Approaches to characterize the transcriptional trajectory of human myogenesis.

Human pluripotent stem cells (hPSCs) have attracted considerable interest in understanding the cellular fate determination processes and modeling a number of intractable diseases. In vitro generation of skeletal muscle tissues using hPSCs provides an essential model to identify the molecular functions and gene regulatory networks controlling the differentiation of skeletal muscle progenitor cells. Such a genetic roadmap is not only beneficial to understanding human myogenesis but also to decipher the molecular pathology of many skeletal muscle diseases. The combination of established human in vitro myogenesis protocols and newly developed molecular profiling techniques offers extensive insight into the molecular signatures for the development of normal and disease human skeletal muscle tissues. In this review, we provide a comprehensive overview of the current progress of in vitro skeletal muscle generation from hPSCs and relevant examples of the transcriptional landscape and disease-related transcriptional aberrations involving signaling pathways during the development of skeletal muscle cells.

Engineering Skeletal Muscle Grafts with PAX7::GFP-Sorted Human Pluripotent Stem Cell-Derived Myogenic Progenitors on Fibrin Microfiber Bundles for Tissue Regeneration.

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

Human pluripotent stem cell-derived myogenic progenitors undergo maturation to quiescent satellite cells upon engraftment.

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following in vivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders.

Novel culture system via wirelessly controllable optical stimulation of the FGF signaling pathway for human and pig pluripotency.

Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Transcriptional landscape of myogenesis from human pluripotent stem cells reveals a key role of TWIST1 in maintenance of skeletal muscle progenitors.

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).

Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder.

Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.

Zika virus directly infects peripheral neurons and induces cell death.

Zika virus (ZIKV) infection is associated with neurological disorders of both the CNS and peripheral nervous systems (PNS), yet few studies have directly examined PNS infection. Here we show that intraperitoneally or intraventricularly injected ZIKV in the mouse can infect and impact peripheral neurons in vivo. Moreover, ZIKV productively infects stem-cell-derived human neural crest cells and peripheral neurons in vitro, leading to increased cell death, transcriptional dysregulation and cell-type-specific molecular pathology.

Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFß signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling.

A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.

The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

Efficient generation human induced pluripotent stem cells from human somatic cells with Sendai-virus.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Profiling individual human embryonic stem cells by quantitative rt-PCR.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Generation of multipotent induced neural crest by direct reprogramming of human postnatal fibroblasts with a single transcription factor.

Neural crest (NC) generates diverse lineages including peripheral neurons, glia, melanocytes, and mesenchymal derivatives. Isolating multipotent human NC has proven challenging, limiting our ability to understand NC development and model NC-associated disorders. Here, we report direct reprogramming of human fibroblasts into induced neural crest (iNC) cells by overexpression of a single transcription factor, SOX10, in combination with environmental cues including WNT activation. iNC cells possess extensive capacity for migration in vivo, and single iNC clones can differentiate into the four main NC lineages. We further identified a cell surface marker for prospective isolation of iNCs, which was used to generate and purify iNCs from familial dysautonomia (FD) patient fibroblasts. FD-iNC cells displayed defects in cellular migration and alternative mRNA splicing, providing insights into FD pathogenesis. Thus, this study provides an accessible platform for studying NC biology and disease through rapid and efficient reprogramming of human postnatal fibroblasts.