Conophylline, is a bis (indole) alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce β-cell differentiation in rat pancreatic acina...Conophylline, is a bis (indole) alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce β-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured rat pancreatic tissue. However, the precise role of conophylline in the growth and survival of immortalized pancreatic mesenchymal stem cells (iPMSCs) derived from fetal porcine pancreas were not understood at present. To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effects of conophylline on iPMSCs. We found that conophylline can robustly stimulate iPMSCs proliferation, even promote their potential differentiation into islet-like clusters analyzed by cell counting, morphology, RT-PCR and real-time PCR, Western blotting, glucose-stimulated insulin release and insulin content analysis. The effects of conophylline were inhibited by LY294002, which is the inhibitor of the PI3K pathway. These results suggest that conophylline plays a key role in the regulation of cell mass proliferation, maintenance of the undifferentiated state of iPMSCs and also promotes iPMSCs differentiated into insulin-producing cells.展开更多
Mesenchymal stem cells (MSCs) derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of ...Mesenchymal stem cells (MSCs) derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs (gMSCs) and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these ceils were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid (RA), as analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.展开更多
Male germline stem cells (mGSCs) are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mG...Male germline stem cells (mGSCs) are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 (SV40) large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell (SSC)-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.展开更多
Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are differ...Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL-1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 μmol L-1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.展开更多
Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic...Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional (3D) agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum (dpp) mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.展开更多
A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition(EMT) via the inhibition of Smad2 phosphorylation.Previous studies have showed ...A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition(EMT) via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells(mGSCs),as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement(KSR) medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies(EBs),which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.展开更多
基金supported by the grants from the National Natural Science Foundation of China(31272518, 31101775, 30972097)the Doctoral Fund of Ministry of Education of China (20100204120020)+4 种基金the Program for New Century Excellent Talents of State Ministry of Education (NCET-09-0654)the Scientific Research Program of Shaanxi Province (2011K02-06, 2008K02-05)the Scientific Research Program of Shaanxi Province, China(2011K02-06)the Fundamental Research Funds for the Central Universities, China (QN2011012)the Graduate Education Innovation Projects of Henan University of Technology, China (11YJCX45)
文摘Conophylline, is a bis (indole) alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce β-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured rat pancreatic tissue. However, the precise role of conophylline in the growth and survival of immortalized pancreatic mesenchymal stem cells (iPMSCs) derived from fetal porcine pancreas were not understood at present. To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effects of conophylline on iPMSCs. We found that conophylline can robustly stimulate iPMSCs proliferation, even promote their potential differentiation into islet-like clusters analyzed by cell counting, morphology, RT-PCR and real-time PCR, Western blotting, glucose-stimulated insulin release and insulin content analysis. The effects of conophylline were inhibited by LY294002, which is the inhibitor of the PI3K pathway. These results suggest that conophylline plays a key role in the regulation of cell mass proliferation, maintenance of the undifferentiated state of iPMSCs and also promotes iPMSCs differentiated into insulin-producing cells.
基金supported by the grants from the National Natural Science Foundation of China(30972097)the Key Program of Ministry of Education of China (109148)+2 种基金the Proram for New Century Excellent Talents in University, China (NCET-09-0654)the Scientific Research Program of Shaanxi Province, China (2011K02-06)the China Postdoctoral Science Foundation(20080431253)
文摘Mesenchymal stem cells (MSCs) derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs (gMSCs) and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these ceils were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid (RA), as analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.
基金supported by the National Major Project for Production of Transgenic Breeding of China(2014ZX08007002)the National Basic Research Program of China(2016YFA0100203)the Program of National Natural Science Foundation of China(31572399,31272518)
文摘Male germline stem cells (mGSCs) are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 (SV40) large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell (SSC)-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.
基金supported by the National Natural Science Foundation of China(30972097,31272518)the Program for the New Century Excellent Talents of State Ministry of Education of P.R.China(NCET-09-0654)+2 种基金the Doctoral Fund of Ministry of Education of P.R.China(RFDP,20120204110030)the Scientific Research Program of Shaanxi Province,China(2011K02-06)the Fundamental Research Funds for the Central Universities,China(QN2011012)
文摘Studies have demonstrated that regulation of GDNF on male germline stem cells (mGSCs) mainly through Ras/Erk1/2, Src family kinase and PI3K/Akt signaling pathways, but the signaling pathways GDNF-mediated are different when the species and cell lines varied. Whether GDNF regulates self-renewal of mGSCs isolated from livestock has not been reported. Here, we purified mGSCs from dairy goat testis using mixed enzymes and fibronectin. Immunofluoresce staining revealed the cultured dairy mGSCs expressed Vasa, Nanos2, Ngn3, Tert, Dazl, Lin28, Oct4, CD49f, Stra8 and GFRa1, reflecting that these cells were mGSCs phenotype. Then we cultured these dairy goat mGSCs in different concentrations of GDNF (0, 5, 10, or 20 ng mL-1) to optimize the best concentration of GDNF to sustain the dairy goat mGSCs self-renewal, after that the inhibitor of PI3K (LY294002, 10 μmol L-1) was added to the medium which contains the optimal concentration of GDNF we obtained by experiments. The mGSCs cultured in different media were compared through the population doubling time (PDT), capacity of cell proliferation evaluated by PCNA and BrdU immunofluorescence staining, RT-PCR, QRT-PCR, Western blotting and flow cytometry. Results showed that 10 ng mL-1 was the optimal concentration of GDNF to maintain goat mGSCs self-renewal and GDNF up-regulates c-Myc transcription via the PI3K/Akt pathway to promote goat mGSCs proliferation. This study provides us an efficient model to study the mechanism in mGSCs proliferation and differentiation in goat, and has important implications in unveiling signaling pathways in livestock GSCs.
基金supported by the National Natural Science Foundation of China(31272518)the program for the New Century Excellent Talents of Ministry of Education of China(NCET-09-0654)+1 种基金the Doctoral Fund of Ministry of Education of P.R.China(RFDP,20120204110030)the Fundamental Research Funds for the Central Universities,China(QN2011012)
文摘Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional (3D) agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum (dpp) mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.
基金supported by grants from the National Natural Science Foundation of China, China (30972097, 31272518)the Program for New Century Excellent Talents in University, China (NCET-09-0654)the Fundamental Research Funds for the Central Universities, China (QN2011012)
文摘A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition(EMT) via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells(mGSCs),as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement(KSR) medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies(EBs),which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.