Collagen X Is Dispensable for Hypertrophic Differentiation and Endochondral Ossification of Human iPSC‐Derived Chondrocytes

• Published in: JBMR plus Vol. 7; no. 5; pp. e10737 - n/a
• Main Authors: Kamakura, Takeshi, Jin, Yonghui, Nishio, Megumi, Nagata, Sanae, Fukuda, Masayuki, Sun, Liping, Kawai, Shunsuke, Toguchida, Junya
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.05.2023; Oxford University Press
• Subjects: Bone growth; BONE MATRIX; Calcification; Cancellous bone; Cartilage; Cell differentiation; Cell lines; CHONDROCYTE AND CARTILAGE BIOLOGY; Chondrocytes; Chondrogenesis; COLLAGEN; Collagen (type X); CRISPR; DISEASES AND DISORDERS OF/RELATED TO BONE; Endochondral bone; GROWTH PLATE; Immunodeficiency; Mutants; Mutation; Ossification; Penicillin; Pluripotency; Transcriptomes; United States > US; Japan; COLLAGEN; DISEASES AND DISORDERS OF/RELATED TO BONE; GROWTH PLATE; BONE MATRIX; CHONDROCYTE AND CARTILAGE BIOLOGY
• ISSN: 2473-4039; 2473-4039
• DOI: 10.1002/jbm4.10737

ABSTRACT 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 Col10a1 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 (COL10A1+/−) or homozygous (COL10A1−/−) deletions of COL10A1 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 COL10A1−/− 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 COL10A1−/− 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, COL10A1−/− iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. Type X collagen (COL X) is a cartilage matrix protein produced by hypertrophic chondrocytes. Human iPS cells with homozygous deletions of COL10A1 gene created by a dual sgRNA CRISPR/Cas9 system differentiated into hypertrophic chondrocytes in vitro and demonstrated the formation of growth plate‐like structure and endochondral ossification in vivo, indicating that COL X is dispensable for these biological processes.