Decellularized extracellular matrices derived from cultured cells at stepwise myogenic stages for the regulation of myotube formation

The regulation of stem cell differentiation is key for muscle tissue engineering and regenerative medicine. To this end, various substrates mimicking the native extracellular matrix (ECM) have been developed with consideration of the mechanical, topological, and biochemical properties. However, mimicking the biochemical properties of the native ECM is difficult due to its compositional complexity. To develop substrates that mimic the native ECM and its biochemical properties, decellularization is typically used. Here, substrates mimicking the native ECM at each myogenic stage are prepared as stepwise myogenesis-mimicking matrices via the in vitro myogenic culture of C2C12 myoblasts and decellularization. Cells adhered to the stepwise myogenesis-mimicking matrices at similar levels. However, the matrices derived from cells at the myogenic early stage suppressed cell growth and promoted myogenesis. This promotion of myogenesis was potentially due to the suppression of the activation of endogenous BMP signaling following the suppression of the expression of the myogenic-inhibitory factors, Id2 and Id3. Our stepwise myogenesis-mimicking matrices will be suitable ECM models for basic biological research and myogenesis of stem cells. Further, these matrices will provide insights that improve the efficacy of decellularized ECM for muscle repair.     

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Skeletal myogenesis by human primordial germ cell-derived progenitors

We have isolated and cultured human primordial germ cells (PGCs) from early embryos. The PGCs expressed embryonic germ (EG) cell-specific surface markers, including Oct4 and Nanos. We derived a cell population from these PGCs that we termed embryoid body-derived (EBD) cells. EBD cells can be extensively expanded in vitro for more than 50 passages and express lineage markers from all three primary germ layers. The myogenic potential of the EBD cells was examined both in vitro and in vivo.In vitro, the EBD cells can be induced to form multinucleated myotubes, which express late skeletal muscle-specific markers, including MHC and dystrophin, when exposed to human galectin-1. In vivo, the EBD cells gave rise to all the myogenic lineages, including the skeletal muscle stem cells known as satellite cells. Strikingly, these cells were able to partially restore degenerated muscles in the SCID/mdx mouse, an animal model of the Duchenne’s muscular dystrophy. These results indicate the EBD cells may be a promising source of myogenic stem cells for cell-based therapies for muscle degenerative disorders.     

The production of fluorescent transgenic trout to study <Emphasis Type="Italic">in vitro</Emphasis> myogenic cell differentiation

Background  

Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.     

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4^Cre and Mrf4^nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Masked mRNA is stored with aggregated nuclear speckles and its asymmetric redistribution requires a homolog of mago nashi

Background

The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors.

Results

Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation.

Conclusion

Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.     

Adult stem cells derived from skeletal muscle — biology and potential

Skeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.     

Effects of trypsinization and of a combined trypsin,collagenase, and DNase digestion on liberation and <Emphasis Type="Italic">in vitro</Emphasis> function of satellite cells isolated from juvenile porcine muscles

Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p?=?0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.     

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

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.     

Loss of RARRES1 function Promotes Follicular Lymphomagenesis and Inhibits B cell Differentiation in Mice

Retinoic acid receptor responder 1 (RARRES1) is among the most commonly methylated loci in multiple cancers. RARRES1 regulates mitochondrial and fatty acid metabolism, stem cell differentiation, and survival of immortalized cell lines in vitro. Here, we created constitutive Rarres1 knockout (Rarres1^-/-) mouse models to study RARRES1 function in vivo. Rarres1^-/- embryonic fibroblasts regulated tubulin glutamylation, cell metabolism, and survival, recapitulating RARRES1 function in immortalized cell lines. In two mouse strains, loss of Rarres1 led to a markedly increased dose-dependent incidence of follicular lymphoma (FL). Prior to lymphoma formation, Rarres1^-/- B cells have compromised activation, maturation, differentiation into antibody-secreting plasma cells, and cell cycle progression. Rarres1 ablation increased B cell survival and led to activation of the unfolded protein response (UPR) and heat shock response (HSR). Rarres1 deficiency had differential effects on cellular metabolism, with increased bioenergetic capacity in fibroblasts, and minor effects on bioenergetics and metabolism in B cells. These findings reveal that RARRES1 is a bona fide tumor suppressor in vivo and the deletion in mice promotes cell survival, and reduces B cell differentiation with B cell autonomous and non-autonomous functions.     

Notch signaling regulates myogenic regenerative capacity of murine and human mesoangioblasts

Somatic stem cells hold attractive potential for the treatment of muscular dystrophies (MDs). Mesoangioblasts (MABs) constitute a myogenic subset of muscle pericytes and have been shown to efficiently regenerate dystrophic muscles in mice and dogs. In addition, HLA-matched MABs are currently being tested in a phase 1 clinical study on Duchenne MD patients (EudraCT #2011-000176-33). Many reports indicate that the Notch pathway regulates muscle regeneration and satellite cell commitment. However, little is known about Notch-mediated effects on other resident myogenic cells. To possibly potentiate MAB-driven regeneration in vivo, we asked whether Notch signaling played a pivotal role in regulating MAB myogenic capacity. Through different approaches of loss- and gain-of-function in murine and human MABs, we determined that the interplay between Delta-like ligand 1 (Dll1)-activated Notch1 and Mef2C supports MAB commitment in vitro and ameliorates engraftment and functional outcome after intra-arterial delivery in dystrophic mice. Furthermore, using a transgenic mouse model of conditional Dll1 deletion, we demonstrated that Dll1 ablation, either on the injected cells, or on the receiving muscle fibers, impairs MAB regenerative potential. Our data corroborate the perspective of advanced combinations of cell therapy and signaling tuning to enhance therapeutic efficaciousness of somatic stem cells.Notch signaling consists of a conserved pathway, triggered by physical interaction between one ligand and one receptor, both transmembrane proteins exposed by contacting cells.¹ Notch signaling has been involved in different stages of muscle formation² and regeneration.^3,4 The canonical signaling encompasses five ligands (Dll1/3/4, Jagged1/2) and four receptors (Notch1–4); however, the axis Dll1-Notch1 appears consistently involved during myogenic fate specification, for example, neural crest-driven somite maturation.⁵ Moreover, murine embryos expressing a hypomorphic allele of the Notch ligand Dll1 displayed marked impairment of skeletal muscle formation.⁶ Interestingly, the Notch pathway may exert different effects according to the cell context. Culture on DLL1-coated plastic improved ex vivo proliferation and in vivo engraftment of canine satellite cells.⁷ Expression of the active Notch1 intracellular domain (NICD) robustly committed murine and rat mesenchymal stem cells toward the myogenic fate both in vitro and in vivo.⁸ However, Notch-mediated effects on the regenerative potential of non-satellite resident myogenic cells are still unknown.Mesoangioblasts (MABs) are non-satellite resident myogenic stem cells, able to circulate and regenerate dystrophic skeletal muscles.^9,10 HLA-matched MABs are currently under phase 1 clinical study on Duchenne muscular dystrophy patients (EudraCT #2011-000176-33). In this view, understanding the cell-specific effects and mechanisms of myogenic cues will help improving clinical translation of MAB-based therapies in vivo. Recently, it has been shown that Notch synergizes with Pdgf-bb to convert fetal myoblasts into myogenic pericytes.¹¹ However, knowledge about Notch-triggered effects on the regenerative potency of somatic MABs is still scant, particularly in the contexts of cell–cell (in vitro) and fiber–cell (in vivo) contact.Therefore, we asked whether the Dll1-Notch1 axis regulates the myogenic potential of murine and human MABs and how to tune this pathway to ameliorate in vivo MAB-driven regeneration.