Human Induced Pluripotent Stem Cells Differentiated into Chondrogenic Lineage Via Generation of Mesenchymal Progenitor Cells
Human Induced Pluripotent Stem Cells Differentiated into Chondrogenic Lineage Via Generation of Mesenchymal Progenitor Cells
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DOI：
10.1089/scd.2012.0127
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发表时间：
2013-01-01
影响因子：
4
通讯作者：
Nakao, Kazuwa
中科院分区：
文献类型：
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作者：
Koyama, Noriaki;Miura, Masako;Nakao, Kazuwa
人诱导多能干细胞（hiPSC）表现出与人胚胎干细胞（hESC）相似的多能性、增殖能力和基因表达。hESC在体内畸胎瘤中容易形成软骨组织;然而，尽管进行了大量努力，迄今为止还没有建立体外诱导成熟软骨细胞的有效方法。hiPSC也可以通过畸胎瘤形成在体内分化成软骨，但是与hESC一样，尚未报道用于hiPSC的体外软骨形成分化的可靠系统。在这里，我们研究了软骨分化能力的hiPSCs使用多步培养方法，包括胚状体（EB）的形成，从EB的细胞生长，从EB的发芽细胞的单层培养，和三维小球培养。在该培养过程中，单层培养的细胞密度对细胞活力和随后的分化能力至关重要。单层培养的细胞表现出成纤维细胞样形态并表达间充质干细胞标志物。沉淀培养2-3周后，沉淀中的细胞表现出典型的软骨细胞的球形形态，并被含有酸性蛋白聚糖的细胞外基质包围。胶原蛋白和聚集蛋白聚糖在颗粒中的表达逐渐增加。组织学分析显示，超过70%的hiPSC衍生的颗粒成功地经历了软骨形成分化。使用相同的培养方法，hESC显示出相似的组织学变化和基因表达，但比hiPSC分化更快，更有效。我们的研究表明，hiPSCs可以有效地分化成软骨细胞系在体外通过产生间充质祖细胞，使用一个简化的，多步骤的培养方法。
Human induced pluripotent stem cells (hiPSCs) exhibit pluripotency, proliferation capability, and gene expression similar to those of human embryonic stem cells (hESCs). hESCs readily form cartilaginous tissues in teratomas in vivo; despite extensive effort, however, to date no efficient method for inducing mature chondrocytes in vitro has been established. hiPSCs can also differentiate into cartilage in vivo by teratoma formation, but as with hESCs, no reliable system for in vitro chondrogenic differentiation of hiPSCs has yet been reported. Here, we examined the chondrogenic differentiation capability of hiPSCs using a multistep culture method consisting of embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and 3-dimensional pellet culture. In this culture process, the cell density of monolayer culture was critical for cell viability and subsequent differentiation capability. Monolayer-cultured cells exhibited fibroblast-like morphology and expressed markers for mesenchymal stem cells. After 2-3 weeks of pellet culture, cells in pellets exhibited a spherical morphology typical of chondrocytes and were surrounded by extracellular matrix that contained acidic proteoglycans. The expression of type II collagen and aggrecan in pellets progressively increased. Histological analysis revealed that over 70% of hiPSC-derived pellets successfully underwent chondrogenic differentiation. Using the same culture method, hESCs showed similar histological changes and gene expression, but differentiated slightly faster and more efficiently than hiPSCs. Our study demonstrates that hiPSCs can be efficiently differentiated into the chondrogenic lineage in vitro via generation of mesenchymal progenitor cells, using a simplified, multistep culture method.