In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells.
OBJECTIVE
To isolate, culture and identify the human fetal pancreatic ductal stem cells in vitro, and to observe the potency of these multipotential cells differentiation into insulin-producing cells.


METHODS
The human fetal pancreas was digested by 1 g/L collagease type IV and then 2.5 g/L trypsin was used to isolate the pancreatic ductal stem cells, followed by culture in serum-free, glucose-free DMEM media with some additional chemical substrates in vitro (according to the different stage). The cells were induced by glucose-free (control), 5 mmol/L, 17.8 mmol/L and 25 mmol/L glucose, respectively. The cell types of differentiated cells were identified using immunocytochemical staining.


RESULTS
The shape of human fetal pancreatic ductal stem cells cultured in vitro was firstly fusiform in the first 2 wk, and became monolayer and cobblestone pattern after another 3 to 4 wk. After induced and differentiated by the glucose of different concentrations for another 1 to 2 wk, the cells formed the pancreatic islet-like structures. The identification and potency of these cells were then identified by using the pancreatic ductal stem cell marker, cytokeratin-19 (CK-19), pancreatic beta cell marker, insulin and pancreatic alpha cell marker, glucagons with immunocytochemical staining. At the end of the second week, 95.2% of the cells were positive for CK-19 immunoreactivity. Up to 22.7% of the cells induced by glucose were positive for insulin immunoreactivity, and less than 3.8% of the cells were positive for glucagon immunoreactivity in pancreatic islet-like structures. The positive ratio of immunoreactive staining was dependent on the concentration of glucose, and it was observed that the 17.8 mmol/L glucose stimulated effectively to produce insulin- and glucagons-producing cells.


CONCLUSIONS
The human fetal pancreatic ductal stem cells are capable of proliferation in vitro. These cells have multidifferentiation potential and can be induced by glucose and differentiated into insulin-producing cells in vitro.
Yao
Zhong-Xiang
ZX
Department of Histology and Embryology, The Third Military Medical University, Chongqing 400038, China. yaozhx@mail.tmmu.com.cn
Qin
Mao-Lin
ML
Liu
Jian-Jun
JJ
Chen
Xing-Shu
XS
Zhou
De-Shan
DS
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D005260:Female; D005333:Fetus / Q000166:cytology; D005865:Gestational Age; D005934:Glucagon / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D010183:Pancreatic Ducts / Q000166:cytology* / Q000187:drug effects; D011247:Pregnancy; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D007328:Insulin; D005934:Glucagon; D005947:Glucose