylation of RCs in the Drosophila male germline. We uncover a hierarchy of tyrosine phosphorylation within germline cysts that positively correlates with RC age. The kinase Src64 is responsible for mediating RC tyrosine phosphorylation, and loss of Src64 causes a reduction in RC diameter within germline cysts. Mechanistically, we show that Src64 controls an actin network around the RCs that depends on Abl and the Rac/SCAR/Arp2/3 pathway. The actin network around RCs is required for correct RC diameter in cysts of developing germ cells. We also identify that Src64 is required for proper germ cell differentiation in the Drosophila male germline independent of its role in RC regulation. In summary, we report that Src64 controls actin dynamics to mediate proper RC formation during incomplete cytokinesis during germline cyst development in vivo.

  14. Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans.

    Science.gov (United States)

    Gracida, Xicotencatl; Eckmann, Christian R

    2013-04-01

    Animals can thrive on variable food resources as a result of autonomous processes and beneficial relationships with their gut microbes [1]. Food intake elicits major physiological changes, which are counteracted by transient systemic responses that maintain homeostasis in the organism. This integration of external information occurs through cellular sensory elements, such as nuclear receptors, which modulate gene expression in response to specific cues [2]. Given the importance of germline stem cells (GSCs) for the development of the germline and the continuity of species, it is reasonable to assume that GSCs might be shielded from the negative influence of environmental perturbations. To our knowledge, however, there are no mechanisms reported that protect GSCs from harmful dietary metabolites. Using Caenorhabditis elegans as a model, we report that the somatic activity of the conserved nuclear receptor nhr-114/HNF4 protects GSC integrity from dietary metabolites. In the absence of nhr-114 and on certain bacterial diets, otherwise somatically normal animals accumulate germ cell division defects during development and become sterile. We found that, in nhr-114(-) animals, the induction of germline defects and sterility depend on bacterial metabolic status, with respect to the essential amino acid tryptophan. This illustrates an animal-microbe interaction in which somatic nuclear receptor activity preserves the germline by buffering against dietary metabolites, most likely through a somatic detoxifying response. Overall, our findings uncover an unprecedented, and presumably evolutionarily conserved, soma-to-germline axis of communication that maintains reproductive robustness on variable food resources.

  15. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

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    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  16. Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation.

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    Anja Schmidt

    2014-07-01

    Full Text Available Seeds of flowering plants can be formed sexually or asexually through apomixis. Apomixis occurs in about 400 species and is of great interest for agriculture as it produces clonal offspring. It differs from sexual reproduction in three major aspects: (1 While the sexual megaspore mother cell (MMC undergoes meiosis, the apomictic initial cell (AIC omits or aborts meiosis (apomeiosis; (2 the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis; and (3 the formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomict. To compare sexual and apomictic development at the cellular level, we used laser-assisted microdissection combined with microarray and RNA-Seq analyses. Conservation of enriched gene ontologies between the AIC and the MMC likely reflects functions of importance to germline initiation, illustrating the close developmental relationship of sexuality and apomixis. However, several regulatory pathways differ between sexual and apomictic germlines, including cell cycle control, hormonal pathways, epigenetic and transcriptional regulation. Enrichment of specific signal transduction pathways are a feature of the apomictic germline, as is spermidine metabolism, which is associated with somatic embryogenesis in various plants. Our study provides a comprehensive reference dataset for apomictic development and yields important new insights into the transcriptional basis underlying apomixis in relation to sexual reproduction.

  17. Use of Germline Polymorphisms in Predicting Concurrent Chemoradiotherapy Response in Esophageal Cancer

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    Chen, Pei-Chun [Department of Statistics and Informatics Science, Providence University, Taiwan (China); Chen, Yen-Ching [Institute of Epidemiology Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Research Center for Gene, Environment, and Human Health, College of Public Health, National Taiwan University, Taiwan (China); Department of Public Health, Institute of Epidemiology, National Taiwan University, Taiwan (China); Lai, Liang-Chuan [Graduate Institute of Physiology, National Taiwan University, Taiwan (China); Tsai, Mong-Hsun [Institute of Biotechnology, National Taiwan University, Taiwan (China); Chen, Shin-Kuang [National Clinical Trial and Research Center, National Taiwan University Hospital, Taiwan (China); Yang, Pei-Wen; Lee, Yung-Chie [Department of Surgery, National Taiwan University Hospital, Taiwan (China); Hsiao, Chuhsing K. [Research Center for Gene, Environment, and Human Health, College of Public Health, National Taiwan University, Taiwan (China); Department of Public Health, Institute of Epidemiology, National Taiwan University, Taiwan (China); Bioinformatics and Biostatistics Core, Research Center for Medical Excellence, National Taiwan University, Taiwan (China); Lee, Jang-Ming, E-mail: jangming@ntuh.gov.tw [Department of Surgery, National Taiwan University Hospital, Taiwan (China); Chuang, Eric Y., E-mail: chuangey@ntu.edu.tw [National Clinical Trial and Research Center, National Taiwan University Hospital, Taiwan (China); Bioinformatics and Biostatistics Core, Research Center for Medical Excellence, National Taiwan University, Taiwan (China); Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taiwan (China)

    2012-04-01

    Purpose: To identify germline polymorphisms to predict concurrent chemoradiation therapy (CCRT) response in esophageal cancer patients. Materials and Methods: A total of 139 esophageal cancer patients treated with CCRT (cisplatin-based chemotherapy combined with 40 Gy of irradiation) and subsequent esophagectomy were recruited at the National Taiwan University Hospital between 1997 and 2008. After excluding confounding factors (i.e., females and patients aged {>=}70 years), 116 patients were enrolled to identify single nucleotide polymorphisms (SNPs) associated with specific CCRT responses. Genotyping arrays and mass spectrometry were used sequentially to determine germline polymorphisms from blood samples. These polymorphisms remain stable throughout disease progression, unlike somatic mutations from tumor tissues. Two-stage design and additive genetic models were adopted in this study. Results: From the 26 SNPs identified in the first stage, 2 SNPs were found to be significantly associated with CCRT response in the second stage. Single nucleotide polymorphism rs16863886, located between SGPP2 and FARSB on chromosome 2q36.1, was significantly associated with a 3.93-fold increase in pathologic complete response to CCRT (95% confidence interval 1.62-10.30) under additive models. Single nucleotide polymorphism rs4954256, located in ZRANB3 on chromosome 2q21.3, was associated with a 3.93-fold increase in pathologic complete response to CCRT (95% confidence interval 1.57-10.87). The predictive accuracy for CCRT response was 71.59% with these two SNPs combined. Conclusions: This is the first study to identify germline polymorphisms with a high accuracy for predicting CCRT response in the treatment of esophageal cancer.

  18. Formation of germline chimera Gaok chicken used circulation primordial germ cells (circulation PGCs fresh and thawed

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    Kostaman T

    2014-03-01

    Full Text Available Formation of germline chimeras by transfer of chicken primordial germ cells (PGCs is one of the effective techniques for preservation and regeneration of genetic resources in chickens. This study attempted to form germline chimeras of Gaok chicken buy purifying circulated PGCs of donor embryo before it is transferred to the recipient (White Leghorn chickens=WL and studied the ability of recipient embryo on survival in incubators, and hatchability. This study used 200 fertile eggs of Gaok and 90 fertile WL breed all of the eggs was incubated at 380C and 60% humidity in a portable incubator. PGCs-circulation of the blood collected Gaok embryos at stage 14-16 were taken from the dorsal aorta, and then purified by centrifugation method using nycodenz. PGCs-circulation results further purification frozen in liquid nitrogen before being transferred to the recipient embryo. The results showed that for the development of embryos transferred to the fresh circulation of PGCs-circulation as many as 25 cells can survive up to day 14, while one of the transferred of 50 and 100 cells into recipient embryos was hatched (10%. On the contrari recipient embryos that are transferred to the frozen PGCs-circulation the embryos development was shorter, and only survived until day 10th (treatment 25 cells, day 14th (treatment of 50 cells and day 17th (treatment of 100 cells. It is concluded that the amount of PGCs-circulation embryos transferred to the recipient is one factor that influence the success of the development germline chimeras.

