Induced Pluripotent Stem Cells for Modeling Physiological and Pathological Striated Muscle Complexity.

Neuromuscular disorders (NMDs) are a large group of diseases associated with either alterations of skeletal muscle fibers, motor neurons or neuromuscular junctions. Most of these diseases is characterized with muscle weakness or wasting and greatly alter the life of patients. Animal models do not always recapitulate the phenotype of patients. The development of innovative and representative human preclinical models is thus strongly needed for modeling the wide diversity of NMDs, characterization of disease-associated variants, investigation of novel genes function, or the development of therapies. Over the last decade, the use of patient's derived induced pluripotent stem cells (hiPSC) has resulted in tremendous progress in biomedical research, including for NMDs. Skeletal muscle is a complex tissue with multinucleated muscle fibers supported by a dense extracellular matrix and multiple cell types including motor neurons required for the contractile activity. Major challenges need now to be tackled by the scientific community to increase maturation of muscle fibers in vitro, in particular for modeling adult-onset diseases affecting this tissue (neuromuscular disorders, cachexia, sarcopenia) and the evaluation of therapeutic strategies. In the near future, rapidly evolving bioengineering approaches applied to hiPSC will undoubtedly become highly instrumental for investigating muscle pathophysiology and the development of therapeutic strategies.

In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype.

Many genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation.

Mesenchymal stem cells derived from patients with premature aging syndromes display hallmarks of physiological aging.

Progeroid syndromes are rare genetic diseases with most of autosomal dominant transmission, the prevalence of which is less than 1/10,000,000. These syndromes caused by mutations in the <i>LMNA</i> gene encoding A-type lamins belong to a group of disorders called laminopathies. Lamins are implicated in the architecture and function of the nucleus and chromatin. Patients affected with progeroid laminopathies display accelerated aging of mesenchymal stem cells (MSCs)-derived tissues associated with nuclear morphological abnormalities. To identify pathways altered in progeroid patients' MSCs, we used induced pluripotent stem cells (hiPSCs) from patients affected with classical Hutchinson-Gilford progeria syndrome (HGPS, c.1824C&gt;T-p.G608G), HGPS-like syndrome (HGPS-L; c.1868C&gt;G-p.T623S) associated with farnesylated prelamin A accumulation, or atypical progeroid syndromes (APS; homozygous c.1583C&gt; T-p.T528M; heterozygous c.1762T&gt;C-p.C588R; compound heterozygous c.1583C&gt;T and c.1619T&gt;C-p.T528M and p.M540T) without progerin accumulation. By comparative analysis of the transcriptome and methylome of hiPSC-derived MSCs, we found that patient's MSCs display specific DNA methylation patterns and modulated transcription at early stages of differentiation. We further explored selected biological processes deregulated in the presence of <i>LMNA</i> variants and confirmed alterations of age-related pathways during MSC differentiation. In particular, we report the presence of an altered mitochondrial pattern; an increased response to double-strand DNA damage; and telomere erosion in HGPS, HGPS-L, and APS MSCs, suggesting converging pathways, independent of progerin accumulation, but a distinct DNA methylation profile in HGPS and HGPS-L compared with APS cells.

Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells.

Skeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both the hESC-SkMC fusion index and the expression levels of various skeletal muscle markers, including the motor protein myosin heavy chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway, and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled with improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery pertaining to further enhancing muscle development or treating muscle diseases.

Human Pluripotent Stem Cells-Based Therapies for Neurodegenerative Diseases: Current Status and Challenges.

Neurodegenerative diseases are characterized by irreversible cell damage, loss of neuronal cells and limited regeneration potential of the adult nervous system. Pluripotent stem cells are capable of differentiating into the multitude of cell types that compose the central and peripheral nervous systems and so have become the major focus of cell replacement therapies for the treatment of neurological disorders. Human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC)-derived cells have both been extensively studied as cell therapies in a wide range of neurodegenerative disease models in rodents and non-human primates, including Parkinson's disease, stroke, epilepsy, spinal cord injury, Alzheimer's disease, multiple sclerosis and pain. In this review, we discuss the latest progress made with stem cell therapies targeting these pathologies. We also evaluate the challenges in clinical application of human pluripotent stem cell (hPSC)-based therapies including risk of oncogenesis and tumor formation, immune rejection and difficulty in regeneration of the heterogeneous cell types composing the central nervous system.

Human induced pluripotent stem cell-derived GABAergic interneuron transplants attenuate neuropathic pain.

Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here, we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated human induced pluripotent stem cell-derived GABAergic (iGABAergic) neurons provide significant, long-term, and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

UNLABELLED: : Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. SIGNIFICANCE: This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development.

A view of bivalent epigenetic marks in two human embryonic stem cell lines reveals a different cardiogenic potential.

