摘要
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。
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.
引文
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