Satellite Cell Transplantation Research Articles

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.

Enhanced Diaphragm Muscle Function upon Satellite Cell Transplantation in Dystrophic Mice.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Transplantation to study satellite cell heterogeneity in skeletal muscle.

Skeletal muscle has a remarkable capacity to regenerate throughout life, which is mediated by its resident muscle stem cells, also called satellite cells. Satellite cells, located periphery to the muscle fibers and underneath the basal lamina, are an indispensable cellular source for muscle regeneration. Satellite cell transplantation into regenerating muscle contributes robustly to muscle repair, thereby indicating that satellite cells indeed function as adult muscle stem cells. Moreover, satellite cells are a heterogenous population in adult tissue, with subpopulations that can be distinguished based on gene expression, cell-cycle progression, ability to self-renew, and bi-potential ability. Transplantation assays provide a powerful tool to better understand satellite cell function in vivo enabling the separation of functionally distinct satellite cell subpopulations. In this review, we focus on transplantation strategies to explore satellite cells’ functional heterogeneity, approaches targeting the recipient tissue to improve transplantation efficiency, and common strategies to monitor the behaviour of the transplanted cells. Lastly, we discuss some recent approaches to overcome challenges to enhance the transplantation potential of muscle stem cells.

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

SummaryGenetic 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.

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Featured characteristics and pivotal roles of satellite cells in skeletal muscle regeneration

Skeletal muscle, the essential organ for locomotion, as well as energy reservoir and expenditure, has robust regenerative capacity in response to mechanical stress and injury. As muscle-specific stem cells, satellite cells are responsible for providing new myoblasts during the process of muscle growth and regeneration. Self-renewal capacity and the fate of satellite cells are highly regulated and influenced by their surrounding factors, such as extracellular matrix and soluble proteins. The strong myogenic potential of satellite cells makes them a potential resource for stem cell therapy to cure genetic muscle disease and repair injured muscle. Here, we both review key features of satellite cells during skeletal muscle development and regeneration and summarize recent outcomes of satellite cell transplantation studies.

High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells.

SummaryInvestigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.

Mouse Satellite Cell Isolation and Transplantation.

Satellite cell (SC) transplantation represents a powerful strategy to investigate SC biology during muscle regeneration. We described here a protocol for SC isolation from green fluorescent protein (GFP)-expressing mice and their transplantation into murine muscles. This procedure was originally used to assess the effects of the hormone unacylated ghrelin on muscle regeneration, in particular evaluating how the increase of unacylated ghrelin in the recipient muscle affected the engraftment of donor SCs ( Reano et al., 2017 ).

Treatment of Vesicovaginal Fistulas With Autologous Cell Injections—A Randomized Study in an Animal Model

Purpose:To evaluate the effect of autologous satellite cell and smooth muscle cell transplantation on vesicovaginal fistulas in a randomized controlled study by comparing the proportion of fistula closure and tissue composition between the 2 groups.Materials and Methods:Eight pigs underwent surgery to establish an experimental induced fistula. One pig was excluded due to insufficient weight gain. Seven pigs were randomized to receive either an injection of satellite cells and smooth muscle cells or no treatment. Cells were isolated from abdominal skeletal muscle and bladder smooth muscle, cultured in vitro, and marked with bromodeoxyuridine. Cell purity, growth kinetics, and differentiation potential were determined. Cells were injected around the fistula using a cystoscope. Five weeks after the injection of cells, the fistula area was examined macroscopically and histologically.Results:The isolated satellite cell population consisted of 76.6% neural cell adhesion molecule positive cell and the smooth muscle cell population of 88.6% α-smooth muscle actin positive cells. All pigs had a persisting fistula before treatment. One of the 4 vesicovaginal fistulas was closed 5 weeks after injection of satellite cells and smooth muscle cells compared to none in the control group. No histological differences in the tissue composition between the 2 groups were observed and no bromodeoxyuridine-labeled cells were detected around the fistula tissue in the histological examination.Conclusion:We established an animal model with a persisting clinical and histological confirmed vesicovaginal fistula. The study indicated that there might be an effect on vesicovaginal fistula closure using an injection of satellite cells/smooth muscle cells and alginate gel.

Transplantation of Skeletal Muscle Stem Cells.

