Myogenic Regeneration Research Articles

Infiltration of plasma rich in growth factors enhances in vivo angiogenesis and improves reperfusion and tissue remodeling after severe hind limb ischemia

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia.

Asynchronous inflammation and myogenic cell migration limit muscle tissue regeneration mediated by a cellular scaffolds.

Volumetric muscle loss (VML) following orthopaedic trauma results in chronic loss of strength and can contribute to disability. Tissue engineering and regenerative medicine approaches to regenerate the lost skeletal muscle and improve functional outcomes are currently under development. At the forefront of these efforts, decellularized extracellular matrices (ECMs) have reached clinical testing and provide the foundation for other approaches that include stem/progenitor cell delivery. ECMs have been demonstrated to possess many qualities to initiate regeneration, to include stem cell chemotaxis and pro-regenerative macrophage polarization. However, the majority of observations indicate that ECM-repair of VML does not promote appreciable muscle fiber regeneration. In a recent study, ECM-repair of VML was compared to classical muscle fiber regeneration (Garg et al., 2014, Cell & Tissue Research) mediated by autologous minced grafts. The most salient findings of this study were: 1) Satellite cells did not migrate into the scaffold beyond ~0.5 mm from the remaining host tissue, although other migratory stem cells (Sca-1+) were observed throughout the scaffold;2) Macrophage migration to the scaffold was over two-times that observed with muscle grafts, but they appeared to be less active, as gene expression of pro- and anti-inflammatory cytokines (TNF-α, IL-12, IL-4, IL-10, VEGF, and TGF-β1) was significantly reduced in scaffold-repaired muscles; And, 3) scaffolds did not promote appreciable muscle fiber regeneration. Collectively, these data suggest that the events following ECM transplantation in VML are either incongruous or asynchronous with classical muscle fiber regeneration.

An in vitro culture system that supports robust expansion and maintenance of in vivo engraftment capabilities for myogenic progenitor cells from adult mice.

Muscle cell therapy and tissue engineering require large numbers of functional muscle precursor/progenitor cells (MPCs), making the in vitro expansion of MPCs a critical step for these applications. The cells must maintain their myogenic properties upon robust expansion, especially for cellular therapy applications, in order to achieve efficacious treatment. A major obstacle associated with MPCs expansion is the loss of “stemness,” or regenerative capacity, of freshly isolated cells, presumably due to the absence of the native cellular niches. In the current study, we developed an in vitro system that allowed for long-term culture and massive expansion of murine MPCs (mMPCs) with the preservation of myogenic regeneration capabilities. Long term in vitro expanded mMPC expressed the myogenic stem cell markers Pax3 and Pax7 and formed spontaneously contracting myotubes. Furthermore, expanded mMPC injected into the tibialis anterior muscle of nude mice engrafted and formed myofibers. Collectively, the method developed in this study can be potentially adapted for the expansion of human MPCs to high enough numbers for treatment of muscle injuries in human patients.

Isolation of Mesenchymal Stem Cells from Shoulder Rotator Cuff: A Potential Source for Muscle and Tendon Repair

The self-healing potential of each tissue belongs to endogenous stem cells residing in the tissue; however, there are currently no reports mentioned for the isolation of human rotator cuff-derived mesenchymal stem cells (RC-MSCs) since. To isolate RC-MSCs, minced rotator cuff samples were first digested with enzymes and the single cell suspensions were seeded in plastic culture dishes. Twenty-four hours later, nonadherent cells were removed and the adherent cells were further cultured. The RC-MSCs had fibroblast-like morphology and were positive for the putative surface markers of MSCs, such as CD44, CD73, CD90, CD105, and CD166, and negative for the putative markers of hematopoietic cells, such as CD34, CD45, and CD133. Similar to BM-MSCs, RC-MSCs were demonstrated to have the potential to undergo osteogenic, adipogenic, and chondrogenic differentiation. Upon induction in the defined media, RC-MSCs also expressed lineage-specific genes, such as Runx 2 and osteocalcin in osteogenic induction, PPAR-γ and LPL in adipogenic differentiation, and aggrecan and Col2a1 in chondrogenic differentiation. The multipotent feature of RC-MSCs in the myogenic injury model was further strengthened by the increase in myogenic potential both in vitro and in vivo when compared with BM-MSCs. These results demonstrate the successful isolation of MSCs from human rotator cuffs and encourage the application of RC-MSCs in myogenic regeneration.

