Generating ectopic subcutaneous bone marrow by reconstitution of endochondral ossification

Abstract 【Introduction】 Bone and marrow formation occur concurrently during endochondral ossification in most bones, except for flat bones. In the process of endochondral ossification, immature chondrocytes residing at the center of primitive avascular cartilage template become hypertrophic and secrete angiogenic factors, which trigger vascular invasion into the cartilage. Our goal is to establish an in vivo model of “neo-bone marrow” (neo-BM). To this end, we developed an improved cartilage organoid (Cart-Org) system that sought to recapitulate endochondral ossification and support marrow formation. In this study, we evaluated the bone marrow-forming potential of the Cart-Orgs generated by our method following subcutaneous transplantation into immunodeficient mice. 【Methods】Cart-Org generation by a conventional 3D pellet Culture method Human BM MSCs (5.0 × 105 cells) were centrifuged at 300 × g for 5 min. After aspiration of the supernatant, cells were resuspended in 0.5 mL of Complete MesenCultTM-ACF Chondrogenic Differentiation Medium (Stem Cell Technology) with antibiotics, and centrifuged again to form a cell pellet. Tubes were incubated vertically at 37℃ and 21% O2 for 21 days, with medium changes every 3days. Cart-Org generation by a improved 3D pellet Culture method (BME-mixed method) To generate bone marrow directional Cart-Orgs, we modified the conventional method by mixing basement membrane extract (BME). After initial centrifugation and supernatant removal, the cell pellet was loosened and mixed with 10 μL of BME. The mixture was incubated to allow gelation at 37 ºC for 15 minutes, after which 0.5 mL of Chondrogenic Differentiation Medium was added. Differentiation proceeded for 21 days with medium exchanges every 3 days. (Notoh H et al. BBRC. 2024)Subcutaneous Transplantation into Immunodeficient Mice Following differentiation, Cart-Orgs were cultured in EGM2 medium (Promocell) for 1 day at 37 ºC, and then transplanted subcutaneously into the dorsal region of immunodeficient mice (C.B-17 SCID; Jaxon Laboratory). Cart-Orgs were harvested after 8 weeks.Histological Analysis Extracted Cart-Orgs were fixed with 4% paraformaldehyde (Wako) at 4 ºC for 2 days, followed by cryoprotection in PBS containing 30% sucrose (Wako). Samples were embedded in OCT (Tissue-Tek), frozen at -80 ºC, and sliced at 10 mm thin-section using a cryostat (Leica). Sections were stained with hematoxylin-eosin, Alcian blue, von Kossa, or subjected to immunohistochemistry (IHC). For immunostaining, blocking procedure was performed with Blocking One Histo (Nakarai Tesque), followed by incubation with primary antibodies: anti-mouse CD31 pAb (AF3628, R&D), anti-mouse PDGFRβ mAb (clone: Y92, abcam), anti-mouse EMCN mAb (clone: V.7C7, Santa Cruz Biotechnology), anti-mouse SLAM/CD150 mAb (clone: 459911, R&D), with appropriate secondary antibodies. 【Results】 Cart-Orgs generated by BME-mixed method exhibited a higher efficiency of bone marrow formation compared to the conventional method (the BME-mixed method: 66.6 %, the conventional method: 33.3 %, n = 6 per group). To characterize the subcutaneously formed neo-BMs, we investigated their tissue structure, vascular composition, and hematopoietic stem cell (HSCs) engraftment. The subcutaneous neo-BM showed von Kossa stain-positive trabecular bones and a vascular network within them. In native developing bone marrow, CD31-high/EMCN-high (Type H/E) vessels expand to form the vascular network. In the subcutaneous neo-BM, the vascular network was predominantly composed of CD31-high/EMCN-high vessels. In addition, CD150-positive HSCs and megakaryocytes were observed within the extravascular spaces of neo-BM, suggesting successful engraftment and hematopoietic support. 【Conclusion】 We generated subcutaneous neo-BM by transplantating Cart-Org into immunodeficient mice. The Cart-Orgs with BME-mixed method showed their enhanced directivity for bone marrow formation in vivo. The subcutaneous neo-BM showed key features of native bone marrow, such as trabecular bone and a Type H/E-like vascular network. Moreover, recipient-derived hematopoietic cells, e.g., HSCs and megakaryocytes, were engrafted within the neo-BM, indicating a functional hematopoietic capacity. This model has a potential to serve a novel platform for studying bone marrow development and regeneration.