[Research on differentiation features of the monoclonal human pancreatic stem cell].
The in vitro and in vivo differentiation features of the monoclonal human pancreatic stem cell (mhPSC) line derived from the pancreatic tissues of a male abortive fetus at 4 month-old were studied. The mhPSCs were plated in culture dishes that had been coated with 0.1% gelatin in phosphate-buffered saline without calcium and magnesium. After proliferated for 3 days, the mhPSCs were induced in modified high-glucose Dulbecco's Modified Eagles's Medium for 25 days. The changes of the cell morphology were observed by phasecontrast microscope during inducement course. The results of the mhPSCs in vitro induced to differentiate into functional pancreatic islets were identified using dithizone staining, RT-PCR and stimulation-glucose secreting insulin and C-peptide radioimmunoassay. The mhPSCs suspension was separately injected under the groin hypoderm of male nude mice. On the 30th day, the grafts were taken off. The immunochemistry reactions were performed by the SP method. The in vivo differentiation ability of the mhPSCs in nude mice was assessed. In vitro proliferation culture, the mhPSCs adhesively grew and showed polygon epithelioid morphology. After proliferation a layer, the mhPSCs showed the gravelstone-like. During in vitro directional inducement, the mhPSCs gradually turned from polygon to round, suspended to grow and assembled pancreatic islets-like clusters. On the 15th inducement day, only a few cells of pancreatic islet-like clusters were induced into the beta cells that became crimson with dithizone staining. However, till the 25th inducement day, most cells of pancreatic islet-like clusters had differentiated into the beta cells, as identified by dithizone staining, which expressed transcription factor of insulin. Respectively stimulated with different concentration glucose, the induced pancreatic islets not only secreted insulin and C-peptide, but also the secretion volumes of the insulin and C-peptide were markedly increased after the stimulation with higher concentration glucose (0.01<P<0.05). In the in vivo differentiation experiment, all nude mice which were injected by the mhPSCs displayed a teratoma-like with white color and rich blood vessels. Immunohistochemistry showed that the teratoma-like expressed the proteins of the pdx1, insulin, glucagon, CK, MBP and NF. This indicated that the mhPSCs not only could be in vitro induced into functional islet-like clusters, but also could in vivo differentiate into the pancreatic islets, epithelium and neural cells.
Xiao
Mei
M
Department of Animal Medicine, Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong.
An
Li Long
LL
Dou
Zhong Ying
ZY
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D005333:Fetus / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010179:Pancreas / Q000166:cytology*; D011863:Radioimmunoassay; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*