Human embryonic stem (HUES) cells are derived from early individual embryos with unique genetic printing. However, how their epigenetic status might affect their potential to differentiate toward specific lineages remains a puzzling question. Using chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP-on-chip, the status of bivalent domains on gene promoters (ie, histone 3 on lysine 4 and histone 3 on lysine 27 trimethylation) was monitored for both undifferentiated and bone morphogenetic protein 2 (BMP2)-induced cardiac-committed cells. A marked difference in the epigenetic profile of HUES cell lines was observed and this was correlated to the pattern of gene expression induced by BMP2 as well as to their potential to generate cardiac progenitors and differentiated myocytes. Thus, the epigenetic H3trimeK4 and H3trimeK27 prints generating bivalent domains on promoters, could be used to predict a preference in their differentiation toward a specific lineage.

Wnt/ß-catenin signaling directs the regional expansion of first and second heart field-derived ventricular cardiomyocytes.

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to ß-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that ß-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/ß-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications.

Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.

The generation and expansion of diverse cardiovascular cell lineages is a critical step during human cardiogenesis, with major implications for congenital heart disease. Unravelling the mechanisms for the diversification of human heart cell lineages has been hampered by the lack of genetic tools to purify early cardiac progenitors and define their developmental potential. Recent studies in the mouse embryo have identified a multipotent cardiac progenitor that contributes to all of the major cell types in the murine heart. In contrast to murine development, human cardiogenesis has a much longer onset of heart cell lineage diversification and expansion, suggesting divergent pathways. Here we identify a diverse set of human fetal ISL1(+) cardiovascular progenitors that give rise to the cardiomyocyte, smooth muscle and endothelial cell lineages. Using two independent transgenic and gene-targeting approaches in human embryonic stem cell lines, we show that purified ISL1(+) primordial progenitors are capable of self-renewal and expansion before differentiation into the three major cell types in the heart. These results lay the foundation for the generation of human model systems for cardiovascular disease and novel approaches for human regenerative cardiovascular medicine.

Marked differences in differentiation propensity among human embryonic stem cell lines.

The differentiation potential of 17 human embryonic stem (hES) cell lines was compared. Some lines exhibit a marked propensity to differentiate into specific lineages, often with >100-fold differences in lineage-specific gene expression. For example, HUES 8 is best for pancreatic differentiation and HUES 3 for cardiomyocyte generation. These non-trivial differences in developmental potential among hES cell lines point to the importance of screening and deriving lines for lineage-specific differentiation.

Islet1 cardiovascular progenitors: a single source for heart lineages?

The creation of regenerative stem cell therapies for heart disease requires that we understand the molecular mechanisms that govern the fates and differentiation of the diverse muscle and non-muscle cell lineages of the heart. Recently, different cardiac cell types have been reported to arise from a common, multipotent Islet1 (Isl1)-positive progenitor, suggesting that a clonal model of heart lineage diversification might occur that is analogous to hematopoiesis. The ability to isolate, renew and differentiate Isl1(+) precursors from postnatal and embryonic hearts and from embryonic stem cells provides a powerful cell-based system for characterizing the signaling pathways that control cardiovascular progenitor formation, renewal, lineage specification and conversion to specific differentiated progeny.

Concise review: regulation of embryonic stem cell lineage commitment by mitogen-activated protein kinases.

Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen-activated protein kinase (MAPK) pathways extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed. Disclosure of potential conflicts of interest is found at the end of this article.

The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway.

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.

Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis.

Mouse embryonic stem (ES) cells can be differentiated, in vitro into a variety of cell types including cardiac cells and neurons. This process is strictly controlled by the potent morphogen retinoic acid (RA). At a concentration of 10(-7) M, RA induces ES cell differentiation into neurons and, conversely, inhibits cardiomyogenesis. We found that p38 mitogen-activated protein kinase (p38MAPK) activity peaked spontaneously, between day 3 and day 5, during ES cell differentiation and that RA completely inhibited this peak of activity. In contrast to wild-type cells, which required RA treatment, p38alpha(-/-) ES cells differentiated spontaneously into neurons and did not form cardiomyocytes. Moreover, inhibition of the peak of p38MAPK activity by a specific inhibitor, PD169316, committed ES cells into the neuronal lineage and blocked cardiomyogenesis. By genetic and biochemical approaches, we demonstrate that, in two different ES cell lines, the control of p38MAPK activity constitutes an early switch, committing ES cells into either neurogenesis (p38 off) or cardiomyogenesis (p38 on).

A new role for the oncogenic high-mobility group A2 transcription factor in myogenesis of embryonic stem cells.

