Role of epigenetics in cellular reprogramming; from iPSCs to disease modeling and cell therapy.

Epigenetics play a fundamental role in induced pluripotent stem cell (iPSC) technology due to their effect on iPSC's reprogramming efficiency and their subsequent role in iPSC differentiation toward a specific lineage. Epigenetics can skew the differentiation course of iPSCs toward a specific lineage based on the epigenetic memory of the source cells, or even lead to acquisition of new cell phenotypes, due to its aberrations during reprogramming. This viewpoint discusses key features of the epigenetic process during iPSC reprogramming/differentiation and outlines important epigenetic factors that need to be considered for successful generation and differentiation of iPSCs for downstream applications.

Evaluation of the Therapeutic Potential of Human iPSCs in a Murine Model of VML.

Volumetric muscle loss injury is a common health problem with long-term disabilities. One common treatment is using muscle flaps from donor site, which has limited potentials due to donor site availability and morbidity. Although several stem cell therapies have been evaluated so far, most suffer from limited availability, immune incompatibility, or differentiation potential. Therefore, induced pluripotent stem cells (iPSCs) have a great promise for this purpose due to their unique differentiation, self-renewal, and immunocompatibility. Current study was designed to determine therapeutic potential of human iPSCs (hiPSCs) in a mouse model of volumetric muscle loss. Muscles were subjected to excision to generate 30%-40% muscle loss. Next, hiPSCs were differentiated toward skeletal myogenic progenitors and used with fibrin hydrogel to reconstruct the lost muscle. Histologic evaluation of the treated muscles indicated abundant engraftment of donor-derived mature fibers expressing human markers. Donor-derived fibers were also positive for the presence of neuromuscular junction (NMJ), indicating their proper innervation. Evaluation of the engrafted region indicated the presence of donor-derived satellite cells expressing human markers and Pax7. Finally, in situ muscle function analysis demonstrated significant improvement of the muscle contractility in muscles treated with hiPSCs. These results therefore provide key evidence for the therapeutic potential of human iPSCs in volumetric muscle loss injuries.

Pluripotent stem cell-derived myogenic progenitors remodel their molecular signature upon in vivo engraftment.

Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7-induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7-induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment.

Stem cell therapies for COVID-19: Strategy and application.

In this perspective, the potential application of stem cells for the treatment of COVID-19 related pneumonia and their potential mechanism of action have been overviewed.

CRISPR/Cas9-Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors.

Although the lack of dystrophin expression in muscle myofibers is the central cause of Duchenne muscular dystrophy (DMD), accumulating evidence suggests that DMD may also be a stem cell disease. Recent studies have revealed dystrophin expression in satellite cells and demonstrated that dystrophin deficiency is directly related to abnormalities in satellite cell polarity, asymmetric division, and epigenetic regulation, thus contributing to the manifestation of the DMD phenotype. Although metabolic and mitochondrial dysfunctions have also been associated with the DMD pathophysiology profile, interestingly, the role of dystrophin with respect to stem cells dysfunction has not been elucidated. In the past few years, editing of the gene that encodes dystrophin has emerged as a promising therapeutic approach for DMD, although the effects of dystrophin restoration in stem cells have not been addressed. Herein, we describe our use of a clustered regularly interspaced short palindromic repeats/Cas9-based system to correct the dystrophin mutation in dystrophic (mdx) muscle progenitor cells (MPCs) and show that the expression of dystrophin significantly improved cellular properties of the mdx MPCs in vitro. Our findings reveal that dystrophin-restored mdx MPCs demonstrated improvements in cell proliferation, differentiation, bioenergetics, and resistance to oxidative and endoplasmic reticulum stress. Furthermore, our in vivo studies demonstrated improved transplantation efficiency of the corrected MPCs in the muscles of mdx mice. Our results indicate that changes in cellular energetics and stress resistance via dystrophin restoration enhance muscle progenitor cell function, further validating that dystrophin plays a role in stem cell function and demonstrating the potential for new therapeutic approaches for DMD. Stem Cells 2019;37:1615-1628.

Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells.

Many obstacles compromise the efficacy of bone marrow mesenchymal stem cells (BM-MSCs) by inducing apoptosis in the grafted BM-MSCs. The current study investigates the effect of melatonin on important mediators involved in survival of BM-MSCs in hydrogen peroxide (H2O2) apoptosis model. In brief, BM-MSCs were isolated, treated with melatonin, and then exposed to H2O2. Their viability was assessed by MTT assay and apoptotic fractions were evaluated through Annexin V, Hoechst staining, and ADP/ATP ratio. Oxidative stress biomarkers including ROS, total antioxidant power (TAP), superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH), thiol molecules, and lipid peroxidation (LPO) levels were determined. Secretion of inflammatory cytokines (TNF-α and IL-6) were measured by ELISA assay. The protein expression of caspase-3, Bax, and Bcl-2, was also evaluated by Western blotting. Melatonin pretreatment significantly increased viability and decreased apoptotic fraction of H2O2-exposed BM-MSCs. Melatonin also decreased ROS generation, as well as increasing the activity of SOD and CAT enzymes and GSH content. Secretion of inflammatory cytokines in H2O2-exposed cells was also reduced by melatonin. Expression of caspase-3 and Bax proteins in H2O2-exposed cells was diminished by melatonin pretreatment. The findings suggest that melatonin may be an effective protective agent against H2O2-induced oxidative stress and apoptosis in MSC.

Physiological and ultrastructural features of human induced pluripotent and embryonic stem cell-derived skeletal myocytes in vitro.

Progress has recently been made toward the production of human skeletal muscle cells from induced pluripotent stem (iPS) cells. However, the functional and ultrastructural characterization, which is crucial for disease modeling and drug discovery, remains to be documented. We show, for the first time to our knowledge, that the electrophysiological properties of human iPS-derived skeletal myocytes are strictly similar to those of their embryonic stem (ES) cell counterparts, and both are typical of aneural mammalian skeletal muscle. In both cell types, intracellular calcium signaling that links membrane depolarization to contraction occurs in the absence of extracellular Ca(2+), a unique feature of skeletal muscle. Detailed analysis of the Ca(2+) signal revealed diverse kinetics of the rising phase, and hence various rates in the release of Ca(2+) from the sarcoplasmic reticulum. This was mirrored by ultrastructural evidence of Ca(2+) release units, which varied in location, shape, and size. Thus, the excitation-contraction coupling machinery of both iPS- and ES-derived skeletal myocytes was functional and specific, but did not reach full maturity in culture. This is in contrast with the myofibrillar network, which displayed the same organization as in adult skeletal muscle. Overall, the present study validates the human iPS-based skeletal myocyte model in comparison with the embryonic system, and provides the functional and ultrastructural basis for its application to human skeletal muscle diseases.

Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of articular cartilage. As chondrocytes are the only cell type forming the articular cartilage, their gradual loss is the main cause of OA. There is a substantial body of published research that suggests reactive oxygen species (ROS) are major causative factors for chondrocyte damage and OA development. Oxidative stress elicited by ROS is capable of oxidizing and subsequently disrupting cartilage homeostasis, promoting catabolism via induction of cell death and damaging numerous components of the joint. IL-1ß and TNF-α are crucial inflammatory factors that play pivotal roles in the pathogenesis of OA. In this process, the mitochondria are the major source of ROS production in cells, suggesting a role of mitochondrial dysfunction in this type of arthritis. This may also be promoted by inflammatory cytokines such as IL-1ß and TNF-α which contribute to chondrocyte death. In patients with OA, the expression of endoplasmic reticulum (ER) stress-associated molecules is positively correlated with cartilage degeneration. Melatonin and its metabolites are broad-spectrum antioxidants and free radical scavengers which regulate a variety of molecular pathways such as inflammation, proliferation, apoptosis, and metastasis in different pathophysiological situations. Herein, we review the effects of melatonin on OA, focusing on its ability to regulate apoptotic processes and ER and mitochondrial activity. We also evaluate likely protective effects of melatonin on OA pathogenesis.

