A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.
A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.
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DOI：
10.1371/journal.pone.0099534
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Azuma T
中科院分区：
文献类型：
--
作者：
Ochiai-Shino H;Kato H;Sawada T;Onodera S;Saito A;Takato T;Shibahara T;Muramatsu T;Azuma T
在这项研究中，我们开发了一种新的方法来刺激组织非特异性碱性磷酸酶(TnAP)阳性细胞的成骨分化，该细胞是从人诱导的多能干细胞来源的类胚体中释放出来的，经过14d的转化生长因子-β/胰岛素样生长因子-1/成纤维细胞生长因子-2处理。TnAP是骨线样细胞的标志性蛋白。我们用荧光激活细胞分选法分析和分离了TNAP阳性和E-钙粘附素阴性的非上皮细胞。转化生长因子-β、胰岛素样生长因子-1和成纤维细胞生长因子-2联合作用14d后，TnAP的表达显著增强，表达频率最高可达77.3%。分离的细胞表达高水平的Osterix，这是一种唯一的成骨标记。在成骨细胞分化培养液(OBM)中培养这些TNAP阳性细胞后，表达了矮小相关转录因子2、I型胶原、骨涎蛋白和骨钙素(OCN)。这些细胞对活化的维生素D3的处理通过上调OCN做出反应。此外，在OBM中，它们能够产生许多矿化结节，并强表达核因子-kappaB受体激活剂配体和硬化素(Sost)。实时定量RT-PCR显示骨细胞标志物基因SOST、神经肽Y和Reelin的表达显著增加。扫描电子显微镜显示出树枝状形态。甲苯胺蓝染色的半薄切片检查显示许多相互连接的树突。因此，在OBM中培养的TNAP阳性细胞最终可能成为终末分化的骨细胞样细胞。综上所述，联合应用转化生长因子-β、胰岛素样生长因子-1和成纤维细胞生长因子-2处理HiPSCs来源的细胞可产生高频率的TnAP阳性细胞。这些TNAP阳性细胞具有较高的成骨潜能，可向成骨细胞样细胞终末分化。这种方法可能揭示新的成骨途径，并为再生医学和药物开发提供新的工具。
In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.
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影响因子：
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作者：
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影响因子：
3.5
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作者：
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