人诱导多能干细胞体外多向分化能力的方法验证
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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.
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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[5]Engle S J,Puppala D.Integrating human pluripotent stem cells into drug development[J].Cell Stem Cell,2013,12(6):669-677.
[6]Sampaziotis F,De Brito M C,Geti I,et al.Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells[J].Nature Protocols,2017,12(4):814-827.
[7]Burridge P W.Li Y F,Matsa E,et al.Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicininducedcardiotoxicity[J].Nat Med,2016,22(5):547-556.
[8]Takasato M,Er P X,Chiu H S.et al.Kidney organoids from human i PS cells contain multiplelineages and model human nephrogenesis[J].Nature,2015,536(7615):564-568.
[9]McCracken K W,CatáE M,Crawford C M,et al.Modelling human development and disease in pluripotent stem-cell-derived gastric organoids[J].Nature,2014,516(7531):400-404.
[10]Watson C L,Mahe M M,Múnera J,et al.An in vivo model of human small intestine using pluripotent stem cells[J].Nat Med,2014,20(11):1310-1314.
[11]Dye B R,Hill D R,Ferguson M A H,et al.In vitro generation of human pluripotent stem cell derived lung organoids[J].e Life,2015,4:e05098.
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[13]Hariharan K,Kurtz A,Schmidtott K M.Assembling kidney tissues from cells:the long road from organoids to organs[J].Front in Cell Dev Biol,2015,3(3 Pt2):1-9.
[14]Mungenast A E,Siegert S,Tsai L H.Modeling Alzheimer's disease with human induced pluripotent stem(i PS)cells[J].Mol Cell Neurosci,2016,73:13-31.
[15]Lee C T,Bendriem R M,Wu W W,et al.3D brain organoids derived from pluripotent stem cells:promising experimental models for brain development and neurodegenerative disorders[J].J Biomed Sci,2017,24(1):59.
[16]Nishimura T,Hatoya S,Kanegi R,et al.Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions[J].Mol Reprod Dev,2017,84(4):329.
[17]Ikeda M,Imaizumi M,Yoshie S,et al.Implantation of induced pluripotent stem cell-derived tracheal epithelial Cells[J].Ann Otol Rhinol Laryngol,2017,126(7):517-524.
[18]Dirja B T,Yoshie S,Ikeda M,et al.Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells[J].Acta Oto-Laryngologica,2016,136(4):6.
[19]Zhu H,Kimura T,Swami S,et al.Pluripotent stem cells as a source of osteoblasts for bone tissue regeneration[J].Biomaterials,2019,196:31-45.
[20]Hashimoto A,Naito A T,Lee J K,et al.Generation of induced pluripotent stem cells from patients with duchenne muscular dystrophy and their induction to cardiomyocytes[J].Int Heart J,2016,57(1):112-117.
[21]Nam Y,Rim Y A,Ju J H.Chondrogenic pellet formation from cord blood-derived induced pluripotent stem cells[J].J Visual Exp Jove,2017,2017(124):58-63.
[22]Chen Y S,Pelekanos R A,Ellis R L,et al.Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells[J].Stem Cells Transl Med,2012,1(2):83-95.
[23]Zheng Y L,Sun Y P,Zhang H,et al.Mesenchymal stem cells obtained from synovial fluid mesenchymal stem cell-derived induced pluripotent stem cells on a matrigel coating exhibited enhanced proliferation and differentiation potential[J].PLo S One,2015,10(12):e0144226.