Kamakura Takeshi, Jin Yonghui, Nishio Megumi, Nagata Sanae, Fukuda Masayuki, Sun Liping, Kawai Shunsuke, Toguchida Junya
Department of Regeneration Sciences and Engineering, Institute for Life and Medical Sciences Kyoto University Kyoto Japan.
Department of Fundamental Cell Technology, Center for iPS Cell Research and Application Kyoto University Kyoto Japan.
JBMR Plus. 2023 Mar 29;7(5):e10737. doi: 10.1002/jbm4.10737. eCollection 2023 May.
Collagen X is a non-fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss of gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous ( ) or homozygous ( ) deletions of gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet-derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger in tissues. Prehypertrophic pellet-derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental- and mutant-derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating-phase genes and a higher expression of calcification-phase genes in pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC-derived chondrocytes, though it may facilitate the differentiation process. Thus, iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
Ⅹ型胶原蛋白是一种由肥大软骨细胞产生的非纤维状胶原蛋白，曾被认为与生长板软骨的钙化过程有关。然而，小鼠中该基因的纯合缺失对生长板形成或骨骼发育并无显著影响。为了研究Ⅹ型胶原蛋白在人软骨细胞中的作用，我们使用双sgRNA CRISPR/Cas9系统建立了具有该基因杂合（ ）或纯合（ ）缺失的人诱导多能干细胞（hiPSC）。建立了多个突变克隆，并通过先前报道的三维诱导方法将其分化为肥大软骨细胞。在亲代细胞系和突变细胞系的分化过程中未观察到明显差异，两者均分化为具有肥大软骨细胞特征的细胞，这表明Ⅹ型胶原蛋白对于人软骨细胞的肥大分化在体外并非必需。为了研究Ⅹ型胶原蛋白缺乏在体内的影响，将增殖期或前肥大期的软骨细胞团块移植到免疫缺陷小鼠体内。增殖期团块来源的组织显示软骨细胞呈带状分布，并向模仿生长板的骨组织过渡，且 组织中的骨比例往往更大。前肥大期团块来源的组织产生了具有软骨内骨化特征的小梁骨结构，亲代来源组织和突变来源组织之间没有明显差异。对肥大期软骨细胞团块的转录组分析显示，与亲代细胞团块相比， 团块中增殖期基因的表达较低，钙化期基因的表达较高。这些体外和体内数据表明，Ⅹ型胶原蛋白对于人iPSC来源软骨细胞的肥大分化和软骨内骨化并非必需，尽管它可能促进分化过程。因此， iPSC系对于研究Ⅹ型胶原蛋白在软骨细胞分化中的生理作用很有用。© 2023作者。由Wiley Periodicals LLC代表美国骨与矿物质研究学会出版。