Skeletal muscle perfusion and stem cell delivery in muscle disorders using intra-femoral artery canulation in mice.

Muscular dystrophies are among major inherited muscle disorders characterized by progressive muscle damage and fibrosis with no definitive cure. Recently, gene or cell based therapies have been developed to restore the missing gene expression or replace the damaged tissues. In order to test the efficiency of these therapies in mice models of muscular dystrophies, the arterial route of delivery is very advantageous as it provides uniform muscle exposure to the therapeutic agents or cells. Although there are few reports of arterial delivery of the therapeutic agents or cells in mice, there is no in-depth description and evaluation of its efficacy in perfusion of downstream muscles. This study is aimed to develop a practical method for intra-femoral artery perfusion in mice and to evaluate perfusion efficiency using near-infrared-fluorescence (NIRF) imaging as well as histology following stem cell delivery. Our results provide a practical guide to perform this delicate method in mice. By using a sensitive fluorescent dye, different muscle groups of the hindlimb have been evaluated for proper perfusion. As the final step, we have validated the efficiency of arterial cell delivery into muscles using human iPS-derived myogenic cells in an immunodeficient mouse model for Duchenne muscular dystrophy (NSG-mdx(4cv)).

A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.

Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice, we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx(4Cv) mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx(4Cv) mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx(4Cv) recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function and the utility of the NSG-mdx(4Cv) model for studies on muscle regeneration and Duchenne muscular dystrophy therapy.

Disease modeling and gene correction of LGMDR21 iPSCs elucidates the role of POGLUT1 in skeletal muscle maintenance, regeneration, and the satellite cell niche.

Autosomal recessive limb-girdle muscular dystrophy 21 (LGMDR21) is caused by pathogenic variants in protein O-glucosyltransferase 1 (POGLUT1), which is responsible for O-glucosylation of specific epidermal growth factor (EGF) repeats found in ∼50 mammalian proteins, including Notch receptors. Previous data from patient biopsies indicated that impaired Notch signaling, reduction of muscle stem cells, and accelerated differentiation are probably involved in disease etiopathology. Using patient induced pluripotent stem cells (iPSCs), their corrected isotypes, and control iPSCs, gene expression profiling indicated dysregulation of POGLUT1, NOTCH, muscle development, extracellular matrix (ECM), cell adhesion, and migration as involved pathways. They also exhibited reduced in vitro POGLUT1 enzymatic activity and NOTCH signaling as well as defective myogenesis, proliferation, migration and differentiation. Furthermore, in vivo studies demonstrated significant reductions in engraftment, muscle stem cell formation, PAX7 expression, and maintenance, along with an increased percentage of mislocalized PAX7+ cells in the interstitial space. Gene correction in patient iPSCs using CRISPR-Cas9 nickase led to the rescue of the main in vitro and in vivo phenotypes. These results demonstrate the efficacy of iPSCs and gene correction in disease modeling and rescue of the phenotypes and provide evidence of the involvement of muscle stem cell niche localization, PAX7 expression, and cell migration as possible mechanisms in LGMDR21.

Assessment of the myogenic stem cell compartment following transplantation of Pax3/Pax7-induced embryonic stem cell-derived progenitors.

