摘要
AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro.METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescenceactivated cell sorting (FACS) and radioimmunoassay (RIA)were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 timesin vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cellmarkers; (3) FACS analysis revealed that CD44, CD90and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreaticduodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intracellular insulin content was detected by RIA after the induction culture.CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulinproducing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.
AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro. METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescence-activated cell sorting (FACS) and radioimmunoassay (RIA) were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 times in vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cell markers; (3) FACS analysis revealed that CD44, CD90 and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreatic-duodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intra-cellular insulin content was detected by RIA after the induction culture. CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulin-producing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.
基金
Supported by the National Natural Science Foundation of China,No. 30170443 Major State Basic Research Development Program of China, No. 2001CB510105 and "211" Project Foundation of Peking University
