[Cultivation and identification of pancreatic endocrine progenitor cells].
OBJECTIVE
To develop an approach to cultivate pancreatic endocrine progenitor cells and evaluate the effect thereof.


METHODS
The pancreatic tissues of newborn SD rats were digested by collagenase. The pancreatic endocrine progenitor cells thus obtained were cultured in medium with glucose of the concentration of 33.2 mmol/L for 3 - 4 generations and then in medium with glucose of the concentration of 13.2 mmol/L for 7 days. Electron microscopy was used to observe the ultramicroscopic structures. Dithizone staining was used to examine the expression of insulin. Immunohistochemistry was used to examine the expression of pancreatic-duodenal homebox 1 (Pdx1) and neurogenic 3 (Ngn3). Athymic nude mice of the line BALB/C underwent intraperitoneal injection of streptozotocin so as to establish model of diabetes. 1 x 10(5) pancreatic endocrine progenitor cells, cultured in the media with low or high glucose levels, were transplanted into the renal subcapsular space of the mice respectively. Mice without transplantation were used as controls (groups 1 - 3). The surviving status and blood glucose level were observed. Two weeks later the mice were killed and their kidneys were taken out, and underwent microscopy and immunohistochemistry to observe the expression of insulin.


RESULTS
Islet of pancreas-like structures were formed with insulin-positive cells, 16% +/- 5% of Pdx1 positive cells, and 69% +/- 8% of Ngn3 positive cells. The rate of diathiazone positive cells in the low glucose level medium was 32% +/- 6%, significantly higher than that in the high glucose medium (10% +/- 3%). When the cells were cultured in the medium with the glucose level of 20 mmol/L, the insulin level in the culture fluid with low glucose level was 26.0 microU/ml +/- 2.2 microU/ml, significantly higher than that with high glucose level (22.7 microU/ml +/- 3.6 microU/ml, P < 0.05). Insulin-positive cells could be found among the transplanted pancreatic cells. The surviving times of the mice transplanted with the pancreatic endocrine progenitor cells, cultured in the media with low or high glucose levels, and the controls, were 10.0 days, 8.4 days, and 5.6 days respectively with a significant difference between the groups 1 and 3 (P < 0.05).


CONCLUSIONS
Culture medium with a certain glucose level, is beneficial for the cultivation of pancreatic endocrine progenitor cells, especially the medium with a low level of glucose that promotes the maturation and differentiation of the pancreatic endocrine progenitor cells.
Wang
Lei
L
Department of General Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China.
Yuan
Chun-hui
CH
Zhao
Yu-pei
YP
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D003921:Diabetes Mellitus, Experimental / Q000473:pathology; D005947:Glucose / Q000008:administration & dosage; D007328:Insulin / Q000032:analysis; D007515:Islets of Langerhans / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells
D017077:Culture Media, Conditioned; D007328:Insulin; D005947:Glucose