[Directed differentiation of Balb/C mouse embryonic stem cells into pancreatic islet-like cell clusters in vitro: observation by atomic force microscope].
OBJECTIVE
To observe the morphological changes of Balb/C mouse embryonic stem cells following directed differentiation into pancreatic islet-like cell clusters (PICC) in vitro using atomic force microscope (AFM).


METHODS
Balb/C mouse embryonic stem cells were first cultured into embryonic bodies (EBs) and allowed to differentiate spontaneously for 4 days. The cells were then transferred to gelatin-coated dishes for the EBs to attach and spread on the tissue culture plates, in the course of which a series of cell growth factors such as basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1) and nicotinamide were added into the culture medium at specific time points to induce directed differentiation of the stem cells into PICC. Immunocytochemistry was employed to detect the cells positive for insulin and glucagon, which were observed with AFM.


RESULTS
The embryonic stem cells developed into cell clusters of different sizes, in which the cells were tightly arranged. Islet B cells were numerous in the center of clusters and darkly stained, but fewer in the peripherals with lighter stains. Islet A cells expressing glucagon were relatively fewer in the cell clusters, found mainly in the peripherals. Scanning of the insulin-positive clusters by AFM revealed large quantity of tissue fibers resembling nerve fibers that formed a reticular structure in disorderly arrangement. Numerous round granules were observed in the cytoplasm of almost identical sizes ranging from 0.5 to 1.0 mum in diameter.


CONCLUSIONS
The cell clusters obtained by directed differentiation are mature in both morphology and function with also well organized structures.
Liu
Mei-Li
ML
Department of Chemistry, College of Life Science and Technology, Ji'nan University, Guangzhou 510632, China.
Xiong
Yi
Y
Cai
Ji-Ye
JY
Pan
Yun-Long
YL
Meng
Fan-Yi
FY
Li
Ling-Song
LS
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force
D003470:Culture Media; D016222:Fibroblast Growth Factor 2; D007334:Insulin-Like Growth Factor I