The role of stem cells in skeletal health and disease

National Institute Of Dental & Craniofacial Research

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Abstract

Biological Activity: Virtually all bone marrow-derived stromal cell (BMSC) chondrogenic induction cultures include greater than 2 weeks exposure to transforming growth factor-beta (TGF-beta), but fail to generate cartilage-like tissue suitable for joint repair. We used a micro-pellet model (5x103 BMSCs per pellet) to determine the duration of TGF-beta exposure required to initiate differentiation machinery, and to characterize the role of intrinsic programming. We found that a single day of TGF-beta exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-beta exposure. Despite cessation of TGF-beta exposure following 24 hours, intrinsic programming mediated further chondrogenic and hypertrophic BMSC differentiation. These important behaviors are clouded by diffusion gradients and heterogeneity in commonly used macro-pellet models (2x105 BMSCs per pellet). Use of more homogenous micro-pellet models will enable identification of the critical differentiation cues required, likely in the first 24-hours, to generate high quality cartilage-like tissue from BMSCs (Futrega et al, Commun Biol, 2021). Diseases: Personalized in vitro models for craniofacial and skeletal disease have been constrained by insufficient differentiation protocols for human pluripotent stem cells (hPSCs) into bona fide bone-forming cells. Previously, we devised a differentiation protocol for hPSCs for osteoprogenitors that are specific for paraxial and lateral plate mesoderm-derived, as well as for neural crest-derived bone. We also generated induced pluripotent stem cells (hiPSCs) from a patient with Muenke syndrome, caused by an activating mutation in the FGFR3 gene that causes a form of craniosynostosis (premature closure of the cranial sutures). This year, studies were initiated to compare the neural crest cells, which give rise to cranial sutures, from normal patients to those derived from patients with Muenke syndrome. In addition, in collaboration with Prof. Dr. Alexander Kleger, Ulm University, we generated hiPSCs from clonal bone marrow stromal cells bearing the activating missense mutation of GNAS from a patient with fibrous dysplasia of bone (Breunig et al, Cell Stem Cell, 2021). Fibrous dysplasia of bone is a disease that that causes replacement of normal bone and marrow with abnormal and weak bone and fibrotic tissue. These cells can now be used for further study of the pathogenetic mechanisms in the development of fibrous dysplastic bone. Tissue Engineering: Although cartilage formed by bone marrow stromal cells (BMSCs) undergoes hypertrophy with time in culture and in vivo, they show promise in cartilage repair if the right conditions can be determined. Sheep are the most common large animal pre-clinical model used to determine efficacy of cartilage repair. In collaboration with Dr. Michael Doran, Queensland Technical University, the objective of this study was to characterize ovine BMSCs (oBMSCs) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSCs or ovine articular chondrocytes (oAChs) to repair osteochondral defects in sheep. oBMSCs were characterized for surface marker expression using flow cytometry and evaluated for differentiation into cartilage, bone and fat, to verify their BMSC nature. Expanded oBMSCs were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSCs, but adipogenesis could be restored by excluding IBMX from the medium. Micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSCs or oAChs appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. The capacity of cartilage micro-pellets manufactured from oBMSCs or oAChs to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. The sensitivity of oBMSCs, compared with human BMSCs, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oAChs were more effective than micro-pellets manufactured from oBMSCs in the repair of osteochondral defects in sheep. While oBMSCs can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration (Futrega et al, Stem Cell Res Ther, 2021). Keeping in mind the results of the ovine study described above, and results from other laboratories, the Section has continued its pre-clinical studies using human BMSCs for regeneration of cartilage by developing an articular cartilage injury model in immunocompromised mice and rats. These injuries are being treated with various combinations of BMSCs and hyaluronic acid-coated fibrin microbeads (HyA-FMBs) that we have previously reported to support formation of stable non-hypertrophic cartilage. A patent has recently been issued for the HyA-FMBs and their use with BMSCs (U.S. Patent #10,940,241, March 9, 2021). Long-term studies in mice and rats aim to determine the ability of the constructs to fill in gaps and to test the durability of the cartilage formed as a function of time in a clinically relevant location. By using appropriate scaffolds, it is anticipated that BMSCs can be used for repairing not only bone defects, but also cartilage defects as well.