An effective long-term cell therapy for skeletal muscle regeneration requires donor contribution to both muscle fibers and the muscle stem cell pool. Although satellite cells have these abilities, their therapeutic potential so far has been limited due to their scarcity in adult muscle. Myogenic progenitors obtained from Pax3-engineered mouse embryonic stem (ES) cells have the ability to generate myofibers and to improve the contractility of transplanted muscles in vivo, however, whether these cells contribute to the muscle stem cell pool and are able to self-renew in vivo are still unknown. Here, we addressed this question by investigating the ability of Pax3, which plays a critical role in embryonic muscle formation, and Pax7, which is important for maintenance of the muscle satellite cell pool, to promote the derivation of self-renewing functional myogenic progenitors from ES cells. We show that Pax7, like Pax3, can drive the expansion of an ES-derived myogenic progenitor with significant muscle regenerative potential. We further demonstrate that a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to reinjury, and contribution to muscle regeneration in secondary transplantation assays. The ability to engraft, self-renew, and respond to injury provide foundation for the future therapeutic application of ES-derived myogenic progenitors in muscle disorders.

Clonal analysis reveals a common progenitor for endothelial, myeloid, and lymphoid precursors in umbilical cord blood.

RATIONALE: several studies demonstrate that hematopoietic tissues are a source of endothelial progenitor cells, which contribute to newly formed blood vessels during tissue repair in adults. However, it is not clear which cell type in these hematopoietic tissues gives rise to endothelial progenitor cells. OBJECTIVE: to identity the origin of endothelial progenitors within the hematopoietic hierarchy and to assess their in vivo revascularization potential. METHODS AND RESULTS: using a single-cell sorting approach and in vitro multilineage differentiation assays, here we show that individual CD34(+)CD45(+)CD133(+)CD38(+) cells from cord blood uniquely have the ability to differentiate into T- and B-lymphoid, myeloid, and endothelial cells. The latter were characterized by the expression of VE-cadherin, KDR, von Willebrand factor, endothelial nitric oxide synthase, the lack of CD45, CD133, and c-fms (colony stimulating factor-1 receptor). Unexpectedly when transplanted into hindlimb ischemic NOD-scid IL2Rγ(null) mice, freshly isolated CD34(+)CD45(+)CD133(+)CD38(+) cells maintained their hematopoietic identity and were rarely found to integrate into host blood vessels. Nevertheless, they significantly improved perfusion, most likely through a paracrine mechanism. On the other hand, CD34(+)CD45(+)CD133(+)CD38(+) cells differentiated in vitro into endothelial cells were able to form vessels in vivo in both Matrigel plug and hindlimb ischemia transplantation assays. CONCLUSIONS: these findings indicate that the CD34(+)CD45(+)CD133(+)CD38(+) cell fraction contains a common progenitor for the hematopoietic and vascular lineages and may represent a valuable cell source for therapeutic applications.

Hormonal therapies up-regulate MANF and overcome female susceptibility to immune checkpoint inhibitor myocarditis.

Immune checkpoint inhibitors (ICIs) have been increasingly used in combination for cancer treatment but are associated with myocarditis. Here, we report that tumor-bearing mice exhibited response to treatment with combinatorial anti-programmed cell death 1 and anti-cytotoxic T lymphocyte antigen-4 antibodies but also presented with cardiovascular toxicities observed clinically with ICI therapy, including myocarditis and arrhythmia. Female mice were preferentially affected with myocarditis compared to male mice, consistent with a previously described genetic model of ICI myocarditis and emerging clinical data. Mechanistically, myocardial tissue from ICI-treated mice, the genetic mouse model, and human heart tissue from affected patients with ICI myocarditis all exhibited down-regulation of MANF (mesencephalic astrocyte-derived neurotrophic factor) and HSPA5 (heat shock 70-kDa protein 5) in the heart; this down-regulation was particularly notable in female mice. ICI myocarditis was amplified by heart-specific genetic deletion of mouse Manf and was attenuated by administration of recombinant MANF protein, suggesting a causal role. Ironically, both MANF and HSPA5 were transcriptionally induced by liganded estrogen receptor ß and inhibited by androgen receptor. However, ICI treatment reduced serum estradiol concentration to a greater extent in female compared to male mice. Treatment with an estrogen receptor ß-specific agonist and androgen depletion therapy attenuated ICI-associated cardiac effects. Together, our data suggest that ICI treatment inhibits estradiol-dependent expression of MANF/HSPA5 in the heart, curtailing the cardiomyocyte response to immune injury. This endocrine-cardiac-immune pathway offers new insights into the mechanisms of sex differences in cardiac disease and may offer treatment strategies for ICI myocarditis.