  19. Germline genetic variation in JAK2 as a prognostic marker in castration-resistant prostate cancer.

    Science.gov (United States)

    Zhang, Ben Y; Riska, Shaun M; Mahoney, Douglas W; Costello, Brian A; Kohli, Rhea; Quevedo, Jorge F; Cerhan, James R; Kohli, Manish

    2017-03-01

    To evaluate the prognostic significance of germline variation in candidate genes in patients with castration-resistant prostate cancer (CRPC). Germline DNA was extracted from peripheral blood mononuclear cells of patients with CRPC enrolled in a clinically annotated registry. Fourteen candidate genes implicated in either initiation or progression of prostate cancer were tagged using single nucleotide polymorphisms (SNPs) from HapMap with a minor allele frequency of >5%. The primary endpoint was overall survival (OS), defined as time from development of CRPC to death. Principal component analysis was used for gene levels tests of significance. For SNP-level results the per allele hazard ratios (HRs) and 95% confidence intervals (CIs) under the additive allele model were estimated using Cox regression, adjusted for age at CRPC and Gleason score (GS). A total of 240 patients with CRPC were genotyped (14 genes; 84 SNPs). The median (range) age of the cohort was 69 (43-93) years. The GS distribution was 55% with GS ≥8, 32% with GS = 7 and 13% with GS <7 or unknown. The median (interquartile range) time from castration resistance to death for the cohort was 2.67 (1.6-4.07) years (144 deaths). At the gene level, a single gene, JAK2 was associated with OS (P < 0.01), and 11 of 18 JAK2 SNPs were individually associated with OS after adjustment for age and GS. A multivariate model consisting of age, GS, rs2149556 (HR 0.67; 95% CI 0.38-1.18) and rs4372063 (HR 2.17; 95% CI 1.25-3.76) was constructed to predict survival in patients with CRPC (concordance of 0.69, P < 3.2 × 10(-9) ). Germline variation in the JAK2 gene was associated with survival in patients with CRPC and warrants further validation as a potential prognostic biomarker. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

  20. Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility

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    Nunes Virginia

    2008-01-01

    Full Text Available Abstract Background Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. Conclusion This study proposes that variation at putative 8q24 cis-regulator(s of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene.

  1. Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.

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    Gabriel Rinaldi

    Full Text Available Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE. After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1 generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance

  2. PUF-8 suppresses the somatic transcription factor PAL-1 expression in C. elegans germline stem cells.

    Science.gov (United States)

    Mainpal, Rana; Priti, Agarwal; Subramaniam, Kuppuswamy

    2011-12-01

    RNA-binding proteins of the PUF family are well conserved post-transcriptional regulators that control a variety of developmental processes. The C. elegans protein PUF-8 is essential for several aspects of germ cell development including the maintenance of germline stem cells (GSCs). To explore the molecular mechanisms underlying its function, we have identified 160 germline-expressed mRNAs as potential targets of PUF-8. We generated GFP::H2B-3' UTR fusions for 17 mRNAs to assay their post-transcriptional regulation in germ cells. Twelve transgenes were not expressed in the mitotic germ cells, and depletion of PUF-8 led to misexpression of six of them in these cells. In contrast, the expression of 3' UTR fusion of hip-1, which encodes the HSP-70 interacting protein, was dependent on PUF-8. These results indicate that PUF-8 may regulate the expression of its targets both negatively as well as positively. We investigated the PUF-8-mediated post-transcriptional control of one mRNA, namely pal-1, which encodes a homeodomain transcription factor responsible for muscle development. Our results show that PUF-8 binds in vitro to specific sequences within pal-1 3' UTR that are critical for post-transcriptional suppression in GSCs. Removal of PUF-8 resulted in PAL-1 misexpression, and PAL-1-dependent misexpression of the myogenic promoter HLH-1 in germ cells. We propose that PUF-8 protects GSCs from the influence of somatic differentiation factors such as PAL-1, which are produced in the maternal germline but meant for embryogenesis.

  3. Increased oxidative damage in carriers of the germline TP53 p.R337H mutation.

    Directory of Open Access Journals (Sweden)

    Gabriel S Macedo

    Full Text Available Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS and Li-Fraumeni-like (LFL Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H. The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17 and non-carriers (NC, n = 17. We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively. Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.20±0.71, C = 160.5±0.88, P<0.0001. Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.

  4. Germline Variation in HSD3B1 as a Novel Biomarker in Prostate Cancer

    Science.gov (United States)

    2016-10-01

    dx.doi.org/10.1016/S1470-2045(16)30227-3 Articles HSD3B1 and resistance to androgen-deprivation therapy in prostate cancer : a retrospective...et al. Intermittent versus continuous androgen deprivation in prostate cancer . N Engl J Med 2013; 368: 1314–25. 20 Articles www.thelancet.com...AWARD NUMBER: W81XWH-15-1-0503 TITLE: Germline Variation in HSD3B1 as a Novel Biomarker in Prostate Cancer PRINCIPAL INVESTIGATOR: Jason

  5. Drosophila germline invasion by the endogenous retrovirus gypsy: involvement of the viral env gene.

    Science.gov (United States)

    Pelisson, A; Mejlumian, L; Robert, V; Terzian, C; Bucheton, A

    2002-10-01

    The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.

  6. Disease evolution and outcomes in familial AML with germline CEBPA mutations

    DEFF Research Database (Denmark)

    Tawana, Kiran; Wang, Jun; Renneville, Aline

    2015-01-01

    therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.......In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were...

  7. Germline large genomic alterations on 7q in patients with multiple primary cancers

    DEFF Research Database (Denmark)

    Villacis, Rolando A R; Basso, Tatiane R; Canto, Luisa M

    2017-01-01

    triple negative breast tumors and no mutations in BRCA1, BRCA2 and TP53 genes. Germline rearrangements on chromosome 7q, involving over 40 Mb of the same region, were found in both patients: one with mosaic loss (80% of cells) and the other with cnLOH (copy-neutral loss of heterozygosity) secondary...... to maternal allele duplication. Five children tested had no alterations on 7q. The patients shared 330 genes in common on 7q22.1-q34, including several tumor suppressor genes (TSGs) previously related to breast cancer risk and imprinted genes. The analysis of the triple negative BC from one patient revealed...

  8. No severe and global X chromosome inactivation in meiotic male germline of Drosophila

    OpenAIRE

    Mikhaylova Lyudmila M; Nurminsky Dmitry I

    2012-01-01

    Abstract This article is a response to Vibranovski et al. See correspondence article http://www.biomedcentral.com/1741-7007/10/49 and the original research article http://www.biomedcentral.com/1741-7007/9/29 We have previously reported a high propensity of testis-expressed X-linked genes to activation in meiotic cells, a similarity in global gene expression between the X chromosome and autosomes in meiotic germline, and under-representation of various types of tissue-specific genes on the X c...

