IN VITRO TRANSDIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS INTO INSULIN-SECRETING CELLS / 解剖学报

ABSTRACT

Objective To investigate the possibility of bone marrow mesenchymal stem cells to transdifferentiate into insulinf-secreting cells or islet progenitors,and to demonstrate whether these islet-like cells can generate insulin and glucagon. Methods BMSCs were isolated from tibia or thighbone of 6-8 weeks normal SD rat,purified on the basis of their ability to adhere to matrix,and expanded through their self-renewal.Two-step strategy was adopted,BMSCs were induced to generate nestin-positive cells under diffferentiation culture medium 1(added 20??g/L bFGF,10??g/L EGF and 2% B-(27) in DMDM/F-(12) medium) in step one;Nestin-positive cells were differentiated and developed into islet-like cells or islet progenitors under complexed culture medium 2(added 10??g/L Betacellulin,10??g/L HCF,10??g/L Activin A,10?mmol/L nicotide and 2% B27 serum-free in HG-DMEM medium) in step two.The differentiated cells were tested by Dithiazone staining;Immunocytochemistry was carried out using standard protocols in which the cells were incubated with Rb-Rat insulin monoclone antibody,mouse-human glucogan polyclone antibody and Rb-Rat nestin monoclone antibody and assessed by examining the cells under a Zeiss microscope.Insulin secretion in response to insulin secretagogues(glucose) was assayed by radioimmunoassay(RIA). Results After 5 days,BMSCs were expressing nestin,a marker shared with neural stem cells and pancreatic stem cells.Dithiazone staining was positive(bright orange).The differentiated cell masses were containing insulin and glycagon as indicated by immunocytochemistry.RIA demonstrated that these cell mass could generate insulin,the responsibility to glucose was weak.Conclusion BMSCs can be induced to differentiate into insulin-secreting cells or islet progenitors by adding various different cell growth factors and the strategy is repeatable.This study may present a potential approach for the treatment of insulin-depended diabetes by interventional therapy.