人诱导多能干细胞体外多向分化能力的方法验证

 目的 探讨人诱导多能干细胞(induced pluripotent stem cells, iPSC)体外多向分化能力的方法。方法 iPSC通过形成拟胚体(embryonic body, EB)的阶段,将其诱导为间充质干细胞(mesenchymal stem cell, MSCs)样细胞,倒置显微镜下观察诱导过程中细胞形态的变化,流式细胞术检测iPSC来源的MSCs(iPS-MSCs)表面标志物的表达,并进一步将iPSC-MSC诱导为成骨、软骨细胞;或直接EB接种,将其诱导成神经细胞。通过上述方法验证iPSC的多向分化能力。结果 诱导后的 iPSC-MSC 逐渐向外生长变为长梭形; CD29、CD105在 iPSC -MSCs 中表达阳性, 而 CD34、CD45则为阴性; 碱性磷酸酶、甲苯氨兰染色结果表明iPSC -MSCs 具有成骨、成软骨的能力;iPSC亦可直接从EB阶段诱导成神经细胞。结论 通过上述方法,可成功诱导iPSC 为成骨、成软骨、成神经细胞,为 iPSC进一步的研究与应用提供了技术基础。

Method validation of multipotential differentiation of human induced pluripotent stem cells in vitro

Objective To study the multi-directional differentiation ability of induced pluripotent stem cells (iPSC). Methods iPSCs were induced into Mesenchymal stem cells (MSCs)-like cells by the formation of Embryonic body EBs. The morphology of cells was observed under inverted microscope. Flow cytometry was performed. Expression of surface markers of iPSC-derived MSCs (iPS-MSCs), and further induction of iPSC-MSCs into osteoblasts, chondrocytes; or direct EB inoculation, which induces them into nerve cells. The multi-directional differentiation ability of iPSC was verified by the above method. Results After induction, iPSC-MSC gradually grew into a long spindle shape; CD29 and CD105 were positive in iPSC-MSCs, while CD34 and CD45 were negative; alkaline phosphatase and toluidine staining showed iPSC- MSCs have the ability to form osteoblasts and cartilage; iPSCs can also be induced into neurons directly from the EB stage. Conclusions Through the above methods, iPSC can be successfully induced into osteoblasts, cartilage and neuroblasts, which provides a technical basis for further research and application of iPSC.