  9. The diverse functions of germline P-granules in Caenorhabditis elegans.

    Science.gov (United States)

    Voronina, Ekaterina

    2013-08-01

    P-granules are conserved cytoplasmic organelles, similar to nuage, that are present in Caenorhabditis elegans germ cells. Based on the prevailing sterility phenotype of the component mutants, P-granules have been seen as regulators of germ cell development and function. Yet, specific germline defects resulting from P-granule failure vary, depending on which component(s) are inactivated, at which stage of development, as well as on the presence of stress factors during animal culture. This review discusses the unifying themes in many P-granule functions, with the main focus on their role as organizing centers nucleating RNA regulation in the germ cell cytoplasm.

  10. NanoTIO2 (UV-Titan) does not induce ESTR mutations in the germline of prenatally exposed female mice

    DEFF Research Database (Denmark)

    Boisen, Anne Mette Zenner; Shipley, Thomas; Hougaard, Karin Sørig

    2012-01-01

    Particulate air pollution has been linked to an increased risk of cardiovascular disease and cancer. Animal studies have shown that inhalation of air particulates induces mutations in the male germline. Expanded simple tandem repeat (ESTR) loci in mice are sensitive markers of mutagenic effects...... on male germ cells resulting from environmental exposures; however, female germ cells have received little attention. Oocytes may be vulnerable during stages of active cell division (e.g., during fetal development). Accordingly, an increase in germline ESTR mutations in female mice prenatally exposed...... exposed by whole-body inhalation to the nanoTiO2 UV-Titan L181 (~42.4 mg UV-Titan/m3) or filtered clean air on gestation days (GD) 8–18. Female C57BL/6 F1 offspring were raised to maturity and mated with unexposed CBA males. The F2 descendents were collected and ESTR germline mutation rates...

  11. Germline variants in Hamartomatous Polyposis Syndrome-associated genes from patients with one or few hamartomatous polyps

    DEFF Research Database (Denmark)

    Jelsig, Anne Marie; Brusgaard, Klaus; Hansen, Tine Plato;

    2016-01-01

    Sequencing, DNA samples from 77 patients with 84 hamartomatous polyps were sequenced. The detected germline variants were classified into pathogenicity classes. RESULTS: We detected several germline variants, among them three in ENG, two in BMPR1A, one in PTEN, and one in SMAD4. Although some of the detected......OBJECTIVE: A subgroup of patients with hamartomatous polyps in the GI tract has a hereditary Hamartomatous Polyposis Syndrome with an increased risk of cancer. The distinction between patients with one or few polyps and patients with a syndrome can be difficult. A pathogenic germline mutation can...... be detected in a majority of HPS patients. This study investigates whether patients with one or few hamartomatous polyps could have a syndrome based on genetic screening of relevant genes. METHODS: We designed a gene panel including 26 hamartomatous polyposis-associated genes. Using targeted Next Generation...

  12. Adaptive evolution of genes involved in the regulation of germline stem cells in Drosophila melanogaster and D. simulans.

    Science.gov (United States)

    Flores, Heather A; DuMont, Vanessa L Bauer; Fatoo, Aalya; Hubbard, Diana; Hijji, Mohammed; Barbash, Daniel A; Aquadro, Charles F

    2015-02-09

    Population genetic and comparative analyses in diverse taxa have shown that numerous genes involved in reproduction are adaptively evolving. Two genes involved in germline stem cell regulation, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), have been shown previously to experience recurrent, adaptive evolution in both Drosophila melanogaster and D. simulans. Here we report a population genetic survey on eight additional genes involved in germline stem cell regulation in D. melanogaster and D. simulans that reveals all eight of these genes reject a neutral model of evolution in at least one test and one species after correction for multiple testing using a false-discovery rate of 0.05. These genes play diverse roles in the regulation of germline stem cells, suggesting that positive selection in response to several evolutionary pressures may be acting to drive the adaptive evolution of these genes.

  13. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes

    NARCIS (Netherlands)

    Ruijs, M.W.G.; Verhoef, S.; Rookus, M.A.; Pruntel, R.; van der Hout, A.H.; Hogervorst, F.B.L.; Kluijt, I.; Sijmons, R.H.; Aalfs, C.M.; Wagner, A.; Ausems, M.G.E.M.; Hoogerbrugge, N.; van Asperen, C.J.; Gómez García, E.B.; Meijers-Heijboer, H.; ten Kate, L.P.; Menko, F.H.; van 't Veer, L.J.

    2010-01-01

    Background Li-Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria a

  14. Prevalence of pathological germline mutations of hMLH1 and hMSH2 genes in colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Dandan Li

    Full Text Available The prevalence of pathological germline mutations in colorectal cancer has been widely studied, as germline mutations in the DNA mismatch repair genes hMLH1 and hMSH2 confer a high risk of colorectal cancer. However, because the sample size and population of previous studies are very different from each other, the conclusions still remain controversial. In this paper, Databases such as PubMed were applied to search for related papers. The data were imported into Comprehensive Meta-Analysis V2, which was used to estimate the weighted prevalence of hMLH1 and hMSH2 pathological mutations and compare the differences of prevalence among different family histories, ethnicities and related factors. This study collected and utilized data from 102 papers. In the Amsterdam-criteria positive group, the prevalence of pathological germline mutations of the hMLH1 and hMSH2 genes was 28.55% (95%CI 26.04%-31.19% and 19.41% (95%CI 15.88%-23.51%, respectively, and the prevalence of germline mutations in hMLH1/hMSH2 was 15.44%/10.02%, 20.43%/13.26% and 15.43%/11.70% in Asian, American multiethnic and European/Australian populations, respectively. Substitution mutations accounted for the largest proportion of germline mutations (hMLH1: 52.34%, hMSH2: 43.25%. The total prevalence of mutations of hMLH1 and hMSH2 in Amsterdam-criteria positive, Amsterdam-criteria negative and sporadic colorectal cancers was around 45%, 25% and 15%, respectively, and there were no obvious differences in the prevalence of germline mutations among different ethnicities.

  15. Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

    Institute of Scientific and Technical Information of China (English)

    Ai-Chun Zhao; Ding-Pei Long; San-Yuan Ma; Long-Xia Xu; Mei-Rong Zhang; Fang-Yin Dai; Qing-You Xia; Cheng Lu; Zhong-Huai Xiang

    2012-01-01

    To overcome the disadvantages of current silkworm Bombyx mori transgenic technology,such as costly and time-consuming to maintain non-diapause transgenic silkworms,we report here on the development of treatments for the germline transformation of diapause silkworm strains.Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains.By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15℃ (15℃-IME),we were able to prevent the diapause of their daughter eggs; a similar strategy (15℃-IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15℃,and the daughter eggs were then microinjected according to the conventional microinjection methods used for non-diapause eggs.By combining temperature and light controls,the improved 15℃-IMES strategy prevented diapause in daughter eggs,and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains.Although each of the strategies developed here has advantages and disadvantages,we suggest that the 15C-IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects.These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also higblight silkworm functional genomics research and its modeling.

  16. Scarce evidence of the causal role of germline mutations in UNC5C in hereditary colorectal cancer and polyposis

    OpenAIRE

    Pilar Mur; Sánchez-Cuartielles Elena; Susanna Aussó; Gemma Aiza; Valdés-Mas Rafael; Marta Pineda; Matilde Navarro; Joan Brunet; Miguel Urioste; Conxi Lázaro; Victor Moreno; Gabriel Capellá; Puente, Xose S; Laura Valle

    2016-01-01

    Germline mutations in UNC5C have been suggested to increase colorectal cancer (CRC) risk, thus causing hereditary CRC. However, the evidence gathered thus far is insufficient to include the study of the UNC5C gene in the routine genetic testing of familial CRC. Here we aim at providing a more conclusive answer about the contribution of germline UNC5C mutations to genetically unexplained hereditary CRC and/or polyposis cases. To achieve this goal we sequenced the coding region and exon-intron ...

