The role of stem cells in skeletal health and disease

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Biological Activity: Human pluripotent stem cells (hPSCs) provide a platform to model bone organogenesis and disease. To reflect development, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: paraxial mesoderm, lateral plate mesoderm, and neural crest. Characterization of genetic and molecular differences of OPs from different embryonic tissues has not been well reported. To generate lineage-specific OPs from hPSCs, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes (Ref. 8). BMSCs/SSCs are essential for the support of hematopoiesis. Erythropoietin (Epo) is essential for bone marrow erythropoiesis and the Epo receptor (EpoR) on non-erythroid cells including BMSCs suggests systemic effects of Epo. Tg6 mice with Epo overexpression have a high hematocrit, reduced trabecular and cortical bone and bone marrow adipocytes, and decreased bone morphogenic protein 2-driven ectopic bone and adipocyte formation. Epo treatment for 10 days similarly exhibit increased hematocrit, reduced bone and bone marrow adipocytes. Interestingly, endogenous Epo is required for normal differentiation of BMSCs to osteoblasts and bone marrow adipocytes. EpoRE mice with erythroid-restricted erythropoietin receptor exhibit reduced trabecular bone, increased bone marrow adipocytes, and decreased BMP2 ectopic bone formation. Epo treated EpoRE mice achieved hematocrit similar to wild-type mice without reduced bone, suggesting that bone reduction with Epo treatment is associated with non-erythropoietic Epo responses. BMSCs from wild-type, Tg6, and EpoRE-mice were transplanted into immunodeficient mice to assess development into a bone/marrow organ. Like endogenous bone formation, Tg6 BMSCs exhibited reduced differentiation to bone and adipocytes indicating that high Epo inhibits osteogenesis and adipogenesis, while EpoRE BMSCs formed ectopic bone with reduced trabecular regions and increased adipocytes, indicating that loss of Epo signaling favors adipogenesis at the expense of osteogenesis. In summary, endogenous Epo signaling regulates BMSC fate and aberrant Epo levels result in impaired differentiation (Ref. 1). Diseases: Fibrous Dysplasia of bone/McCune-Albright Syndrome (FD/MAS, OMIM#174800) is a complex disease of the skeleton caused by dominant activating mutations of the GNAS locus encoding for the subunit of the G protein-coupled receptor complex (Gs). The mutation involves a substitution of arginine at position 201 by histidine or cysteine (Gsa-R201H or R201C), which leads to overproduction of cAMP. Several signaling pathways are implicated downstream of excess cAMP in the manifestation of disease. However, the pathogenesis of FD remains largely unknown. The overall FD phenotype can be attributed to alterations of skeletal stem/progenitor cells which normally develop into osteogenic or adipogenic cells (in cis), and are also known to provide support to angiogenesis, hematopoiesis, and osteoclastogenesis (in trans). We engineered human skeletal stem/progenitor cells with the Gsa-R201C mutation and performed transcriptomic analysis. Our data suggest that this FD mutation pushes them towards formation of disorganized bone with a concomitant alteration of adipogenic differentiation, and creates an altered in trans environment that induces neovascularization, cytokine/chemokine changes and osteoclastogenesis. In silico comparison of our data with the signature of FD craniofacial samples highlighted common traits, such as the upregulation of ADAM (A Disintegrin and Metalloprotease) proteins and other matrix-related factors, and of PDE7B (Phosphodiesterase 7B), which can be considered as a buffering process, activated to compensate for excess cAMP. We also observed high levels of factors related to browning of white fat. We believe this study provides a useful background for further studies on the molecular basis of the disease and for the identification of novel potential therapeutic targets (Ref. 6). Gain-of-function mutations of GNAS cause a spectrum of clinical phenotypes, ranging from McCune-Albright syndrome (MAS) to isolated disease of bone, endocrine glands and skin. The heterogeneity of organ involvement, age of onset, and clinical severity of the disease reflect the variable size and random distribution of the mutated cell clone arising from the postzygotic mutation. We reported a case of neonatal MAS with hypercortisolism and cholestatic hepatobiliary dysfunction in which pulmonary embolism of marrow and bone fragments secondary to rib fractures was the immediate cause of death. Bones were free of changes of typical FD and fractures appeared to be the result of a mild-to-moderate degree of osteopenia. The mutation was abundant in the adrenal glands and liver, but not in skin, muscle, and fractured ribs, where it could only be demonstrated using a much more sensitive PNA hybridization probe-based FRET technique. It appears that gain-of-function mutations of GNAS underlie a unique syndromic profile in neonates characterized by CAL skin spots, hypercortisolism, hyperthyroidism, hepatic and cardiac dysfunction, and an absence (or latency) of FD, often with a lethal outcome. Our and previous cases highlight the phenotypic severity and the diagnostic and therapeutic challenges of neonatal MAS. Furthermore, our case shows how secondary bone changes, unrelated to the direct impact of the mutation, may contribute to the unfavorable outcome of very early-onset MAS (Ref. 4). The Section has initiated studies on Muenke syndrome, caused by a p.Pro250Arg (c.749C>G) gain-of-function mutation in the FGFR3 gene. It is the leading genetic cause of craniosynostosis and results in a variety of disabling clinical phenotypes. To model the disease and study the pathogenic mechanisms, a human induced pluripotent stem cell (hiPSC) line was generated from a patient diagnosed with Muenke syndrome. Successful reprogramming was validated by morphological features, karyotyping, loss of reprogramming factors, expression of pluripotency markers, mutation analysis and teratoma formation. These cells will be used in future studies to study the differentiation of the mutant iPSCs into neural crest-derived bone and uncover pathogenetic mechanism that result in the craniosynostosis phenotype (Ref. 7). Tissue Engineering: 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 fibrin microbeads that we have reported previously to support formation of stable non-hypertrophy cartilage. Long-term studies in mice and rats aim to determine the ability of the constructs to fill in gaps and the durability of the cartilage formed as a function of time in a clinically relevant location. In addition to developing pre-clinical data for cartilage repair, the Section has extensive preclinical data on the feasibility of using BMSCs along with an appropriate scaffold for bone regeneration. Procedures were previously developed for the ex vivo expansion of clinical grade BMSCs, appropriate scaffolds have been identified, and mouse and rat models of jawbone defects are being developed with the goal of formulating a clinical protocol and an IND for submission to regulatory bodies.