Aged skeletal muscle retains the ability to fully regenerate functional architecture

While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.

Effects of genistein and estrogen on the genioglossus in rats exposed to chronic intermittent hypoxia may be HIF‐1α dependent

Chronic intermittent hypoxia (CIH) is a frequent feature of OSAHS. The present study was designed to evaluate the effects of genistein and estrogen on genioglossus contractile and regeneration properties in CIH rats and investigate the involvement of HIF-1α. Ovariectomized female rats were exposed to CIH for 5 weeks. Genistein and estrogen were administered by intraperitoneal injection. The genioglossus myoblasts of rat were also isolated and cultured in vitro, and the HIF-1α shRNA lentivirus was used. Muscle fatigue resistance and myogenic regeneration were significantly decreased after CIH but were partially reversed by estrogen and genistein treatment. The effect of estrogen was more powerful than that of genistein. Compared with control group, RT-PCR and western blotting showed higher levels of HIF-1α mRNA and protein in the CIH group, but estrogen and genistein treatment reduced the levels of HIF-1α mRNA and protein in rats exposed to CIH. In genioglossus myoblasts, the expression of HIF-1α was up-regulated under hypoxia rather than normoxia and decreased over time under both hypoxia and normoxia during myogenic differentiation. HIF-1α knockdown relieved myogenesis inhibition under hypoxia. We concluded that genistein and estrogen may inhibit the overexpression of HIF-1α induced by CIH and improve the endurance and regeneration of the genioglossus muscle.

Hypoxia Increases Mouse Satellite Cell Clone Proliferation Maintaining both In Vitro and In Vivo Heterogeneity and Myogenic Potential

Satellite cells (SCs) are essential for postnatal muscle growth and regeneration, however, their expansion potential in vitro is limited. Recently, hypoxia has been used to enhance proliferative abilities in vitro of various primary cultures. Here, by isolating SCs from single mouse hindlimb skeletal myofibers, we were able to distinguish two subpopulations of clonally cultured SCs (Low Proliferative Clones - LPC - and High Proliferative Clones - HPC), which, as shown in rat skeletal muscle, were present at a fixed proportion. In addition, culturing LPC and HPC at a low level of oxygen we observed a two fold increased proliferation both for LPC and HPC. LPC showed higher myogenic regulatory factor (MRF) expression than HPC, particularly under the hypoxic condition. Notably, a different myogenic potential between LPC and HPC was retained in vivo: green fluorescent protein (GFP)+LPC transplantation in cardiotoxin-injured Tibialis Anterior led to a higher number of new GFP+muscle fibers per transplanted cell than GFP+HPC. Interestingly, the in vivo myogenic potential of a single cell from an LPC is similar if cultured both in normoxia and hypoxia. Therefore, starting from a single satellite cell, hypoxia allows a larger expansion of LPC than normal O2 conditions, obtaining a consistent amount of cells for transplantation, but maintaining their myogenic regeneration potential.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

Administration of a Soluble Activin Type IIB Receptor Promotes the Transplantation of Human Myoblasts in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits the introduction of the dystrophin gene into dystrophic muscle fibers. However, this strategy has so far produced limited results. Modulation of transforming growth factor-β (TGF-β) superfamily signaling promotes skeletal muscle differentiation and growth and myogenic regeneration. We investigated the possibility that the combination of TGF-β superfamily signaling inhibition with myoblast transplantation might be an effective therapeutic approach in dystrophin-deficient patients. In vitro, blocking myostatin and other ligands with a soluble form of the extracellular domain of the activin IIB receptor (ActRIIB/Fc) upregulated the expression of myogenic differentiation factors and increased human myoblast fusion. In vivo, systemic inhibition of activin IIB receptor signaling by delivery of ActRIIB/Fc increased the success of the myoblast transplantation. This effect was further increased by forcing the mice to swim weekly to induce cycles of muscle degeneration and regeneration. Treatment of dystrophic mice with ActRIIB/Fc led to increased body weight, increased skeletal muscle mass, and improved myoblast transplantation. Thus, ActRIIB/Fc represents an effective therapeutic strategy for muscular dystrophies, and its effects are enhanced when combined with muscle exercise.