  17. Germline mutation in NLRP2 (NALP2 in a familial imprinting disorder (Beckwith-Wiedemann Syndrome.

    Directory of Open Access Journals (Sweden)

    Esther Meyer

    2009-03-01

    Full Text Available Beckwith-Wiedemann syndrome (BWS is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2. However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.

  18. Familial adenomatous polyposis patients without an identified APC germline mutation have a severe phenotype

    DEFF Research Database (Denmark)

    Bisgaard, M L; Ripa, R; Knudsen, Anne Louise;

    2004-01-01

    BACKGROUND: Development of more than 100 colorectal adenomas is diagnostic of the dominantly inherited autosomal disease familial adenomatous polyposis (FAP). Germline mutations can be identified in the adenomatous polyposis coli (APC) gene in approximately 80% of patients. The APC protein compri...... they do not themselves more often represent an isolated case. CONCLUSIONS: The severe phenotype should be considered when counselling FAP families in which attenuated FAP is excluded and in which a causative APC mutation has not been identified.......BACKGROUND: Development of more than 100 colorectal adenomas is diagnostic of the dominantly inherited autosomal disease familial adenomatous polyposis (FAP). Germline mutations can be identified in the adenomatous polyposis coli (APC) gene in approximately 80% of patients. The APC protein...... in patients with a known APC mutation and with the phenotypes characteristic of patients with mutations in specific APC regions and domains. PATIENTS: Data on 121 FAP probands and 149 call up patients from 70 different families were extracted from the Danish Polyposis register. METHODS: Differences in 16...

  19. Identification of 3 novel VHL germ-line mutations in Danish VHL patients

    Directory of Open Access Journals (Sweden)

    Dandanell Mette

    2012-07-01

    Full Text Available Abstract Background von Hippel-Lindau (VHL disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene. Methods VHL mutational analysis was carried out by sequencing of the coding sequence and by multiplex ligation-dependent probe amplification analysis. The functional consequence of the variants was investigated using in silico prediction tools. Results A total of 289 probands suspected of having VHL syndrome have been screened for mutations in the VHL gene. Twenty-six different VHL mutations were identified in 36 families including one in-frame duplication, two frame-shift mutations, four nonsense mutations, twelve missense mutations, three intronic mutations and four large genomic rearrangements. Three of these mutations (c.319 C > T, c.342_343dupGGT and c.520_521dupAA were novel. Conclusions In this study we report the VHL germ-line mutations found in Danish families. We found three novel VHL mutations where two were classified as pathogenic and the latter was classified as a variant of unknown significance. Together, our findings contribute to the interpretation of the potential pathogenicity of VHL germ-line mutations.

  20. The arrest gene is required for germline cyst formation during Drosophila oogenesis.

    Science.gov (United States)

    Parisi, M J; Deng, W; Wang, Z; Lin, H

    2001-04-01

    In Drosophila, oogenesis is initiated when a germline stem cell produces a differentiating daughter cell called the cystoblast. The cystoblast undergoes four rounds of synchronous divisions with incomplete cytokinesis to generate a syncytial cyst of 16 interconnected cystocytes, in which one cystocyte differentiates into an oocyte. Strong mutations of the arrest (aret) gene disrupt cyst formation and cause the production of clusters of ill-differentiated germline cells that retain cellular and molecular characteristics of cystoblasts. These mutant germ cells express high levels of BAM-C and SXL proteins in the cytoplasm but do not accumulate markers for advanced cystocytes or differentiating oocytes, such as the nuclear localization of SXL or the accumulation of osk mRNA, orb mRNA, and cytoplasmic dynein. However, the mutant germ cells do not contain spectrosomes, the cytoplasmic structure that objectifies the divisional asymmetry of the cystoblast. The aret mutant germ cells undergo active mitosis with complete cytokinesis. Their mitosis is accompanied by massive necrosis, so that the number of germ cells in a stem cell-derived cluster ranges from one to greater than 70. These defects of aret mutants reveal a novel function of aret as the first gene with a defined function in the cystoblast to cyst transition during early oogenesis.

  1. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary.

    Science.gov (United States)

    Malone, Colin D; Brennecke, Julius; Dus, Monica; Stark, Alexander; McCombie, W Richard; Sachidanandam, Ravi; Hannon, Gregory J

    2009-05-01

    In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.

  2. Non-Canonical and Sexually Dimorphic X Dosage Compensation States in the Mouse and Human Germline.

    Science.gov (United States)

    Sangrithi, Mahesh N; Royo, Helene; Mahadevaiah, Shantha K; Ojarikre, Obah; Bhaw, Leena; Sesay, Abdul; Peters, Antoine H F M; Stadler, Michael; Turner, James M A

    2017-02-06

    Somatic X dosage compensation requires two mechanisms: X inactivation balances X gene output between males (XY) and females (XX), while X upregulation, hypothesized by Ohno and documented in vivo, balances X gene with autosomal gene output. Whether X dosage compensation occurs in germ cells is unclear. We show that mouse and human germ cells exhibit non-canonical X dosage states that differ from the soma and between the sexes. Prior to genome-wide reprogramming, X upregulation is present, consistent with Ohno's hypothesis. Subsequently, however, it is erased. In females, erasure follows loss of X inactivation, causing X dosage excess. Conversely, in males, erasure leads to permanent X dosage decompensation. Sex chromosomally abnormal models exhibit a "sex-reversed" X dosage state: XX males, like XX females, develop X dosage excess, while XO females, like XY males, develop X dosage decompensation. Thus, germline X dosage compensation states are determined by X chromosome number, not phenotypic sex. These unexpected differences in X dosage compensation states between germline and soma offer unique perspectives on sex chromosome infertility.

  3. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl.

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Randal Voss, S; Gardiner, David M; Hunter, Tony

    2012-10-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

  4. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

    2013-01-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration – wound healing, cellular dedifferentiation and blastemal formation – will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration. PMID:22841627

  5. Pediatric intracranial clear cell meningioma associated with a germline mutation of SMARCE1: a novel case.

    Science.gov (United States)

    Raffalli-Ebezant, Helen; Rutherford, Scott A; Stivaros, Stavros; Kelsey, Anna; Smith, Miriam; Evans, D Gareth; Kilday, John-Paul

    2015-03-01

    Intracranial clear cell meningioma (CCM) represents a rare and potentially more aggressive subgroup of meningioma that is observed more frequently in children and adolescents. Despite its characterization as a histological entity, there is little evidence identifying tumorigenic etiologies. Recently, a novel mutation in SMARCE1, encoding a subunit of the SWI/SNF chromatin remodeling complex, was identified in a cohort of spinal CCMs. To date, no intracranial CCM has been subjected to analysis. We report the case of an isolated intracranial CCM in a 14-year-old girl. Gross total resection was achieved following a two-stage approach with no evidence of tumor recurrence 8 months following presentation. Exon sequencing identified a germline mutation in SMARCE1, which was also present in tumor DNA. Extensive literature review confirmed our study is the first to seek and report a genetic anomaly for childhood intracranial CCMs outside of the NF2 gene locus, and the first to make an association between a germline SMARCE1 mutation and childhood intracranial CCMs. Together with the previous description of SMARCE1 mutations in spinal CCMs, our report suggests that SMARCE1 aberrations may be implicated in establishing a clear cell histology irrespective of meningioma location. We would advocate that, where feasible, genetic sequencing is performed on future new cases of childhood neuraxial CCMs and includes interrogation of the SMARCE1 gene.