Hypoxia in Cell Reprogramming and the Epigenetic Regulations.

Cellular reprogramming is a fundamental topic in the research of stem cells and molecular biology. It is widely investigated and its understanding is crucial for learning about different aspects of development such as cell proliferation, determination of cell fate and stem cell renewal. Other factors involved during development include hypoxia and epigenetics, which play major roles in the development of tissues and organs. This review will discuss the involvement of hypoxia and epigenetics in the regulation of cellular reprogramming and how interplay between each factor can contribute to different cellular functions as well as tissue regeneration.

Directed Differentiation of Human Pluripotent Stem Cells toward Skeletal Myogenic Progenitors and Their Purification Using Surface Markers.

Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45-65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24-). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.

Engraftment of mesenchymal stem cells into dystrophin-deficient mice is not accompanied by functional recovery.

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.

Isolation, culture, characterization and optimization of human corneal stem cells.

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85 +/- 4.6%), and a correspondingly low proportion K3 positive cells (15 +/- 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean +/- SE, n=10). Cultures reached full confluency after 17.3 +/- 1.2 days when the medium was supplemented with human EGF, while 21.7 +/- 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.

The lncRNA H19 alleviates muscular dystrophy by stabilizing dystrophin.

Dystrophin proteomic regulation in muscular dystrophies (MDs) remains unclear. We report that a long noncoding RNA (lncRNA), H19, associates with dystrophin and inhibits E3-ligase-dependent polyubiquitination at Lys 3584 (referred to as Ub-DMD) and its subsequent protein degradation. In-frame deletions in BMD and a DMD non-silent mutation (C3340Y) resulted in defects in the ability of the protein to interact with H19, which caused elevated Ub-DMD levels and dystrophin degradation. Dmd C3333Y mice exhibited progressive MD, elevated serum creatine kinase, heart dilation, blood vessel irregularity and respiratory failure with concurrently reduced dystrophin and increased Ub-DMD status. H19 RNA oligonucleotides conjugated with agrin (AGR-H19) and nifenazone competed with or inhibited TRIM63. Dmd C3333Y animals, induced-pluripotent-stem-cell-derived skeletal muscle cells from patients with Becker MD and mdx mice subjected to exon skipping exhibited inhibited dystrophin degradation, preserved skeletal and cardiac muscle histology, and improved strength and heart function following AGR-H19 or nifenazone treatment. Our study paves the way for meaningful targeted therapeutics for Becker MD and for certain patients with Duchenne MD.

iPSCs as a Platform for Disease Modeling, Drug Screening, and Personalized Therapy in Muscular Dystrophies.

Induced pluripotent stem cells (iPSCs) are the foundation of modern stem cell-based regenerative medicine, especially in the case of degenerative disorders, such as muscular dystrophies (MDs). Since their introduction in 2006, many studies have used iPSCs for disease modeling and identification of involved mechanisms, drug screening, as well as gene correction studies. In the case of muscular dystrophies, these studies commenced in 2008 and continue to address important issues, such as defining the main pathologic mechanisms in different types of MDs, drug screening to improve skeletal/cardiac muscle cell survival and to slow down disease progression, and evaluation of the efficiency of different gene correction approaches, such as exon skipping, Transcription activator-like effector nucleases (TALENs), Zinc finger nucleases (ZFNs) and RNA-guided endonuclease Cas9 (CRISPR/Cas9). In the current short review, we have summarized chronological progress of these studies and their key findings along with a perspective on the future road to successful iPSC-based cell therapy for MDs and the potential hurdles in this field.