Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells.

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

A molecular roadmap of reprogramming somatic cells into iPS cells.

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.

Small molecules facilitate rapid and synchronous iPSC generation.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-ß inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells.

Transcription factor-based cellular reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.

Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells.

Transient MyoD overexpression in concert with small molecule treatment reprograms mouse fibroblasts into induced myogenic progenitor cells (iMPCs). However, the molecular landscape and mechanisms orchestrating this cellular conversion remain unknown. Here, we undertook an integrative multiomics approach to delineate the process of iMPC reprogramming in comparison to myogenic transdifferentiation mediated solely by MyoD. Using transcriptomics, proteomics, and genome-wide chromatin accessibility assays, we unravel distinct molecular trajectories that govern the two processes. Notably, only iMPC reprogramming is characterized by gradual up-regulation of muscle stem cell markers, unique signaling pathways, and chromatin remodelers in conjunction with exclusive chromatin opening in core myogenic promoters. In addition, we determine that the Notch pathway is indispensable for iMPC formation and self-renewal and further use the Notch ligand Dll1 to homogeneously propagate iMPCs. Collectively, this study charts divergent molecular blueprints for myogenic transdifferentiation or reprogramming and underpins the heightened capacity of iMPCs for capturing myogenesis ex vivo.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells.

Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.

Aberrant epigenetic silencing of tumor suppressor genes is reversed by direct reprogramming.

Direct reprogramming procedures reset the epigenetic memory of cells and convert differentiated somatic cells into pluripotent stem cells. In addition to epigenetic memory of cell identity, which is established during development, somatic cells can accumulate abnormal epigenetic changes that can contribute to pathological conditions. Aberrant promoter hypermethylation and epigenetic silencing of tumor suppressor genes (TSGs) are now recognized as an important mechanism in tumor initiation and progression. Here, we have studied the fate of the silenced TSGs p16(CDKN2A) during direct reprogramming. We find that following reprogramming, p16 expression is restored and is stably maintained even when cells are induced to differentiate. Large-scale methylation profiling of donor cells identified aberrant methylation at hundreds of additional sites. Methylation at many, but not all these sites was reversed following reprogramming. Our results suggest that reprogramming approaches may be applied to repair the epigenetic lesions associated with cancer.

Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells.

Genomic imprinting is an epigenetic phenomenon whereby genes are expressed in a monoallelic manner, which is inherited either maternally or paternally. Expression of imprinted genes has been examined in human embryonic stem (ES) cells, and the cells show a substantial degree of genomic imprinting stability. Recently, human somatic cells were reprogrammed to a pluripotent state using various defined factors. These induced pluripotent stem (iPS) cells are thought to have a great potential for studying genetic diseases and to be a source of patient-specific stem cells. Thus, studying the expression of imprinted genes in these cells is important. We examined the allelic expression of various imprinted genes in several iPS cell lines and found polymorphisms in four genes. After analyzing parent-specific expression of these genes, we observed overall normal monoallelic expression in the iPS cell lines. However, we found biallelic expression of the H19 gene in one iPS cell line and biallelic expression of the KCNQ10T1 gene in another iPS cell line. We further analyzed the DNA methylation levels of the promoter region of the H19 gene and found that the cell line that showed biallelic expression had undergone extensive DNA demethylation. Additionally we studied the imprinting gene expression pattern of multiple human iPS cell lines via DNA microarray analyses and divided the pattern of expression into three groups: (a) genes that showed significantly stable levels of expression in iPS cells, (b) genes that showed a substantial degree of variability in expression in both human ES and iPS cells, and (c) genes that showed aberrant expression levels in some human iPS cell lines, as compared with human ES cells. In general, iPS cells have a rather stable expression of their imprinted genes. However, we found a significant number of cell lines with abnormal expression of imprinted genes, and thus we believe that imprinted genes should be examined for each cell line if it is to be used for studying genetic diseases or for the purpose of regenerative medicine.

Screening method to identify hydrogel formulations that facilitate myotube formation from encapsulated primary myoblasts.

Hydrogel-based three-dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence-based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

Exercise promotes satellite cell contribution to myofibers in a load-dependent manner.

BACKGROUND: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown. RESULTS: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise. CONCLUSIONS: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.

Voluntary Resistance Running as a Model to Induce mTOR Activation in Mouse Skeletal Muscle.

Long-term voluntary resistance running has been shown to be a valid model to induce muscle growth in rodents. Moreover, the mammalian target of rapamycin complex 1 (mTORC1) is a key signaling complex regulating exercise/nutrient-induced alterations in muscle protein synthesis. How acute resistance running affects mTORC1 signaling in muscle and if resistance applied to the wheel can modulate mTORC1 activation has not yet been fully elucidated. Here, we show that both acute resistance running and acute free running activated mTORC1 signaling in the m. gastrocnemius, m. soleus, and m. plantaris, but not in m. tibialis anterior of mice when compared to sedentary controls. Furthermore, only the low threshold oxidative part in the m. gastrocnemius showed increased mTORC1 signaling upon running and acute heavy-load resistance running evoked higher downstream mTORC1 signaling in both m. soleus and m. plantaris than free running without resistance, pointing toward mechanical load as an important independent regulator of mTORC1. Collectively, in this study, we show that voluntary resistance running is an easy-to-use, time-efficient and low stress model to study acute alterations in mTORC1 signaling upon high-load muscular contractions in mice.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

Probabilistic Modeling of Reprogramming to Induced Pluripotent Stem Cells.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is typically an inefficient and asynchronous process. A variety of technological efforts have been made to accelerate and/or synchronize this process. To define a unified framework to study and compare the dynamics of reprogramming under different conditions, we developed an in silico analysis platform based on mathematical modeling. Our approach takes into account the variability in experimental results stemming from probabilistic growth and death of cells and potentially heterogeneous reprogramming rates. We suggest that reprogramming driven by the Yamanaka factors alone is a more heterogeneous process, possibly due to cell-specific reprogramming rates, which could be homogenized by the addition of additional factors. We validated our approach using publicly available reprogramming datasets, including data on early reprogramming dynamics as well as cell count data, and thus we demonstrated the general utility and predictive power of our methodology for investigating reprogramming and other cell fate change systems.

A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2.

The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.

Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage.

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, has been reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4(+) cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.

Nanog is dispensable for the generation of induced pluripotent stem cells.

Cellular reprogramming from somatic cells to induced pluripotent stem cells (iPSCs) can be achieved through forced expression of the transcription factors Oct4, Klf4, Sox2, and c-Myc (OKSM) [1-4]. These factors, in combination with environmental cues, induce a stable intrinsic pluripotency network that confers indefinite self-renewal capacity on iPSCs. In addition to Oct4 and Sox2, the homeodomain-containing transcription factor Nanog is an integral part of the pluripotency network [5-11]. Although Nanog expression is not required for the maintenance of pluripotent stem cells, it has been reported to be essential for the establishment of both embryonic stem cells (ESCs) from blastocysts and iPSCs from somatic cells [10, 12]. Here we revisit the role of Nanog in direct reprogramming. Surprisingly, we find that Nanog is dispensable for iPSC formation under optimized culture conditions. We further document that Nanog-deficient iPSCs are transcriptionally highly similar to wild-type iPSCs and support the generation of teratomas and chimeric mice. Lastly, we provide evidence that the presence of ascorbic acid in the culture media is critical for overcoming the previously observed reprogramming block of Nanog knockout cells.

Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.

Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming.

The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin "interactome" for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.