  6. Characterization of cryptic splicing in germline PTEN intronic variants in Cowden syndrome.

    Science.gov (United States)

    Chen, Hannah Jinlian; Romigh, Todd; Sesock, Kaitlin; Eng, Charis

    2017-10-01

    Germline mutations in the tumor-suppressor gene PTEN predispose to subsets of Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and autism. Evidence-based classification of PTEN variants as either deleterious or benign is urgently needed for accurate molecular diagnosis and gene-informed genetic counseling. We studied 34 different germline PTEN intronic variants from 61 CS patients, characterized their PTEN mRNA processing, and analyzed PTEN expression and downstream readouts of P-AKT and P-ERK1/2. While we found that many mutations near splice junctions result in exon skipping, we also identified the presence of cryptic splicing that resulted in premature termination or a shift in isoform usage. PTEN protein expression is significantly lower in the group with splicing changes while P-AKT, but not P-ERK1/2, is significantly increased. Our observations of these PTEN intronic variants should contribute to the determination of pathogenicity of PTEN intronic variants and aid in genetic counseling. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  7. Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Yergeau Donald A

    2011-11-01

    Full Text Available Abstract Background The Sleeping Beauty (SB transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

  8. Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons.

    Science.gov (United States)

    Ivics, Zoltán; Garrels, Wiebke; Mátés, Lajos; Yau, Tien Yin; Bashir, Sanum; Zidek, Vaclav; Landa, Vladimír; Geurts, Aron; Pravenec, Michal; Rülicke, Thomas; Kues, Wilfried A; Izsvák, Zsuzsanna

    2014-04-01

    The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase, together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer (SCNT), and it offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing and the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in 1 d; generation of germline-transgenic lines by using this protocol takes ∼1 year.

  9. Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons.

    Science.gov (United States)

    Ivics, Zoltán; Hiripi, László; Hoffmann, Orsolya I; Mátés, Lajos; Yau, Tien Yin; Bashir, Sanum; Zidek, Vaclav; Landa, Vladimír; Geurts, Aron; Pravenec, Michal; Rülicke, Thomas; Bösze, Zsuzsanna; Izsvák, Zsuzsanna

    2014-04-01

    The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

  10. Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons.

    Science.gov (United States)

    Ivics, Zoltán; Mátés, Lajos; Yau, Tien Yin; Landa, Vladimír; Zidek, Vaclav; Bashir, Sanum; Hoffmann, Orsolya I; Hiripi, László; Garrels, Wiebke; Kues, Wilfried A; Bösze, Zsuzsanna; Geurts, Aron; Pravenec, Michal; Rülicke, Thomas; Izsvák, Zsuzsanna

    2014-04-01

    We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

  11. Identification of genes for engineering the male germline of Aedes aegypti and Ceratitis capitata.

    Science.gov (United States)

    Sutton, Elizabeth R; Yu, Yachuan; Shimeld, Sebastian M; White-Cooper, Helen; Alphey, And Luke

    2016-11-21

    Synthetic biology approaches are promising new strategies for control of pest insects that transmit disease and cause agricultural damage. These strategies require characterised modular components that can direct appropriate expression of effector sequences, with components conserved across species being particularly useful. The goal of this study was to identify genes from which new potential components could be derived for manipulation of the male germline in two major pest species, the mosquito Aedes aegypti and the tephritid fruit fly Ceratitis capitata. Using RNA-seq data from staged testis samples, we identified several candidate genes with testis-specific expression and suitable expression timing for use of their regulatory regions in synthetic control constructs. We also developed a novel computational pipeline to identify candidate genes with testis-specific splicing from this data; use of alternative splicing is another method for restricting expression in synthetic systems. Some of the genes identified display testis-specific expression or splicing that is conserved across species; these are particularly promising candidates for construct development. In this study we have identified a set of genes with testis-specific expression or splicing. In addition to their interest from a basic biology perspective, these findings provide a basis from which to develop synthetic systems to control important pest insects via manipulation of the male germline.

  12. No severe and global X chromosome inactivation in meiotic male germline of Drosophila

    Directory of Open Access Journals (Sweden)

    Mikhaylova Lyudmila M

    2012-06-01

    Full Text Available Abstract This article is a response to Vibranovski et al. See correspondence article http://www.biomedcentral.com/1741-7007/10/49 and the original research article http://www.biomedcentral.com/1741-7007/9/29 We have previously reported a high propensity of testis-expressed X-linked genes to activation in meiotic cells, a similarity in global gene expression between the X chromosome and autosomes in meiotic germline, and under-representation of various types of tissue-specific genes on the X chromosome. Based on our findings and a critical review of the current literature, we believe that there is no global and severe silencing of the X chromosome in the meiotic male germline of Drosophila. The term 'meiotic sex chromosome inactivation' (MSCI therefore seems misleading when used to describe the minor underexpression of the X chromosome in the testis of Drosophila, because this term erroneously implies a profound and widespread silencing of the X-linked genes, by analogy to the well-studied MSCI system in mammals, and therefore distracts from identification and analysis of the real mechanisms that orchestrate gene expression and evolution in this species.

  13. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W. (Toronto); (UBC)

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  14. A DTC niche plexus surrounds the germline stem cell pool in Caenorhabditis elegans.

    Science.gov (United States)

    Byrd, Dana T; Knobel, Karla; Affeldt, Katharyn; Crittenden, Sarah L; Kimble, Judith

    2014-01-01

    The mesenchymal distal tip cell (DTC) provides the niche for Caenorhabditis elegans germline stem cells (GSCs). The DTC has a complex cellular architecture: its cell body caps the distal gonadal end and contacts germ cells extensively, but it also includes multiple cellular processes that extend along the germline tube and intercalate between germ cells. Here we use the lag-2 DTC promoter to drive expression of myristoylated GFP, which highlights DTC membranes and permits a more detailed view of DTC architecture. We find that short processes intercalating between germ cells contact more germ cells than seen previously. We define this region of extensive niche contact with germ cells as the DTC plexus. The extent of the DTC plexus corresponds well with the previously determined extent of the GSC pool. Moreover, expression of a differentiation marker increases as germ cells move out of the plexus. Maintenance of this DTC plexus depends on the presence of undifferentiated germ cells, suggesting that germ cell state can influence niche architecture. The roles of this DTC architecture remain an open question. One idea is that the DTC plexus delivers Notch signaling to the cluster of germ cells comprising the GSC pool; another idea is that the plexus anchors GSCs at the distal end.

  15. Identification of Two Novel HOXB13 Germline Mutations in Portuguese Prostate Cancer Patients

    Science.gov (United States)

    Maia, Sofia; Cardoso, Marta; Pinto, Pedro; Pinheiro, Manuela; Santos, Catarina; Peixoto, Ana; Bento, Maria José; Oliveira, Jorge; Henrique, Rui; Jerónimo, Carmen; Teixeira, Manuel R.