Transplanting adult stem cells provides a stringent test for self-renewal and the assessment of comparative engraftment in competitive transplant assays. Transplantation of satellite cells into mammalian skeletal muscle provided the first critical evidence that satellite cells function as adult muscle stem cells. Transplantation of a single satellite cell confirmed and extended this hypothesis, providing proof that the satellite cell is a bona fide adult skeletal muscle stem cell as reported by Sacco et al. (Nature 456(7221):502-506). Satellite cell transplantation has been further leveraged to identify culture conditions that maintain engraftment and to identify self-renewal deficits in satellite cells from aged mice. Conversion of iPSCs (induced pluripotent stem cells) to a satellite cell-like state, followed by transplantation, demonstrated that these cells possess adult muscle stem cell properties as reported by Darabi et al. (Stem Cell Rev Rep 7(4):948-957) and Mizuno et al. (FASEB J 24(7):2245-2253). Thus, transplantation strategies involving either satellite cells derived from adult muscles or derived from iPSCs may eventually be exploited as a therapy for treating patients with diseased or failing skeletal muscle. Here, we describe methods for isolating dispersed adult mouse satellite cells and satellite cells on intact myofibers for transplantation into recipient mice to study muscle stem cell function and behavior following engraftment .

Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor.

Background and Objectives: Cell transplantation is a promising therapy for several muscle diseases, including Duchenne muscular dystrophy. Satellite cells are stem cells in skeletal muscle that provide an important cell source for transplantation therapy. However, culture of satellite cells in vitro causes them to lose their undifferentiated state, associated with reduced transplantation efficiency. It is therefore necessary to develop optimal culture conditions for maintaining the undifferentiated state of satellite cells.Methods: Primary satellite cells were cultured with or without leukemia inhibitory factor (LIF). The expression of undifferentiation and differentiation markers, and the transplantation efficiency were analyzed.Results: LIF-treated satellite cells showed increased expression of Pax7, and enhanced transplantation efficiency in a mouse model of Duchenne muscular dystrophy.Conclusions: Our study showed that the treatment with LIF effectively maintained the undifferentiated state of satellite cells, and enhanced their transplantation efficiency. These results will contribute to the optimization of culture conditions for cell transplantation therapy.

The ins and outs of muscle stem cell aging.

Skeletal muscle has a remarkable capacity to regenerate by virtue of its resident stem cells (satellite cells). This capacity declines with aging, although whether this is due to extrinsic changes in the environment and/or to cell-intrinsic mechanisms associated to aging has been a matter of intense debate. Furthermore, while some groups support that satellite cell aging is reversible by a youthful environment, others support cell-autonomous irreversible changes, even in the presence of youthful factors. Indeed, whereas the parabiosis paradigm has unveiled the environment as responsible for the satellite cell functional decline, satellite cell transplantation studies support cell-intrinsic deficits with aging. In this review, we try to shed light on the potential causes underlying these discrepancies. We propose that the experimental paradigm used to interrogate intrinsic and extrinsic regulation of stem cell function may be a part of the problem. The assays deployed are not equivalent and may overburden specific cellular regulatory processes and thus probe different aspects of satellite cell properties. Finally, distinct subsets of satellite cells may be under different modes of molecular control and mobilized preferentially in one paradigm than in the other. A better understanding of how satellite cells molecularly adapt during aging and their context-dependent deployment during injury and transplantation will lead to the development of efficacious compensating strategies that maintain stem cell fitness and tissue homeostasis throughout life.

The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.

Skeletal Muscle Stem Cells: Effects of Aging and Metabolism on Muscle Regenerative Function

Homeostatic and regenerative replacement of skeletal muscle fibers requires the activity of a dedicated pool of myogenic stem cells, called satellite cells, that are activated by muscle injury and act as a renewable source of muscle-forming cells throughout adult life. Satellite cell function is controlled by both intrinsic and extrinsic regulatory cues, whose integration determines the success of muscle regenerative responses. Pathological deregulation of satellite cell function through perturbation of these signaling pathways appears to play an important role in age-dependent deterioration of muscle function and in muscle dystrophic disease. The regenerative activity of skeletal muscle also appears to be tightly linked to metabolism, and alterations in metabolic state can directly influence the activity of these tissue-specific stem cells. Here, we review recent and emerging insights into the molecular and biochemical signals that control satellite cell function and discuss these in the context of muscle degenerative diseases such as dystrophy and sarcopenia. Novel discoveries from this ongoing work bring new opportunities to enhance or restore muscle repair and are likely to facilitate satellite cell transplantation in clinical applications.

Prevention of Muscle Aging by Myofiber-Associated Satellite Cell Transplantation

Skeletal muscle is dynamic, adapting to environmental needs, continuously maintained, and capable of extensive regeneration. These hallmarks diminish with age, resulting in a loss of muscle mass, reduced regenerative capacity, and decreased functionality. Although the mechanisms responsible for this decline are unclear, complex changes within the local and systemic environment that lead to a reduction in regenerative capacity of skeletal muscle stem cells, termed satellite cells, are believed to be responsible. We demonstrate that engraftment of myofiber-associated satellite cells, coupled with an induced muscle injury, markedly alters the environment of young adult host muscle, eliciting a near-lifelong enhancement in muscle mass, stem cell number, and force generation. The abrogation of age-related atrophy appears to arise from an increased regenerative capacity of the donor stem cells, which expand to occupy both myonuclei in myofibers and the satellite cell niche. Further, these cells have extensive self-renewal capabilities, as demonstrated by serial transplantation. These near-lifelong, physiological changes suggest an approach for the amelioration of muscle atrophy and diminished function that arise with aging through myofiber-associated satellite cell transplantation.

