Oral Virtual Presentation (Virtual only) ESA-SRB-ANZBMS 2021

Elucidating human skeletal development using single-cell analyses with a model of endochondral bone formation using human pluripotent stem cells     (#13)

Shoichiro Tani 1 2 , Hiroyuki Okada 1 2 , Masahide Seki 3 , Yutaka Suzuki 3 , Taku Saito 2 , Sakae Tanaka 2 , Ung-il Chung 1 4 , Hironori Hojo 1 4 , Shinsuke Ohba 5
  1. Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo, Japan
  2. Sensory & Motor System Medicine, The University of Tokyo, Tokyo, Japan
  3. Department of Computational Biology and Medical Sciences, The University of Tokyo, Chiba, Japan
  4. Department of Bioengineering, The University of Tokyo, Tokyo, Japan
  5. Department of Cell Biology, Nagasaki University, Nagasaki, Japan

Modeling human skeletal development is an essential step in elucidating detailed mechanisms underlying this process. Although human pluripotent stem cells (hPSCs) can differentiate into any embryonic cell type, it is challenging to recapitulate 3D bone tissues composed of multiple cell types. To generate human bone tissues, we induced the in vitro differentiation of hPSCs into sclerotome and implanted them beneath the renal capsules of immunodeficient mice. RNA-seq analysis demonstrated cell type-specific gene expression, indicating a stepwise differentiation of hPSCs into the sclerotome. In vivo micro-CT images obtained after implantation showed the growth of mineralized tissues over time. Histological analyses revealed endochondral bone-like structures with specific marker expression patterns: columnar structure of chondrocytes, bone collar, and bone marrow. The induced bone tissues were then analyzed by single-cell RNA-seq, and the obtained data were integrated with the publicly available gene expression profiles of human embryonic long bones at 8 weeks post conception (He J. et al., Cell Res. 2021). Clustering analysis identified multiple skeletal cell types with distinct gene expression signatures; the gene expression profiles of the hPSC-derived bone tissues overlapped with those of the embryonic long bones. Pseudotime analysis predicted a bifurcating trajectory from skeletal progenitors to osteoblasts or chondrocytes. By integrating differential gene expression analysis, gene regulatory network analysis, and ligand-receptor analysis, we extracted novel transcriptional regulators that may play important roles in human osteogenesis. In situ hybridization of the hPSC-derived bone tissues showed a partial co-expression of the identified regulators with RUNX2 and SP7. The knockdown of these regulators downregulated osteoblast marker genes in a human osteosarcoma cell line (Saos2), indicating the involvement of these regulators in human osteogenesis. Collectively, our bone induction method may provide a valuable model of human endochondral bone formation, enabling us to investigate hard-to-access human skeletal development.

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