Mesenchymal stem cells can give rise to several cell types, but variations depending on isolation method and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an ALK2 receptor-dependent mechanism. In lesions from patients with Fibrodysplasia Ossificans Progressiva, a disease where heterotopic ossification occurs as a result of activating ALK2 mutations, or from a mutant ALK2 transgenic mouse model, chondrocytes and osteoblasts express endothelial markers. Tie2-Cre lineage tracing also suggests an endothelial origin of these cells. Expressing mutant ALK2 in endothelial cells, or treatment with the ALK2 ligands TGF-β2 or BMP4, causes endothelial-mesenchymal transition and acquisition of a stem cell-like phenotype. In selective media, these cells differentiate into osteoblasts, chondrocytes, or adipocytes. The process is inhibited by ALK2-specific siRNA. Conversion of endothelial cells to stem-like cells may provide a novel approach to tissue engineering.

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Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive BMP signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however null mutations of the preprotachykinin gene encoding SP prevent HO. Importantly, ablation of SP+ sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibits injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.

Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF-1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF-1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF-1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)-mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF-1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1Q207D/+ mouse model of FOP. Thus, an inflammation and cellular oxygen-sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF-1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP, and further identifies HIF-1α as a therapeutic target for FOP and perhaps non-genetic forms of HEO.

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.

Metamorphosis, the transformation of one normal tissue or organ system into another, is a biological process rarely studied in higher vertebrates or mammals, but exemplified pathologically by the extremely disabling autosomal dominant disorder fibrodysplasia ossificans progressiva (FOP). The recurrent single nucleotide missense mutation in the gene encoding activin receptor IA/activin-like kinase-2 (ACVR1/ALK2), a bone morphogenetic protein type I receptor that causes skeletal metamorphosis in all classically affected individuals worldwide, is the first identified human metamorphogene. Physiological studies of this metamorphogene are beginning to provide deep insight into a highly conserved signaling pathway that regulates tissue stability following morphogenesis, and that when damaged at a highly specific locus (c.617G > A; R206H), and triggered by an inflammatory stimulus permits the renegade metamorphosis of normal functioning connective tissue into a highly ramified skeleton of heterotopic bone. A comprehensive understanding of the process of skeletal metamorphosis, as revealed by the rare condition FOP, will lead to the development of more effective treatments for FOP and, possibly, for more common disorders of skeletal metamorphosis.

Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gsα, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gsα signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gsα, is imprinted. Mice with paternal allele deletion of Gnas (Gnas+/p−) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas+/p− mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas+/p− cells. During differentiation, Gnas+/p− cells showed diminished pCREB, β-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas+/p− by reducing Nfatc1 levels. Cortical bone of Gnas+/p− mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/β-catenin signaling. Our data identify a new role for Gsα signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/β-catenin pathways.

The most important milestone in understanding a genetic disease is the identification of the causative mutation. However, such knowledge is often insufficient to decipher the pathophysiology of the disorder or to effectively treat those affected. Fibrodysplasia ossificans progressiva (FOP) is a rare, disabling, genetic disease of progressive heterotopic endochondral ossification (HEO) enabled by missense mutations that promiscuously and provisionally activate ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor, in all affected individuals. While activating mutations of the ACVR1/ALK2 receptor are necessary, disease activity and progression also depend on altered cell and tissue physiology. Recent findings identify inflammatory and immunological factors, vascular-derived mesenchymal stem cells, and a hypoxic lesional microenvironment that trigger, promote, and enable episodic progression of FOP in the setting of the genetic mutation. Effective therapies for FOP will need to consider these seminal pathophysiologic interactions.