    2015-01-01

    The HOXB13 germline variant G84E (rs138213197) was recently described in men of European descent, with the highest prevalence in Northern Europe. The G84E mutation has not been found in patients of African or Asian ancestry, which may carry other HOXB13 variants, indicating allelic heterogeneity depending on the population. In order to gain insight into the full scope of coding HOXB13 mutations in Portuguese prostate cancer patients, we decided to sequence the entire coding region of the HOXB13 gene in 462 early-onset or familial/hereditary cases. Additionally, we searched for somatic HOXB13 mutations in 178 prostate carcinomas to evaluate their prevalence in prostate carcinogenesis. Three different patients were found to carry in their germline DNA two novel missense variants, which were not identified in 132 control subjects. Both variants are predicted to be deleterious by different in silico tools. No somatic mutations were found. These findings further support the hypothesis that different rare HOXB13 mutations may be found in different ethnic groups. Detection of mutations predisposing to prostate cancer may require re-sequencing rather than genotyping, as appropriate to the population under investigation. PMID:26176944

  16. piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline.

    Science.gov (United States)

    Chambeyron, Séverine; Popkova, Anna; Payen-Groschêne, Geneviève; Brun, Christine; Laouini, Dorsaf; Pelisson, Alain; Bucheton, Alain

    2008-09-30

    Germline silencing of transposable elements is essential for the maintenance of genome integrity. Recent results indicate that this repression is largely achieved through a RNA silencing pathway that involves Piwi-interacting RNAs (piRNAs). However the repressive mechanisms are not well understood. To address this question, we used the possibility to disrupt the repression of the Drosophila I element retrotransposon by hybrid dysgenesis. We show here that the repression of the functional I elements that are located in euchromatin requires proteins of the piRNA pathway, and that the amount of ovarian I element piRNAs correlates with the strength of the repression in the female germline. Antisense RNAs, which are likely used to produce antisense piRNAs, are transcribed by heterochromatic defective I elements, but efficient production of these antisense small RNAs requires the presence in the genome of euchromatic functional I elements. Finally, we demonstrate that the piRNA-induced silencing of the functional I elements is at least partially posttranscriptional. In a repressive background, these elements are still transcribed, but some of their sense transcripts are kept in nurse cell nuclear foci together with those of the Doc retrotransposon. In the absence of I element piRNAs, either in dysgenic females or in mutants of the piRNA silencing pathway, sense I element transcripts are transported toward the oocyte where retrotransposition occurs. Our results indicate that piRNAs are involved in a posttranscriptional gene-silencing mechanism resulting in RNA nuclear accumulation.

  17. Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.

    Science.gov (United States)

    Yergeau, Donald A; Kelley, Clair M; Kuliyev, Emin; Zhu, Haiqing; Johnson Hamlet, Michelle R; Sater, Amy K; Wells, Dan E; Mead, Paul E

    2011-11-24

    The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

  18. Little evidence for demasculinization of the Drosophila X chromosome among genes expressed in the male germline.

    Science.gov (United States)

    Meiklejohn, Colin D; Presgraves, Daven C

    2012-01-01

    Male-biased genes-those expressed at higher levels in males than in females-are underrepresented on the X chromosome of Drosophila melanogaster. Several evolutionary models have been posited to explain this so-called demasculinization of the X. Here, we show that the apparent paucity of male-biased genes on the X chromosome is attributable to global X-autosome differences in expression in Drosophila testes, owing to a lack of sex chromosome dosage compensation in the male germline, but not to any difference in the density of testis-specific or testis-biased genes on the X chromosome. First, using genome-wide gene expression data from 20 tissues, we find no evidence that genes with testis-specific expression are underrepresented on the X chromosome. Second, using contrasts in gene expression profiles among pairs of tissues, we recover a statistical underrepresentation of testis-biased genes on the X but find that the pattern largely disappears once we account for the lack of dosage compensation in the Drosophila male germline. Third, we find that computationally "demasculinizing" the autosomes is not sufficient to produce an expression profile similar to that of the X chromosome in the testes. Our findings thus show that the lack of sex chromosome dosage compensation in Drosophila testes can explain the apparent signal of demasculinization on the X, whereas evolutionary demasculinization of the X cannot explain its overall reduced expression in the testes.

  19. A recurrent germline BAP1 mutation and extension of the BAP1 tumor predisposition spectrum to include basal cell carcinoma

    DEFF Research Database (Denmark)

    Wadt, K A W; Aoude, L G; Johansson, P;

    2015-01-01

    We report four previously undescribed families with germline BAP1 mutations and expand the clinical phenotype of this tumor syndrome. The tumor spectrum in these families is predominantly uveal malignant melanoma (UMM), cutaneous malignant melanoma (CMM) and mesothelioma, as previously reported f...

  20. Accuracy of Hereditary Diffuse Gastric Cancer Testing Criteria and Outcomes in Patients With a Germline Mutation in CDH1

    NARCIS (Netherlands)

    van der Post, Rachel S.; Vogelaar, Ingrid P.; Manders, Peggy; van der Kolk, Lizet E.; Cats, Annemieke; van Hest, Liselotte P.; Sijmons, Rolf; Aalfs, Cora M.; Ausems, Margreet G. E. M.; Garcia, Encarna B. Gomez; Wagner, Anja; Hes, Frederik J.; Arts, Neeltje; Mensenkamp, Arjen R.; van Krieken, J. Han; Hoogerbrugge, Nicoline; Ligtenberg, Marjolijn J. L.

    2015-01-01

    BACKGROUND & AIMS: Germline mutations in the cadherin 1, type 1, E-cadherin gene (CDH1) cause a predisposition to gastric cancer. We evaluated the ability of the internationally accepted hereditary diffuse gastric cancer (HDGC) criteria to identify individuals with pathogenic mutations in CDH1, and

  1. Accuracy of Hereditary Diffuse Gastric Cancer Testing Criteria and Outcomes in Patients With a Germline Mutation in CDH1

    NARCIS (Netherlands)

    Van Der Post, Rachel S.; Vogelaar, Ingrid P.; Manders, Peggy; Van Der Kolk, Lizet E.; Cats, Annemieke; Van Hest, Liselotte P.; Sijmons, Rolf; Aalfs, Cora M.; Ausems, Margreet G E M; Gómez García, Encarna B.; Wagner, Anja; Hes, Frederik J.; Arts, Neeltje; Mensenkamp, Arjen R.; Van Krieken, J. Han; Hoogerbrugge, Nicoline; Ligtenberg, Marjolijn J L

    2015-01-01

    Background & Aims Germline mutations in the cadherin 1, type 1, E-cadherin gene (CDH1) cause a predisposition to gastric cancer. We evaluated the ability of the internationally accepted hereditary diffuse gastric cancer (HDGC) criteria to identify individuals with pathogenic mutations in CDH1, and a

  2. Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility

    NARCIS (Netherlands)

    Astuti, Dewi; Morris, Mark R.; Cooper, Wendy N.; Staals, Raymond H. J.; Wake, Naomi C.; Fews, Graham A.; Gill, Harmeet; Gentle, Dean; Shuib, Salwati; Ricketts, Christopher J.; Cole, Trevor; van Essen, Anthonie J.; van Lingen, Richard A.; Neri, Giovanni; Opitz, John M.; Rump, Patrick; Stolte-Dijkstra, Irene; Mueller, Ferenc; Pruijn, Ger J. M.; Latif, Farida; Maher, Eamonn R.

