The role of stem cells in skeletal health and disease

National Institute Of Dental & Craniofacial Research

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Abstract

BIOLOGICAL ACTIVITY The role of SP7 and RUNX2 in the hypertrophic conversion of bone marrow stromal cells (BMSC) during chondrogenesis: For bone marrow stromal cells (BMSCs) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentiation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies (Franco et al, J Tiss Eng 14:1-16 (2023). Diseases Gs-alph-R201C and estrogen reveals different subsets of bone marrow adiponectin expressing osteogenic cells: The Gs/cAMP signaling pathway mediates the effect of a variety of factors that regulate the homeostasis of the post-natal skeleton. Dysregulated activity of Gs due to gain-of-function mutations (R201C/R201H) results in severe derangements of the entire bone/bone marrow organ. The consequences of gain-of-function mutations of Gs-alpha in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of G-salpha-R201C driven by the Adiponectin (Adq) promoter. In the metaphysis, GsaR201C caused an early phase of bone resorption followed by bone deposition. Metaphyseal bone formation was sustained by cells that were traced by Adq-Cre and eventually resulted in a high trabecular bone mass phenotype. In the diaphysis, GsaR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, GsaR201C caused the development of a lytic phenotype that affected both cortical (increased porosity) and trabecular (tunneling resorption) bone. These results provide the first evidence that the Adq-cell network in the skeleton not only regulates bone resorption but also contributes to bone formation, and that the Gs/cAMP pathway is a major modulator of both functions (Palmisano et al, Bone Research, 2022). Changes in collagen fibril organization in different forms of Osteogenesis imperfecta: We are currently collaborating with Dr. Joan Marini, NICHD, on several models of Osteogenesis Imperfecta type V and VI. These forms of OI are caused by mutations in non-collagenous proteins (mutations in IFITM5 (interferon induced transmembrane 5) and SERPINF1 (serpin family F member 1), respectively. We are also characterizing a high bone mass disease caused by a BMP1 mutation. We are performing a histological characterization to determine the collagen organization by using polarized light microscopy. Tissue Engineering: Induced pluripotent stem cell technology in bone biology: Technologies on the development and differentiation of human induced pluripotent stem cells (hiPSCs) are rapidly improving and have been applied to create cell types relevant to the bone field. We have published a brief history of cell and stem cell biology that led to development of iPSCs, and provded current best practices on how iPSCs are made, characterized, and modified. Differentiation protocols to form bona fide bone-forming cells from iPSCs, as verified by in vivo transplantation, are now available and can be used to probe details of differentiation and function in depth. When applied to iPSCs bearing disease-causing mutations, the pathogenetic mechanisms of diseases of the skeleton can be elucidated, along with the development of novel therapeutics. These cells can also be used for development of cell therapies for cell and tissue replacement. Hematopoietic organoids: Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. HPC formation can be induced in a simple way by coculturing with bone marrow stromal cells. We first attempted to determine whether coculturing with healthy donor-derived hBMSCs in a 2D condition can generate CD34+CD43+ cells, which contain multipotent HPCs with lympho-myeloid potential. However, hiPSCs grown on hBMSCs in 2D formed flattened colonies and failed to develop into cystic structures after 13 days of coculture. Thus, we attempted to convert to a 3D organoid type culture. We found that hiPSC cells could be differentiated into stromal cells with BMSC-like characteristics. We found that hiPSCs cocultured with these hiPSC-derived stromal cells as spheroids (hematopoietic spheroids Hp-spheroids) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lymphomyeloid potential.