The role of stem cells in skeletal health and disease

National Institute Of Dental & Craniofacial Research

Investigators

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Abstract

Biological Activity The biological basis of heterogeneity SSCs/BMSCs: There is strong evidence that the self-renewing skeletal lineages derived from bone marrow stroma can be an ideal source for skeletal tissue engineering. However, the heterogeneous nature SSCs/BMSCs needs to be clarified. We highlighted: i) the importance of distinguishing SSCs/BMSCs from other "mesenchymal stem/stromal cells", and ii) factors that contribute to their heterogeneity. The possible role of SSC enrichment, their expansion and its impact on SSC phenotype was explored. Emphasis was also given to emerging single cell RNA sequencing approaches. Understanding the factors involved in heterogeneity will help to improve strategies to isolate, characterize and adopt best operating protocols for developing reproducible stem cells grafts. However, more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations (Arora and Robey, Biomater Transl, 2022). Secreted frizzled related protein 2 (Sfrp2) deficiency decreases adult skeletal stem cell function in mice: SFRP2 was highly over-represented in a subset of human SSCs. SFRPs are thought to modulate WNT signaling, which is essential to maintain skeletal homeostasis, but the specific role of SFRP2 in BMSCs/SSCs was unclear. We evaluated Sfrp2 deficiency on BMSC/SSC function in models of skeletal organogenesis and regeneration. Sfrp2-deficient (KO) mice are overtly normal; but their BMSCs/SSCs exhibited reduced colony formation. Sfrp2 KO BMSCs/SSCs formed less trabecular bone in an ectopic bone formation assay, and regeneration of a cortical drilled hole defect was dramatically impaired in Sfrp2 KO mice. Sfrp2-KO BMSCs/SSCs exhibited poor in vitro osteogenic differentiation. Activation of the Wnt co-receptor, Lrp6, and expression of Wnt target genes, Axin2, C-myc and Cyclin D1, were reduced. Addition of recombinant Sfrp2 restored most of these activities, suggesting that Sfrp2 acts as a Wnt agonist. Sfrp2 plays a role in self-renewal of SSCs and in the recruitment and differentiation of adult SSCs during bone healing. SFRP2 is also a useful marker of BMSC/SSC multipotency, and a factor to potentially improve the quality of ex vivo expanded BMSC/SSC products (de Castro et al, Bone Research, 2021). A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise: The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. We found that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-2 (GFR2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing bone loss and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects (Gadomski et al. Cell Stem Cell, 2022). Diseases Quantitative craniofacial analysis and generation of human inuduced pluripotent stem cells for Muenke Syndrome: In a recent report, we focused on Muenke syndrome (MS), a disease caused by the p.Pro250Arg variant in fibroblast growth factor receptor 3 (FGFR3), characterized by coronal suture synostosis, macrocephaly without craniosynostosis, dysmorphic craniofacial features, and dental malocclusion. The clinical findings of MS are further complicated by variable expression of phenotypic traits and incomplete penetrance. Unraveling the mechanisms behind MS requires a comprehensive and systematic phenotyping patients to precisely identify the impact of the mutation variant. To establish this framework, we quantitatively delineated the craniofacial phenotype of an individual with MS and compared with his unaffected parents using three-dimensional cephalometric analysis of cone beam computed tomography scans and geometric morphometric analysis, in addition to an extensive clinical evaluation. Secondly, we also generated the first hiPSCs derived from this family trio, with detailed characterization of all cell lines. This report provides a starting point for evaluating the mechanistic underpinning of the craniofacial development in MS with the goal of linking specific clinical manifestations to molecular insights gained from hiPSC-based disease modeling (Kidwai et al, J Dev Biol, 2021 GsaR201C 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 in adipogenically-committed bone marrow stromal cells has remained unaddressed. We generated a mouse model with expression of GsR201C driven by the Adiponectin (Adq) promoter. In the metaphysis, GsR201C 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, GsR201C, in combination with estrogen, triggered the osteogenic activity of Adq-Cre-targeted perivascular bone marrow stromal cells leading to intramedullary bone formation. Finally, GsR201C 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). Tissue Engineering Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis: Bone morphogenetic protein (BMP)-mediated cascades are upregulated during BMSC chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures, we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy (Franco et al, Stem Cell Reports, 2022).