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Review
. 2023 Mar 29:14:1143344.
doi: 10.3389/fphys.2023.1143344. eCollection 2023.

Analysis of skeletal stem cells by renal capsule transplantation and ex vivo culture systems

Wei Hsu  1   2   3 , Takamitsu Maruyama  1
Affiliations
Review

Analysis of skeletal stem cells by renal capsule transplantation and ex vivo culture systems

Wei Hsu et al. Front Physiol. .

Abstract

Skeletal stem cells residing in the suture mesenchyme are responsible for proper development, homeostasis, and injury repair of the craniofacial skeleton. These naïve cells are programmed to differentiate into osteoblast cell types and mediate bone formation via an intramembranous ossification mechanism. The simplicity of this system also offers great advantages to studying osteoblastogenesis compared to the appendicular and axial skeletons. Recent studies utilizing genetically based cell tracing have led to the identification of skeletal stem cell populations in craniofacial and body skeletons. Although the genetic analysis indicates these cells behave like stem cells in vivo, not all of them have been thoroughly examined by stem cell isolation and stem cell-mediated tissue generation. As regeneration is an integral part of stem cell characteristics, it is necessary to further analyze their ability to generate tissue at the ectopic site. The establishment of an ex vivo culture system to maintain the stemness properties for extended periods without losing the regenerative ability is also pertinent to advance our knowledge base of skeletal stem cells and their clinical applications in regenerative medicine. The purpose of this review is to discuss our recent advancements in analyses of skeletal stem cells using renal capsule transplantation and sphere culture systems.

Keywords: bone; calvaria; cartilage; cell-based therapy; cranial suture; mesenchymal stem cell; skeletogenic mesenchyme; tissue regeneration.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Post-transplantation analyses of tissue generation. (A) Wholemount analysis of the kidney transplanted (right) and non-transplanted (left) by 104 cells isolated from C57/BL6 mouse sutures in bright field (top) and von Kossa (vK) staining (bottom). Enlargements of the inset indicate bone formation underneath the kidney capsule. (B) Wholemount analysis of the kidney transplanted by suture cells infected by lentivirus expressing the RFP reporter (Lenti-RFP). Analysis of renal sections containing the transplant of 5 × 104 suture cells with or without infection of Lenti-RFP. (C) Diagrams illustrate the use of the confetti mouse allele to examine potential mechanisms for bone formation by the skeletal stem cell (SSC) or progenitor cells upon renal capsule transplantation. The representative images show the bone is formed by single-color cells indicating its generation by a single suture stem cell via the clonal expansion mechanism. (D) Examining lineage specification of skeletal stem cells by transplantation. Analysis of the renal capsule transplanted by 5 × 104 control (A, C) or β-cat-null (B, C) suture cells by immunostaining of Osterix (Osx) and type 2 collagen (Col2). Note skeletal stem cells residing in the suture (SuSCs) are developmentally programmed to become osteoblasts positive for Osx but the loss of β-catenin alters their fate to form Col2 positive chondrocytes Scale bars, 5 mm (A); 200 μm (B); 400 μm (C); 50 μm (D).
FIGURE 2
FIGURE 2
Preserving the stemness of SuSCs by sphere culture system. (A) Schematic representations illustrate the sphere culture of suture cells isolated from the calvarial suture mesenchyme in primary (10), followed by replating and subsequent culture for secondary (20), tertiary (30), and up to 40–50 passages. The representative image of mouse and human spheres in culture. (B) Wholemount imaging of kidney implanted with ex vivo cultured spheres derived from SuSCs positive for Tomato fluorescence 4 weeks after renal capsule transplantation. (C) Representative images of the transplanted kidney evaluated by von Kossa (vK) and hematoxylin and eosin (H and E) staining 6 weeks after the transplantation of freshly isolated suture cells (Suture cells), or cortical cells isolated from the limb (Limb cells), or 8 weeks after the transplantation of spheres formed by the culture of suture cells (Spheres). Note bones generated by spheres show identical features to those generated by freshly isolated cells. Scale bars, 50 μm (A); 2 mm (B); 100 μm (C).
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References

    1. Ambrosi T. H., Sinha R., Steininger H. M., Hoover M. Y., Murphy M. P., Koepke L. S., et al. (2021). Distinct skeletal stem cell types orchestrate long bone skeletogenesis. Elife 10, 66063. 10.7554/eLife.66063 - DOI - PMC - PubMed
    1. Bianco P., Cao X., Frenette P. S., Mao J. J., Robey P. G., Simmons P. J., et al. (2013). The meaning, the sense and the significance: Translating the science of mesenchymal stem cells into medicine. Nat. Med. 19, 35–42. 10.1038/nm.3028 - DOI - PMC - PubMed
    1. Bianco P., Robey P. G., Simmons P. J. (2008). Mesenchymal stem cells: Revisiting history, concepts, and assays. Cell Stem Cell 2, 313–319. 10.1016/j.stem.2008.03.002 - DOI - PMC - PubMed
    1. Biressi S., Miyabara E. H., Gopinath S. D., Carlig P. M., Rando T. A. (2014). A Wnt-TGFβ2 axis induces a fibrogenic program in muscle stem cells from dystrophic mice. Sci. Transl. Med. 6, 267ra176. 10.1126/scitranslmed.3008411 - DOI - PMC - PubMed
    1. Brack A. S., Conboy M. J., Roy S., Lee M., Kuo C. J., Keller C., et al. (2007). Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Sci. (New York, N. Y. 317, 807–810. 10.1126/science.1144090 - DOI - PubMed