The high mobility group type A-2 (HMGA 2) transcription factor is involved in proliferation and differentiation, mainly during embryogenesis. Its activated form (HMGA 2/T) presents oncogenic activities both in vivo and in vitro. However, its precise role during embryogenesis is unknown. We investigated its role during the commitment of mouse embryonic stem (ES) cells by constructing cell lines expressing either wild type (wt) or HMGA 2/T forms of the gene. Following differentiation, control and wt HMGA 2 ES cells did not display myotubes; whereas HMGA 2/T ES cell lines massively formed contractile myotubes. Furthermore, as opposed to control cells, HMGA 2/T ES cells highly expressed the muscle myosin heavy chain (MHC) marker. Interestingly, in experimental conditions inhibitory for myogenesis, we observed a strong expression of MyoD and myogenin in HMGA 2/T cells. By contrast, commitment into adipocyte, neuron, and cardiomyocyte lineages was not affected. Teratocarcinomas induced by HMGA 2/T ES cell lines presented numerous skeletal muscle-differentiated tissues that were not observed in wt HMGA 2 or control tumours. Finally, rapamycin, an inhibitor of the mTOR kinase, downregulated endogenous HMGA-2 expression and inhibited myogenesis. This effect was prevented by overexpression of exogenous HMGA-2. Our results reveal a novel function of HMGA-2 in skeletal muscle differentiation.

The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis.

Hyperplasia of adipose tissue is critical for the development of obesity, but molecular mechanisms governing normal or pathological recruitment of new adipocytes remain unclear. The extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in many essential cellular functions, such as proliferation and differentiation. Using ERK1(-/-) mice, we investigated the role of this isoform in adipose tissue development. Mice lacking ERK1 have decreased adiposity and fewer adipocytes than wild-type animals. Furthermore, ERK1(-/-) mice challenged with high-fat diet are resistant to obesity, are protected from insulin resistance, and have a higher postprandial metabolic rate. To get insights into cellular mechanisms implicated in reduced adiposity in ERK1(-/-) animals, we analyzed adipocyte differentiation in ERK1(-/-) cells. Compared with wild-type control cells, mouse embryo fibroblasts and cultures of adult preadipocytes isolated from ERK1(-/-) adult animals exhibit impaired adipogenesis. An inhibitor of the ERK pathway does not affect the residual adipogenesis of the ERK1(-/-) cells, suggesting that ERK2 is not implicated in adipocyte differentiation. Our results clearly link ERK1 to the regulation of adipocyte differentiation, adiposity, and high-fat diet-induced obesity. This suggests that a therapeutic approach of obesity targeting specifically the ERK1 isoform and not ERK2 would be of particular interest.

Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage.

Mouse embryonic stem (ES) cells are pluripotent cells that differentiate into multiple cell lineages. The commitment of ES cells into the adipocyte lineage is dependent on an early 3-day treatment with all-trans retinoic acid (RA). To characterize the molecular mechanisms underlying this process, we examined the contribution of the extracellular-signal-regulated kinase (ERK) pathway. Treatment of ES cell-derived embryoid bodies with RA resulted in a prolonged activation of the ERK pathway, but not the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase or phosphoinositide 3-kinase pathways. To investigate the role of ERK activation, co-treatment of RA with PD98059, a specific inhibitor of the ERK signalling pathway, prevented both adipocyte formation and expression of the adipogenic markers, adipocyte lipid-binding protein and peroxisome-proliferator-activated receptor gamma. Furthermore, we show that ERK activation is required only during RA treatment. PD98059 does not interfere with the commitment of ES cells into other lineages, such as neurogenesis, myogenesis and cardiomyogenesis. As opposed to the controversial role of the ERK pathway in terminal differentiation, our results clearly demonstrate that this pathway is specifically required at an early stage of adipogenesis, corresponding to the RA-dependent commitment of ES cells.

Bcl-2 inhibits glucocorticoid-induced apoptosis but only partially blocks calcium ionophore or cycloheximide-regulated apoptosis in S49 cells.

Many non-Hodgkins B-cell lymphomas possess a deregulated bcl-2 gene resulting in a phenotype that is apparently resistant to programmed cell death (apoptosis). We have used a mouse lymphoma cell line (S49.1) that undergoes apoptosis in response to a variety of stimuli to determine the effect of bcl-2 expression on induction of apoptosis. S49 cells were stably transfected with recombinant amphotrophic retroviruses carrying either a G418 antibiotic resistance gene alone (S49-NEO) or this gene in combination with a bcl-2 complementary DNA (S49-Bcl-2). Three different agents previously shown to activate apoptosis by different pathways in S49 cells (dexamethasone, the calcium ionophore A23187, and cycloheximide) were used to examine the effect of bcl-2 expression on cell growth and apoptosis caused by multiple signal transduction pathways. Dexamethasone (DEX) treatment inhibited cell growth and stimulated cell death in S49-NEO cells. Although S49-Bcl-2 cells exhibited a similar antiproliferative response, they failed to die in response to steroid treatment. Western blot analysis revealed no difference in the levels of glucocorticoid receptor protein in the two cell lines, and both responded to glucocorticoid with a profound inhibition of protein synthesis. Cycloheximide (CX) and A23187 also had antiproliferative and cell killing effects in both cell types, although higher concentrations of each agent were needed to kill S49-Bcl-2 cells. To determine whether the loss of viability in response to these drugs was due to apoptosis, cells were examined morphologically and DNA integrity was examined by gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)