Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(−) CD108(−) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.
Human pluripotent stem cells (hPSCs) proliferate in vitro for long periods without losing pluripotency and can be induced to differentiate into various cell types including skeletal muscle cells (SMCs). Human embryonic stem cells (hESCs) are generated from a preimplantation-stage embryo. Human-induced pluripotent stem cells (hiPSCs) are derived from somatic cells of both healthy donors and patients with muscle diseases of any age using reprogramming factors. Currently, there are two kinds of protocols to induce skeletal muscle from hPSCs. One type utilizes overexpression of a potent myogenic master regulator, MyoD, to directly induce skeletal muscle. Stepwise induction of skeletal muscle has also been reported by many research groups, but hiPSC-based cell therapy for muscular dystrophy is still experimental. On the other hand, hiPSCs derived from patients with muscle disease are widely used for disease modeling in vitro. Here, we review the recent literature on derivation of skeletal muscle from human pluripotent stem cells and discuss their application.
Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a protocol and provide experimental proof for building a digital 3D-atlas (here, for zebrafish hindbrain) that integrates spatial and temporal data for neuronal differentiation and brain morphogenesis, through a combination of in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a reference 3D hindbrain from several imaged specimens and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain vs. the progenitor domain in the whole hindbrain. Third, we used in vivo Kaede-photoconversion experiments to generate a temporal heatmap of the neuronal growth in the whole hindbrain, revealing the spatiotemporal dynamics of neuronal differentiation upon morphogenesis. Last, as proof-of-concept, we assessed the birthdate order of GABAergic-neurons using our temporal registration map. As this protocol uses open-access tools and algorithms, it can be shared for standardized and accessible tissue-wide cell population atlas construction.
Reconstruction of prototypic three-dimensional (3D) atlases at the scale of whole tissues or organs requires specific methods to be developed. We have established a digital 3D-atlas maker (DAMAKER) and built a digital 3D-atlas to monitor the changes in the growth of the neuronal differentiation domain in the zebrafish hindbrain upon time. DAMAKER integrates spatial and temporal data of cell populations, neuronal differentiation and brain morphogenesis, through in vivo imaging techniques paired with image analyses and segmentation tools. First, we generated a 3D-reference from several imaged hindbrains and segmented them using a trainable tool; these were aligned using rigid registration, revealing distribution of neuronal differentiation growth patterns along the axes. Second, we quantified the dynamic growth of the neuronal differentiation domain by in vivo neuronal birthdating experiments. We generated digital neuronal birthdating 3D-maps and revealed that the temporal order of neuronal differentiation prefigured the spatial distribution of neurons in the tissue, with an inner-outer differentiation gradient. Last, we applied it to specific differentiated neuronal populations such as glutamatergic and GABAergic neurons, as proof-of-concept that the digital birthdating 3D-maps could be used as a proxy to infer neuronal birthdate. As this protocol uses open-access tools and algorithms, it can be shared for standardized, accessible, tissue-wide cell population atlas construction.
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