Inducing differentiation of human amnion-derived mesenchymal stem cells into insulin-secreting cells in vitro

Abstract

Objective To investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.Methods The hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results ( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).Conclusion The hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes. Key words: Human amnion-derived mesenchymal stem cells; Insulin secreting cells ; Differentiation