No abstract

Statistics. All studies were performed with biological replicates as described in Supplemental Tables 1-11. The data were analyzed with GraphPad Prism v.7 software using 2-tailed Student's t test and 2-way ANOVA Sidak or Tukey's multiple comparison tests. The Sidak test was used when comparing means between WT and FOP, and the Tukey test was used when means of both WT and FOP were compared together with other groups. The software R was used for heatmaps and PCA. P < 0.05 were considered statistically significant. Study approvals. All of the human study and sample collection procedures were reviewed and approved by the UCSF Committee on Human Research. All subjects provided informed consent prior to their participation in the study.

BackgroundThe Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways, as in conditions of congenital heterotopic ossification, are thought to activate an osteogenic program in endothelial cells. However, if and how this occurs in human endothelial cells remains unclear.MethodsWe used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP), a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand, and tested for their ability to activate osteogenesis, extracellular matrix production, and differential downstream signaling in the BMP/Activin A pathways.ResultsWe found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining, suggesting formation of immature osteoblasts, but failed to show mature osteoblastic features. However, FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs, suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally, FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs, FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation, suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition, we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs, which could explain the cell type-specific SMAD signaling.ConclusionsOur results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction, increase expression of fibrogenic matrix proteins, and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0372-6) contains supplementary material, which is available to authorized users.

To introduce a functional vascular network into tissue‐engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow‐derived MSCs and cord blood‐derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)‐derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10‐day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 μm diffusion limit. In addition, iPSC‐derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.

No abstract