Changes of insulin and nestin expression during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Domestic and international studies have confirmed that human umbilical cord mesenchymal stem cel s could be induced to differentiate into islet-like cel s, but little is reported about the changes of insulin and nestin expressions during the differentiation phase. OBJECTIVE:To observe the changes of insulin and nestin expressions during the differentiation of human umbilical cord mesenchymal stem cel s into islet-like cel s. METHODS:Human umbilical cord mesenchymal stem cel s were cultured using UltraCULTURE medium in vitro. Stem cel s were cultured for three generations to observe cel morphological changes under an inverted microscope, to test immunophenotype by flow cytometry, and to identify the capacity of osteogenesis and adipogenic differentiation. Induction protocol was divided into two stages. In stage 1, stem cel s were induced for 14 days in the UltraCULTURE medium with 4 nmol/L activin A, 25μg/L epidermal growth factor, 100μg/Lβ-nerve growth factor, 10 mmol/L nicotinamide. In stage 2, the cel s were cultured in the UltraCULTURE medium with 1%insulin-transferin-selenium, 10 mmol/L nicotinamide, 10μg/L basic fibroblast growth factor for an additional 14 days. The expressions of nestin and insulin in those differentiated cel s were tested by flow cytometry, and zinc ion expression in the islet-like cel clusters was identified by dithizone staining. RESULTS AND CONCLUSION:During the differentiation process, the insulin level was increased gradual y in the induction group and reached a higher level on day 28, but the insulin expression showed negative in the control group. In addition, on day 14 of induced differentiation, the nestin expression reached the peak and then gradual y reduced along with the prolonged inductive time. On day 28 of induction, islet-like cel clusters formed and were positive for dithizone staining. In this experiment, the umbilical cord mesenchymal stem cel s were successful y induced and differentiated into islet-like cel s, accompanied with the variation of insulin and nestin expression.