Autologus satellite cell transplantation into the striated sphincter in exstrophy-epispadias children: an evolving modality in incontinence management?

Purpose Incontinence is still a challenging problem in children with the exstrophy-epispadias complex (EEC) and satisfactory results using conventional management has not yet been achieved. Recent advances in cell therapy and tissue engineering techniques have been promising. We investigated autologous, heterotopic targeted muscle precursor cell donation in the recovery of external urethral sphincter function in children with the EEC and urinary incontinence (UI). Material and methods Following extensive animal model studies at our laboratory and approval of the study protocol by the local bioethical committee, 5 children (4 boys and 1 girl, mean age: 9.6 years) with severe urinary incontinence due to exstrophy/epispadias were enrolled in May 2006. Autologous skeletal muscle precursor cells were obtained from the pelvic floor and cultured in vitro for cell expansion. Before transurethral injection of the cells into the sphincter, microbiological control and karyotyping were performed. The continence status of the patients prior to and following satellite injection was evaluated according to incontinence score, biochemical parameters, urodynamic studies and changes in quality of life. Results An average of 0.2-8x10 7 cells (>70% Pax7+/ MyoD+/ Desmin+) were obtained after a mean period of 18 days and implanted uneventfully. Neither microbial infection nor chromosomal change was detected in the specimens prior to cell implantation. During 4 months follow-up the incontinence score improved in 4 patients but 1 female patient with a very short urethral length and fibrosis had no change in continence status. Conclusions This data confirms the potential application and outcome of satellite cell transplantation in functional reconstruction in UI by compensating for the cell loss. Although it can provide a new therapeutic approach for incontinence therapy with a reasonable success rate, long-term follow-up is still required.

Transplantation of Adipose Tissue-Derived Stromal Cells Increases Mass and Functional Capacity of Damaged Skeletal Muscle

The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, β-galactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

Long-term cell survival and hemodynamic improvements after neonatal cardiomyocyte and satellite cell transplantation into healed myocardial cryoinfarcted lesions in rats.

Cell engraftment is a new strategy for the repair of ischemic myocardial lesions. The hemodynamic effectiveness of this strategy, however, is not completely elucidated yet. In a rat model of cryothermia-induced myocardial dysfunction, we investigated whether syngeneic transplantation of neonatal cardiomyocytes or satellite cells is able to improve left ventricular performance. Myocardial infarction was induced in female Lewis rats by a standardized cryolesion to the obtuse margin of the left ventricle. After 4 weeks, 5 x 10(6) genetically male neonatal cardiomyocytes (n = 16) or satellite cells (n = 16) were engrafted into the myocardial scar. Sham-transplanted animals (n = 15) received injections with cell-free medium. Sham-operated animals (n = 15) served as controls. Left ventricular performance was analyzed 4 months after cell engraftment. Chimerism after this sex-mismatched transplantation was evaluated by detection of PCR-amplified DNA of the Y chromosome. The average heart weight of the infarcted animals significantly exceeded that of controls (p < 0.05). In sham-transplanted animals, mean aortic pressure, left ventricular systolic pressure, aortic flow (indicator of cardiac output), and left ventricular systolic reserve were significantly lower (p < 0.05) compared with sham-operated controls. This was associated with deterioration of ventricular diastolic function (maximal negative dP/dt, time constants of isovolumic relaxation; p < 0.05). Transplantation of satellite cells was found more effective than transplantation of neonatal cardiomyocytes, resulting in i) normalization of mean aortic pressure compared with sham-operated controls, and ii) significantly improved left ventricular systolic pressure and aortic flow (p < 0.05) compared with sham-transplanted animals. Left ventricular systolic reserve and diastolic function, however, were improved by neither satellite cell nor neonatal cardiomyocyte transplantation. Analysis of male genomic DNA revealed 3.98 +/- 2.70 ng in hearts after neonatal cardiomyocyte engraftment and 6.16 +/- 4.05 ng in hearts after satellite cell engraftment, representing approximately 10(3) viable engrafted cells per heart. Our study demonstrates i) long-term survival of both neonatal cardiomyocytes and satellite cells after transplantation into cryoinfarcted rat hearts, ii) slight superiority of satellite cells over neonatal cardiomyocytes in improving global left ventricular pump performance, and iii) no effect of both transplant procedures on diastolic dysfunction.