    2012-01-01

    Perlman syndrome is a congenital overgrowth syndrome inherited in an autosomal recessive manner that is associated with Wilms tumor susceptibility. We mapped a previously unknown susceptibility locus to 2q37.1 and identified germline mutations in DIS3L2, a homolog of the Schizosaccharomyces pombe di

  3. Germline and somatic NF1 mutations in sporadic and NF1-associated malignant peripheral nerve sheath tumours

    NARCIS (Netherlands)

    Bottillo, Irene; Ahiquist, Terje; Brekke, Helge; Danielsen, Stine A.; van den Berg, Eva; Mertens, Fredrik; Lothe, Ragnhild A.; Dallapiccola, Bruno

    2009-01-01

    Malignant peripheral nerve sheath tumours (MPNSTs) are a malignancy occurring with increased frequency in patients with neurofibromatosis type 1 (NF1). In contrast to the well-known spectrum of germline NF1 mutations, the information on somatic mutations in MPNSTs is limited. In this study, we scree

  4. High burden of copy number alterations and c-MYC amplification in prostate cancer from BRCA2 germline mutation carriers

    NARCIS (Netherlands)

    Castro, E.; Jugurnauth-Little, S.; Karlsson, Q.; Al-Shahrour, F.; Pineiro-Yanez, E.; Van de Poll, F.; Leongamornlert, D.; Dadaev, T.; Govindasami, K.; Guy, M.; Eeles, R.; Kote-Jarai, Z.; Oosterwijk, J. C.

    2015-01-01

    Background: Germline BRCA2 mutations are associated with poorer outcome prostate cancer ( PCa) compared with sporadic tumours but this association remains to be characterised. In this study, we aim to assess if there is a signature set of copy number alterations ( CNA) that could aid to the identifi

  5. PALB2 germline mutations in familial breast cancer cases with personal and family history of pancreatic cancer

    OpenAIRE

    2010-01-01

    PALB2 germline mutations in familial breast cancer cases with personal and family history of pancreatic cancer phone: +39-02-23903224 (Radice, Paolo) (Radice, Paolo) IFOM, Fondazione Istituto FIRC di Oncologia Molecolare - Milan - ITALY (Peterlongo, Paolo) Department of Preventive and Predictive Medicine, Unit of Molecular Bases of Genetic Risk and Genetic Testing, Fondazione IRCCS Istituto Nazionale dei Tumori - Milan - ITALY (Peterlongo, Pao...

  6. Discovery of germline-related genes in Cephalochordate amphioxus: A genome wide survey using genome annotation and transcriptome data.

    Science.gov (United States)

    Yue, Jia-Xing; Li, Kun-Lung; Yu, Jr-Kai

    2015-12-01

    The generation of germline cells is a critical process in the reproduction of multicellular organisms. Studies in animal models have identified a common repertoire of genes that play essential roles in primordial germ cell (PGC) formation. However, comparative studies also indicate that the timing and regulation of this core genetic program vary considerably in different animals, raising the intriguing questions regarding the evolution of PGC developmental mechanisms in metazoans. Cephalochordates (commonly called amphioxus or lancelets) represent one of the invertebrate chordate groups and can provide important information about the evolution of developmental mechanisms in the chordate lineage. In this study, we used genome and transcriptome data to identify germline-related genes in two distantly related cephalochordate species, Branchiostoma floridae and Asymmetron lucayanum. Branchiostoma and Asymmetron diverged more than 120 MYA, and the most conspicuous difference between them is their gonadal morphology. We used important germline developmental genes in several model animals to search the amphioxus genome and transcriptome dataset for conserved homologs. We also annotated the assembled transcriptome data using Gene Ontology (GO) terms to facilitate the discovery of putative genes associated with germ cell development and reproductive functions in amphioxus. We further confirmed the expression of 14 genes in developing oocytes or mature eggs using whole mount in situ hybridization, suggesting their potential functions in amphioxus germ cell development. The results of this global survey provide a useful resource for testing potential functions of candidate germline-related genes in cephalochordates and for investigating differences in gonad developmental mechanisms between Branchiostoma and Asymmetron species.

  7. Germline mutations in BMP9 are not identified in a series of Danish and French patients with hereditary hemorrhagic telangiectasia

    DEFF Research Database (Denmark)

    Tørring, P. M.; Dupuis-Girod, S.; Giraud, S

    2016-01-01

    had a different vascular-anomaly syndrome, the suspicion that BMP9 mutations might cause HHT remained. To evaluate if germline mutations in BMP9 can be identified in HHT patients, we investigated the Danish and the French Lyon cohort of mutation-negative and clinically definite HHT patients. Exons...

  8. Absence of loss of heterozygosity of BRCA1 in a renal tumor from a BRCA1 germline mutation carrier

    OpenAIRE

    Alanee, Shaheen; Shah, Sohela; Murali, Rajmohan; Rau-Murthy, Rohini; Kasmintan A Schrader; Offit, Kenneth

    2013-01-01

    BRCA1 functions as a tumor suppressor gene and germline and somatic mutations in this gene have been shown to be associated with many types of cancer. We report the first tumor study of renal cell carcinoma in a carrier of the deleterious BRCA1 mutation-c.68_69delAG.

  9. Germline Polymorphisms of the VEGF Pathway Predict Recurrence in Nonadvanced Differentiated Thyroid Cancer.

    Science.gov (United States)

    Marotta, Vincenzo; Sciammarella, Concetta; Capasso, Mario; Testori, Alessandro; Pivonello, Claudia; Chiofalo, Maria Grazia; Gambardella, Claudio; Grasso, Marica; Antonino, Antonio; Annunziata, Annamaria; Macchia, Paolo Emidio; Pivonello, Rosario; Santini, Luigi; Botti, Gerardo; Losito, Simona; Pezzullo, Luciano; Colao, Annamaria; Faggiano, Antongiulio

    2017-02-01

    Tumor angiogenesis is determined by host genetic background rather than environment. Germline single nucleotide polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) pathway have demonstrated prognostic value in different tumors. Our main objective was to test the prognostic value of germline SNPs of the VEGF pathway in nonadvanced differentiated thyroid cancer (DTC). Secondarily, we sought to correlate analyzed SNPs with microvessel density (MVD). Multicenter, retrospective, observational study. Four referral centers. Blood samples were obtained from consecutive DTC patients. Genotyping was performed according to the TaqMan protocol, including 4 VEGF-A (-2578C>A, -460T>C, +405G>C, and +936C>T) and 2 VEGFR-2 (+1192 C>T and +1719 T>A) SNPs. MVD was estimated by means of CD34 staining. Rate of recurrent structural disease/disease-free survival (DFS). Difference in MVD between tumors from patients with different genotype. Two hundred four patients with stage I-II DTC (mean follow-up, 73 ± 64 months) and 240 patients with low- to intermediate-risk DTC (mean follow-up, 70 ± 60 months) were enrolled. Two "risk" genotypes were identified by combining VEGF-A SNPs -2578 C>A, -460 T>C, and +405 G>C. The ACG homozygous genotype was protective in both stage I-II (odds ratio [OR], 0.08; 95% confidence interval [CI], 0.01 to 1.43; P = 0.018) and low- to intermediate-risk (OR, 0.14; 95% CI, 0.01 to 1.13; P = 0.035) patients. The CTG homozygous genotype was significantly associated with recurrence in stage I-II (OR, 5.47; 95% CI, 1.15 to 26.04; P = 0.018) and was slightly deleterious in low- to intermediate-risk (OR, 3.39; 95% CI, 0.8 to 14.33; P = 0.079) patients. MVD of primary tumors from patients harboring a protective genotype was significantly lower (median MVD, 76.5 ± 12.7 and 86.7 ± 27.9, respectively; P = 0.024). Analysis of germline VEGF-A SNPs could empower a prognostic approach to DTC.

