Interactions between cells help to elaborate pattern within the vertebrate central nervous system (CNS). The genes Wnt-1 and Wnt-3a, which encode members of the Wnt family of cysteine-rich secreted signals, are coexpressed at the dorsal midline of the developing neural tube, coincident with dorsal patterning. Each signal is essential for embryonic development, Wnt-1 for midbrain patterning, and Wnt-3a for formation of the paraxial mesoderm, but the absence of a dorsal neural-tube phenotype in each mutant suggests that Wnt signalling may be redundant. Here we demonstrate that in the absence of both Wnt- and Wnt-3a there is a marked deficiency in neural crest derivatives, which originate from the dorsal neural tube, and a pronounced reduction in dorsolateral neural precursors within the neural tube itself. These phenotypes do not seem to result from a disruption in the mechanisms responsible for establishing normal dorsoventral polarity. Rather, our results are consistent with a model in which local Wnt signalling regulates the expansion of dorsal neural precursors. Given the widespread expression of different Wnt genes in discrete areas of the mammalian neural tube, this may represent a general model for the action of Wnt signalling in the developing CNS.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and liganddependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOPiPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.iPSC | fibrodysplasia ossificans progressiva | heterotopic ossification | BMP | TGF H eterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1-6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycineand serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8-10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11,12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12-25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12-20), mouse embryonic fibroblasts derived from Alk2 R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2 R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22,26). In addition, despite the essential role of BMP signaling in development (27-31), the pre-and postnatal development and growth of FOP patients are almost normal, and H...

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Recent reports on ES cell differentiation have suggested the possibility that information on in vivo neurogenesis might be systematically linked to stem cell technology (3, 4). However, it remains to be known whether ES cell-derived neural precursors generated in vitro can produce the full dorsal-ventral range of neuroectodermal derivatives in response to embryonic positional information. To address this question, we have tested in this study whether SDIA-treated ES cells have the competence of differentiating into the dorsal-(neural crest) and ventralmost (floor plate) cells under embryologically relevant conditions. Materials and MethodsCell Culture and Treatment with Patterning Factors. Mouse ES cells (EB5), primate ES cells (cynomolgus monkey-derived; purchased from Asahi Technoglass, Funabashi, Japan), and PA6 cells were maintained and used for induction as described (1, 2, 5). Human bone morphogenetic protein (BMP)4 and mouse were purchased from R&D Systems and freshly added at each medium change. The day on which ES cells are seeded on PA6 is defined as day 0.Immunocytochemistry, Statistics, and RT-PCR. Cells were fixed with 4% paraformaldehyde, and immunostaining was performed with secondary antibodies conjugated with FITC, cy3, or cy5. For statistics, Ϸ100 colonies were observed in each experiment, and three or more experiments were performed. P values for statistical significance (t test) are described in the corresponding figure legends. The values shown in graphs represent the mean Ϯ SD. RT-PCR was performed with ES cell colonies detached from feeder cells as described (1). The primary antibodies and primers used are described in Supporting Materials and Methods, which are published as supporting information on the PNAS web site, www.pnas.org.Colony Isolation and Axon Guidance Assays. The 3D collagen gel assay for axon guidance was performed by using isolated ES cell colonies (day 8; ref. 1) and the cerebellar plate region excised from embryonic day 13 Wistar rats as a responder (6). Results Positional Identity of Neural Tissues Induced from ES Cells by SDIA.We first examined the expression of rostral-caudal CNS markers by RT-PCR (Fig. 1A). SDIA-treated mouse ES cells express the forebrain markers Otx2 and Six3, the ventral diencephalon marker Rx, the ventral forebrain marker Nkx2.1, the midbrainhindbrain border marker En2, and the hindbrain marker Gbx2, but not the spinal cord markers Hoxb4 and b9 (lane 2). These results show that a majority of neural cells induced by SDIA express rostral neural markers. This idea is consistent with our previous report that dopaminergic neurons generated by the SDIA method are those of the midbrain type (1).We next attempted to alter the rostral-caudal identity of SDIA-induced neural cells by the caudalizing factor retinoic acid (RA; ref. 7). RA treatment (0.2 M all-trans RA; Fig. 1 A, lane 3) suppressed the forebrain markers Otx2, Six3, Rx, and Nkx2.1, whereas it induced the hindbrain marker Gbx2 and the spinal cord markers Hoxb4 and b9. RA treatment did not sign...