Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells
Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells
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DOI：
10.1073/pnas.1210422109
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发表时间：
2012-11-20
影响因子：
11.1
通讯作者：
Guilak, Farshid
中科院分区：
文献类型：
--
作者：
Diekman, Brian O.;Christoforou, Nicolas;Guilak, Farshid
软骨损伤的再生疗法的发展已大大有助于干细胞生物学的最新进展。诱导多能干细胞（iPSC）有可能为组织工程提供丰富的细胞来源，并产生患者匹配的体外模型，以研究软骨修复和骨关节炎中的遗传和环境因素。然而，细胞治疗和建模方法都需要纯化和均匀分化的细胞群，以可预测地概括软骨的生理特征。在此，通过II型胶原（Col 2）驱动的绿色荧光蛋白（GFP）表达，将源自成年小鼠成纤维细胞的iPSC软骨分化并纯化。与GFP-细胞相比，Col 2和聚集蛋白聚糖基因表达水平在GFP+细胞中显著上调，并且随着单层扩增而降低。体外软骨缺损模型用于证明通过接种在琼脂糖中的GFP+细胞的整合修复，支持其在软骨治疗中的潜在用途。在软骨细胞团培养中，细胞合成软骨特异性基质，如高水平的糖胺聚糖和II型胶原蛋白以及低水平的I型和X型胶原蛋白所示。初始分化后细胞扩增的可行性通过来自两次传代的GFP+细胞的沉淀中的均匀基质沉积来说明。最后，原子力显微镜分析表明，增加的微尺度弹性模量与胶原对齐在周边的小球，模仿原生软骨的带状变化。这项研究证明了iPSCs在软骨缺损修复和创建可与特定遗传背景相匹配的软骨组织模型方面的潜在用途。
The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitromodels to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP-cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.