AIM: To determine the maturity-promoting protocols for directing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)towards islet-like clusters (ILCs) expressing prohormone conver-tase (PC) 1 and PC2 through different combinations of biological products. METHODS: Primary hUC-MSCs were isolated from the whole human umbilical cord by digestion (with collagenase Ⅱ) , filtration and centrifugation, and then were purified by incomplete digestion. The stem cell-specific markers were detected by the methods of flow cytometry, RT-PCR and immunocytofluorescence. The protocols were adopted for inducing the differentiation of hUC-MSCs towards ILCs. Protocol A consisted of IMDM culture medium containing 10 μg/L basic fibroblast growth factor (bFGF) , 10 μg/L epidermal growth factor ( EGF) , 10 mg/L Ginkgo biloba extract ( GBE) and 2% fetal calf serum ( FCS) . Protocol B was based on the protocol A plus 10 μg/L nicotinamide. Protocol C was based on the protocol B plus 10 μg/L hepatocyte growth factor ( HGF). Before and after induction, the morphological changes of hUC-MSCs were observed under inverted microscope. The islet-related mRNA and proteins especially PCI and PC2 were measured by RT-PCR, qPCR and Western blotting. RESULTS: The cell morphology, surface antigen and stem cell-specific markers indicated that hUC-MSCs were successfully isolated and purified from human umbilical cord. hUC-MSCs expressed nestin broadly and Isll to a less degree. ILCs induced by protocol A expressed Glut-2 and MafA mRNA. ILCs induced by protocol B expressed Glut-2, MafA, Nkx6. 1 and PC2 mRNA. ILCs induced by protocol C expressed Glut-2, MafA, Nkx6. 1, PC2, Ngn3, Pdxl, PCI and insulin mRNA. The expression of PCI mRNA was only observed in ILCs induced by protocol C. ILCs induced by protocol B and protocol C both expressed PC2 mRNA and the expression in the cells induced by protocol C was significantly higher than that in the cells induced by protocol B. ILCs induced by protocol C expressed PCI protein. ILCs induced by protocol B and protocol C both expressed ProPC2 protein and PC2 protein, and the former (protocol B) was less than the latter (protocol C) with statistically difference ( P < 0. 01) . CONCLUSION: hUC-MSCs possess the potential to differentiate towards the islet progenitor cells and/or islet cells. Compared to protocol A and B, protocol C offers a maturity-promoting method to induce hUC-MSCs differentiation to ILCs with the expression of PCI and PC2.%目的:通过各种生物化学制剂的不同组合在体外诱导人脐带间充质干细胞(hUC-MSCs)向胰岛样细胞团(ILCs)分化,筛选出能表达激素原转化酶(PC)1和PC2的促成熟方案.方法:用胶原酶Ⅱ消化、过滤离心和不完全消化传代的方法从人的完整脐带中分离和纯化hUC-MSCs,流式细胞术、RT-PCR和免疫细胞荧光技术检测其干细胞标志物.选用A、B、C 3种方案诱导hUC-MSCs向ILCs分化:方案A为含10 μg/L 碱性成纤维细胞生长因子(bFGF)、10 μg/L 表皮生长因子(EGF)、10 mg/L银杏提取液(GBE)及2%胎牛血清(FCS)的高糖培养基IMDM;在方案A基础上加入10 μg/L尼克酰胺为方案B;在方案B基础上加入10 μg/L肝细胞生长因子(HGF)为方案C.在hUC-MSCs诱导前后,通过倒置显微镜观察其形态变化,采用RT-PCR、qPCR和Western blotting检测胰岛相关mRNA和蛋白,特别是PC1和 PC2的表达情况.结果:(1)细胞形态、表面相关抗原、干细胞特异基因的表达等情况表明hUC-MSCs可以从人脐带中被成功地分离和纯化;hUC-MSCs普遍表达nestin,轻微表达Isl1.(2) RT-PCR检测发现方案A诱导的ILCs有Glut-2和MafA mRNA表达,方案B诱导的ILCs有Glut-2、MafA、Nkx6.1和PC2 mRNA表达,方案C诱导的ILCs有Glut-2、MafA、Nkx6.1、PC2、Ngn3、Pdx1、PC1和胰岛素mRNA表达,提示A、B和C 3种方案诱导的细胞有逐渐成熟的趋势.qPCR检测也证实了PC1 mRNA仅在方案C中有表达,PC2 mRNA在方案B和方案C中均有表达,但方案C中PC2 mRNA的表达量显著高于方案B(P<0.01);Western blotting结果也表明PC1蛋白只在方案C中有表达;PC2酶原(ProPC2)与PC2在方案B和方案C中都有表达,且它们在方案C中的表达量均显著高于方案B(P<0.01).结论:(1)hUC-MSCs具有向胰岛祖细胞或胰岛细胞分化的潜能;(2)方案C中尼克酰胺和HGF可促进hUC-MSCs向ILCs分化,使ILCs表达PC1并上调PC2;相对于方案A和B,方案C是一种促成熟方案.
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