Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies.

Myosin phosphatase modulates the cardiac cell fate by regulating the subcellular localization of Nkx2.5 in a Wnt/Rho-associated protein kinase-dependent pathway.

RATIONALE: Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level. OBJECTIVE: We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity. METHODS AND RESULTS: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1ß and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 ß/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process. CONCLUSIONS: We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.

Gli2 and MEF2C activate each other's expression and function synergistically during cardiomyogenesis in vitro.

The transcription factors Gli2 (glioma-associated factor 2), which is a transactivator of Sonic Hedgehog (Shh) signalling, and myocyte enhancer factor 2C (MEF2C) play important roles in the development of embryonic heart muscle and enhance cardiomyogenesis in stem cells. Although the physiological importance of Shh signalling and MEF2 factors in heart development is well known, the mechanistic understanding of their roles is unclear. Here, we demonstrate that Gli2 and MEF2C activated each other's expression while enhancing cardiomyogenesis in differentiating P19 EC cells. Furthermore, dominant-negative mutant proteins of either Gli2 or MEF2C repressed each other's expression, while impairing cardiomyogenesis in P19 EC cells. In addition, chromatin immunoprecipitation (ChIP) revealed association of Gli2 to the Mef2c gene, and of MEF2C to the Gli2 gene in differentiating P19 cells. Finally, co-immunoprecipitation studies showed that Gli2 and MEF2C proteins formed a complex, capable of synergizing on cardiomyogenesis-related promoters containing both Gli- and MEF2-binding elements. We propose a model whereby Gli2 and MEF2C bind each other's regulatory elements, activate each other's expression and form a protein complex that synergistically activates transcription, enhancing cardiac muscle development. This model links Shh signalling to MEF2C function during cardiomyogenesis and offers mechanistic insight into their in vivo functions.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes.

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.

Combinatorial Utilization of Murine Embryonic Stem Cells and In Vivo Models to Study Human Congenital Heart Disease.

We have established an in vitro model of the human congenital heart defect (CHD)-associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation. We describe the isolation of RNA from embryonic day 8.5 (E8.5) embryos and E9.5 hearts of wild-type and mutant mice. We found that the in vitro model shows reduced cardiomyogenesis, similar to Nkx2.5R141C/+ embryos at E8.5, indicating a transient loss of cardiomyogenesis at this time point. These results suggest that our in vitro model can be used to study very early changes in heart development that cause CHD. © 2018 by John Wiley & Sons, Inc.

Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression.

Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential.

Exploring De Novo metabolic pathways from pyruvate to propionic acid.

Industrial biotechnology provides an efficient, sustainable solution for chemical production. However, designing biochemical pathways based solely on known reactions does not exploit its full potential. Enzymes are known to accept non-native substrates, which may allow novel, advantageous reactions. We have previously developed a computational program named Biological Network Integrated Computational Explorer (BNICE) to predict promiscuous enzyme activities and design synthetic pathways, using generalized reaction rules curated from biochemical reaction databases. Here, we use BNICE to design pathways synthesizing propionic acid from pyruvate. The currently known natural pathways produce undesirable by-products lactic acid and succinic acid, reducing their economic viability. BNICE predicted seven pathways containing four reaction steps or less, five of which avoid these by-products. Among the 16 biochemical reactions comprising these pathways, 44% were validated by literature references. More than 28% of these known reactions were not in the BNICE training dataset, showing that BNICE was able to predict novel enzyme substrates. Most of the pathways included the intermediate acrylic acid. As acrylic acid bioproduction has been well advanced, we focused on the critical step of reducing acrylic acid to propionic acid. We experimentally validated that Oye2p from Saccharomyces cerevisiae can catalyze this reaction at a slow turnover rate (10(-3) s(-1) ), which was unknown to occur with this enzyme, and is an important finding for further propionic acid metabolic engineering. These results validate BNICE as a pathway-searching tool that can predict previously unknown promiscuous enzyme activities and show that computational methods can elucidate novel biochemical pathways for industrial applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:303-311, 2016.

Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells.

Cell therapies treating pathological muscle atrophy or damage requires an adequate quantity of muscle progenitor cells (MPCs) not currently attainable from adult donors. Here, we generate cultures of approximately 90% skeletal myogenic cells by treating human embryonic stem cells (ESCs) with the GSK3 inhibitor CHIR99021 followed by FGF2 and N2 supplements. Gene expression analysis identified progressive expression of mesoderm, somite, dermomyotome, and myotome markers, following patterns of embryonic myogenesis. CHIR99021 enhanced transcript levels of the pan-mesoderm gene T and paraxial-mesoderm genes MSGN1 and TBX6; immunofluorescence confirmed that 91% ± 6% of cells expressed T immediately following treatment. By 7 weeks, 47% ± 3% of cells were MYH(+ve) myocytes/myotubes surrounded by a 43% ± 4% population of PAX7(+ve) MPCs, indicating 90% of cells had achieved myogenic identity without any cell sorting. Treatment of mouse ESCs with these factors resulted in similar enhancements of myogenesis. These studies establish a foundation for serum-free and chemically defined monolayer skeletal myogenesis of ESCs.

ß-catenin is essential for efficient in vitro premyogenic mesoderm formation but can be partially compensated by retinoic acid signalling.

Previous studies have shown that P19 cells expressing a dominant negative ß-catenin mutant (ß-cat/EnR) cannot undergo myogenic differentiation in the presence or absence of muscle-inducing levels of retinoic acid (RA). While RA could upregulate premyogenic mesoderm expression, including Pax3/7 and Meox1, only Pax3/7 and Gli2 could be upregulated by RA in the presence of ß-cat/EnR. However, the use of a dominant negative construct that cannot be compensated by other factors is limiting due to the possibility of negative chromatin remodelling overriding compensatory mechanisms. In this study, we set out to determine if ß-catenin function is essential for myogenesis with and without RA, by creating P19 cells with reduced ß-catenin transcriptional activity using an shRNA approach, termed P19[shß-cat] cells. The loss of ß-catenin resulted in a reduction of skeletal myogenesis in the absence of RA as early as premyogenic mesoderm, with the loss of Pax3/7, Eya2, Six1, Meox1, Gli2, Foxc1/2, and Sox7 transcript levels. Chromatin immunoprecipitation identified an association of ß-catenin with the promoter region of the Sox7 gene. Differentiation of P19[shß-cat] cells in the presence of RA resulted in the upregulation or lack of repression of all of the precursor genes, on day 5 and/or 9, with the exception of Foxc2. However, expression of Sox7, Gli2, the myogenic regulatory factors and terminal differentiation markers remained inhibited on day 9 and overall skeletal myogenesis was reduced. Thus, ß-catenin is essential for in vitro formation of premyogenic mesoderm, leading to skeletal myogenesis. RA can at least partially compensate for the loss of ß-catenin in the expression of many myogenic precursor genes, but not for myoblast gene expression or overall myogenesis.

The interaction of subsyllabic encoding and stress assignment: A new examination of an old problem in Spanish.

This study employs a naming task to examine the role of the syllable in speech production, focusing on a lesser-studied aspect of syllabic processing, the interaction of subsyllabic patterns (i.e. syllable phonotactics) and higher-level prosody, in this case, stress assignment in Spanish. Specifically, we examine a controversial debate in Spanish regarding the interaction of syllable weight and stress placement, showing that traditional representations of weight fail to predict the differential modulation of stress placement by rising versus falling diphthongs in Spanish nonce forms. Our results also suggest that the internal structure of the syllable plays a larger role than is assumed in the processing literature in that it modulates higher-level processes such as stress encoding. Our results thus inform the debate regarding syllable weight in Spanish and linguistic theorizing more broadly, as well as expand our understanding of the importance of the syllable, and more specifically its internal structure, in modulating word processing.