Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase dige...Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.展开更多
AIM： To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS： Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started prolifera...AIM： To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS： Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and sub- cultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CDl17. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature （α-fetoprotein） but also differentiated hepatocyte （albumin and CK-18） and biliary markers （CK-7 and 19）, whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma. CONCLUSION： According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.展开更多
Objective To study the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods A culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells sep...Objective To study the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods A culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by Percoll Centrifugation and maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% selected fetal calf serum. Cell growth pattern and its responses to cytokines were evaluated by trypan blue exclusion and MTT test, respectively. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Cytochemistry characteristics of MSCs were determined. Results Easy-handling methods to isolate and culture expand MSCs were developed in this study. MSCs were unique in their phenotypes. They were positive for CD29, CD44, CD166, and negative for CD34, CD45, HLA-DR and Ulex europaeus. Cytochemistry evaluation showed that MSCs were homogeneously positive for acid α-naphthl acetate esterase (ANAE), glycogen (periodic acid Schiff reaction, PAS), and negative for acid phosphatase (ACP) and the Sudan black reaction (SB). Around 5% of them were positive for alkaline phosphatase (ALP). The cells had a population doubling time of 30 hours and cell cycle analysis showed that approximately 10% of them were in S phase. MSCs grew at significantly different rates when incubated in the presence of various recombinant human cytokines, of which interferon γ, tumor necrosis factor α, stem cell factor and insulin-like growth factor promoted the proliferation of MSCs dramatically, while others tested had no effects on cell growth. Conclusions MSCs are a homogenous population of cells that have unique growth, phenotypical and cytochemical characteristics. Furthermore, the diverse responses of MSCs to different cytokines provide a clue for the selection of optimal expansion and maintenance of MSCs.展开更多
Pluripotent or multipotent stem cells are involved in development and tissue homeostasis;they have the ability to self-renew and differentiate into various types of functional cells.To maintain these properties,stem c...Pluripotent or multipotent stem cells are involved in development and tissue homeostasis;they have the ability to self-renew and differentiate into various types of functional cells.To maintain these properties,stem cells must undergo sustained or unlimited proliferation that requires the stabilization of telomeres,which are essential for chromosome end protection.Telomerase,an RNA-dependent DNA polymerase,synthesizes telomeric DNA.Through the lengthening of telomeres the lifespans of cells are extended,or indefinite proliferation is conferred;this is intimately associated with stem cell phenotype.This review highlights our current understanding of telomerase as a"stemness"enzyme and discusses the underlying implications.展开更多
基金Supported by Science and Technology Program of Shenyang (2009-090063, 2011-F10-222-4-00)
文摘Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.
基金Supported by DGTRE,Région Wallonne (Grant Waleo HEPATERA) and by the Fondation saint-Luc. The first author is a recipient of fellowship from FNRS and the Université Catholique de Louvain
文摘AIM： To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS： Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and sub- cultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CDl17. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature （α-fetoprotein） but also differentiated hepatocyte （albumin and CK-18） and biliary markers （CK-7 and 19）, whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma. CONCLUSION： According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.
基金ThisstudywassupportedbytheNational"973"Program China (No G19990 5 430 2 )
文摘Objective To study the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods A culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by Percoll Centrifugation and maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% selected fetal calf serum. Cell growth pattern and its responses to cytokines were evaluated by trypan blue exclusion and MTT test, respectively. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Cytochemistry characteristics of MSCs were determined. Results Easy-handling methods to isolate and culture expand MSCs were developed in this study. MSCs were unique in their phenotypes. They were positive for CD29, CD44, CD166, and negative for CD34, CD45, HLA-DR and Ulex europaeus. Cytochemistry evaluation showed that MSCs were homogeneously positive for acid α-naphthl acetate esterase (ANAE), glycogen (periodic acid Schiff reaction, PAS), and negative for acid phosphatase (ACP) and the Sudan black reaction (SB). Around 5% of them were positive for alkaline phosphatase (ALP). The cells had a population doubling time of 30 hours and cell cycle analysis showed that approximately 10% of them were in S phase. MSCs grew at significantly different rates when incubated in the presence of various recombinant human cytokines, of which interferon γ, tumor necrosis factor α, stem cell factor and insulin-like growth factor promoted the proliferation of MSCs dramatically, while others tested had no effects on cell growth. Conclusions MSCs are a homogenous population of cells that have unique growth, phenotypical and cytochemical characteristics. Furthermore, the diverse responses of MSCs to different cytokines provide a clue for the selection of optimal expansion and maintenance of MSCs.
基金supported by the National Basic Research Program of China(2012CB911202)National Natural Science Foundation of China(81071721)+4 种基金the Swedish Cancer Societythe Swedish Research Councilthe Cancer Society in Stockholmthe Stockholm County CouncilKarolinska Institutet
文摘Pluripotent or multipotent stem cells are involved in development and tissue homeostasis;they have the ability to self-renew and differentiate into various types of functional cells.To maintain these properties,stem cells must undergo sustained or unlimited proliferation that requires the stabilization of telomeres,which are essential for chromosome end protection.Telomerase,an RNA-dependent DNA polymerase,synthesizes telomeric DNA.Through the lengthening of telomeres the lifespans of cells are extended,or indefinite proliferation is conferred;this is intimately associated with stem cell phenotype.This review highlights our current understanding of telomerase as a"stemness"enzyme and discusses the underlying implications.