  10. Germline DNA copy number aberrations identified as potential prognostic factors for breast cancer recurrence.

    Directory of Open Access Journals (Sweden)

    Yadav Sapkota

    Full Text Available Breast cancer recurrence (BCR is a common treatment outcome despite curative-intent primary treatment of non-metastatic breast cancer. Currently used prognostic and predictive factors utilize tumor-based markers, and are not optimal determinants of risk of BCR. Germline-based copy number aberrations (CNAs have not been evaluated as determinants of predisposition to experience BCR. In this study, we accessed germline DNA from 369 female breast cancer subjects who received curative-intent primary treatment following diagnosis. Of these, 155 experienced BCR and 214 did not, after a median duration of follow up after breast cancer diagnosis of 6.35 years (range = 0.60-21.78 and 8.60 years (range = 3.08-13.57, respectively. Whole genome CNA genotyping was performed on the Affymetrix SNP array 6.0 platform. CNAs were identified using the SNP-Fast Adaptive States Segmentation Technique 2 algorithm implemented in Nexus Copy Number 6.0. Six samples were removed due to poor quality scores, leaving 363 samples for further analysis. We identified 18,561 CNAs with ≥1 kb as a predefined cut-off for observed aberrations. Univariate survival analyses (log-rank tests identified seven CNAs (two copy number gains and five copy neutral-loss of heterozygosities, CN-LOHs showing significant differences (P<2.01×10(-5 in recurrence-free survival (RFS probabilities with and without CNAs.We also observed three additional but distinct CN-LOHs showing significant differences in RFS probabilities (P<2.86×10(-5 when analyses were restricted to stratified cases (luminal A, n = 208 only. After adjusting for tumor stage and grade in multivariate analyses (Cox proportional hazards models, all the CNAs remained strongly associated with the phenotype of BCR. Of these, we confirmed three CNAs at 17q11.2, 11q13.1 and 6q24.1 in representative samples using independent genotyping platforms. Our results suggest further investigations on the potential use of germline DNA

  11. Genome engineering through CRISPR/Cas9 technology in the human germline and pluripotent stem cells.

    Science.gov (United States)

    Vassena, R; Heindryckx, B; Peco, R; Pennings, G; Raya, A; Sermon, K; Veiga, A

    2016-06-01

    With the recent development of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing technology, the possibility to genetically manipulate the human germline (gametes and embryos) has become a distinct technical possibility. Although many technical challenges still need to be overcome in order to achieve adequate efficiency and precision of the technology in human embryos, the path leading to genome editing has never been simpler, more affordable, and widespread. In this narrative review we seek to understand the possible impact of CRISR/Cas9 technology on human reproduction from the technical and ethical point of view, and suggest a course of action for the scientific community. This non-systematic review was carried out using Medline articles in English, as well as technical documents from the Human Fertilisation and Embryology Authority and reports in the media. The technical possibilities of the CRISPR/Cas9 technology with regard to human reproduction are analysed based on results obtained in model systems such as large animals and laboratory rodents. Further, the possibility of CRISPR/Cas9 use in the context of human reproduction, to modify embryos, germline cells, and pluripotent stem cells is reviewed based on the authors' expert opinion. Finally, the possible uses and consequences of CRISPR/cas9 gene editing in reproduction are analysed from the ethical point of view. We identify critical technical and ethical issues that should deter from employing CRISPR/Cas9 based technologies in human reproduction until they are clarified. Overcoming the numerous technical limitations currently associated with CRISPR/Cas9 mediated editing of the human germline will depend on intensive research that needs to be transparent and widely disseminated. Rather than a call to a generalized moratorium, or banning, of this type of research, efforts should be placed on establishing an open, international, collaborative and regulated research

  12. PAB-1, a Caenorhabditis elegans poly(A-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Directory of Open Access Journals (Sweden)

    Sunhee Ko

    Full Text Available Poly(A-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  13. PAB-1, a Caenorhabditis elegans poly(A)-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Science.gov (United States)

    Ko, Sunhee; Kawasaki, Ichiro; Shim, Yhong-Hee

    2013-01-01

    Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  14. Lack of GNAQ and GNA11 germ-line mutations in familial melanoma pedigrees with uveal melanoma or blue nevi

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    Jason Ezra Hawkes

    2013-06-01

    Full Text Available Approximately 10% of melanoma cases are familial, but only 25-40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A - the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q subunit alpha (GNAQ, Guanine nucleotide-binding protein G(q subunit alpha-11 (GNA11 and BRCA1 associated protein-1 (BAP1. Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.

  15. A Patient with an Extra-adrenal Pheochromocytoma and Germ-line SDHB Mutation Accompanied by an Atypical Meningioma.

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    Shiwa, Tsuguka; Oki, Kenji; Yoneda, Masayasu; Arihiro, Koji; Ohno, Haruya; Kishimoto, Rui; Kohno, Nobuoki

    2015-01-01

    The gene succinate dehydrogenase subunit B (SDHB) encodes a protein comprising part of the mitochondrial complex II, which links the Krebs cycle and the electron-transport chain. Heterozygous germ-line SDHB mutations causes familial pheochromocytoma-paraganglioma syndrome and has also been linked to gastrointestinal stromal tumors, as well as renal cell carcinomas. We herein report a patient with a germ-line SDHB mutation who presented with an atypical meningioma that was identified as originating from a somatic SDHB mutation. The 41-year-old man, who had a surgical history of extra-adrenal pheochromocytoma at 23 years of age, recently developed gait disorder and hypertension. At the radiological examination, a tumor was detected in the cervical spinal cord at the C6-7 intervertebral level. The pathological findings of the isolated tumor were atypical meningioma assessed as grade II according to the World Health Organization criteria. Inherited neoplasia syndrome was suspected because of the patient's history of early-onset extra-adrenal pheochromocytoma and the development of meningioma. We therefore performed molecular genetic analyses. A direct sequence analysis revealed a heterozygous germ-line frameshift mutation in SDHB, specifically an 11-nucleotide deletion, c.305-315delCAATGAACATC, in exon 4, resulting in a frameshift p.A102EfsX12. Additionally, the sequence analysis of the tumor DNA revealed only a mutated allele with a frameshift mutation in the germ-line SDHB. Our findings suggest that SDHB plays an important role in the pathogenesis of meningiomas as well as pheochromocytomas. Therefore, a differential diagnosis for metastatic pheochromocytoma and other new onset tumors, including meningioma, particularly in patients with germ-line SDHB mutations and a previous history of pheochromocytoma should be carefully made.

  16. Sex Chromosome-wide Transcriptional Suppression and Compensatory Cis-Regulatory Evolution Mediate Gene Expression in the Drosophila Male Germline.

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    Emily L Landeen

    2016-07-01

    Full Text Available The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

  17. Sex Chromosome-wide Transcriptional Suppression and Compensatory Cis-Regulatory Evolution Mediate Gene Expression in the Drosophila Male Germline.

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    Landeen, Emily L; Muirhead, Christina A; Wright, Lori; Meiklejohn, Colin D; Presgraves, Daven C

    2016-07-01

    The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

  18. Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

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    Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

    2015-12-04

    In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

  19. Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

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    Man Cheng

    Full Text Available Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3. The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx. Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N protein of SARS-associated coronavirus (SCoV were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.

  20. C. elegans DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation.

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    Wenjing Qi

    2017-05-01

    Full Text Available Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS is considered to be beneficial in C. elegans due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß/bone morphogenic protein (BMP signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/hpo-11 gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.