Losartan Enhances the Success of Myoblast Transplantation

Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor-β (TGF-β) is a negative regulator of skeletal muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin-deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that downregulates TGF-β expression. In vitro, blocking TGF-β activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in tibialis anterior cross sections 1 month posttransplantation revealed more human dystrophin-positive myofibers in these mice than in nontreated dystrophic mice. Thus, blocking the TGF-β signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation, and changing the expression of myogenic regulator factors.

Fetal muscle contains different CD34 + cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34 + cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34 + cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34 + subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34 + population comprised a CD31 +/CD45 − cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34 +/CD31 −/CD45 −/Sca1 + subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34 +/CD31 −/CD45 −/Sca1 − cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34 + cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.

Impact of resistance exercise training on interleukin‐6 and JAK/STAT in young men

The JAK/STAT signaling pathway is essential for myogenic regeneration and is regulated by a diverse range of ligands, including interleukin-6 (IL-6) and platelet-derived growth factor-BB (PDGF-BB). Our aim was to evaluate the responsiveness of IL-6 and PDGF-BB to intense exercise, along with STAT3 activation, before and after 12 weeks of resistance training. In young men, IL-6 and PDGF-BB protein concentrations were quantified in biopsied muscle and increased at 3 h post-exercise (17.5-fold and 3-fold, respectively). The response was unaltered by 12 weeks of training. Similarly, STAT3 phosphorylation was elevated post-exercise (12.5-fold), irrespective of training status, as was the expression of downstream targets c-MYC (8-fold), c-FOS (4.5-fold), and SOCS3 (2.3-fold). Thus, intense exercise transiently increases IL-6 and PDGF-BB proteins, and STAT3 phosphorylation is increased. These responses are preserved after intense exercise. This suggests they are not modified by training and may be an essential component of the adaptive responses to intense exercise.

Assessment of cell proliferation and muscular structure following surgical tongue volume reduction in pigs

Tongue volume reduction is an adjunct treatment in several orofacial orthopaedic procedures for various craniofacial deformities; it may affect structural reconstitution and functional recovery as a result of the repair process. The aim of this study was to investigate myogenic regeneration and structural alteration of the tongue following surgical tongue volume reduction. Five 12-week-old sibling pairs of Yucatan minipigs (three males and two females) were used. Midline uniform glossectomy was performed on one of each pair (reduction); siblings had identical incisions without tissue removal (sham). All pigs were raised for a further 4 weeks and received 5-bromo-2-deoxyuridine (BrdU) injection intravenously 1 day before killing. Tissue sections of tongues were stained with anti-BrdU antibody to evaluate numbers of replicating cells. Haematoxylin and eosin plus trichrome staining were performed to assess muscular structure. Reduction tongues contained significantly more BrdU+ cells compared to sham tongues (P < 0.01). However, these BrdU+ cells were mostly identified in reparative connective tissues (fibroblasts) rather than in regenerating muscle tissue (myoblasts). Trichrome-stained sections showed disorganized collagen fibres linked to few intermittent muscle fibres in the reduction tongues. These myofibres presented signs of atrophy with reduced perimysium and endomysium. Matrix between reduced perimysium and endomysium was filled with fibrous tissue. Fibrosis without predominant myogenic regeneration was the major histological consequence of surgical tongue volume reduction.

Laminin α4 and Integrin α6 Are Upregulated in Regenerating dy/dy Skeletal Muscle: Comparative Expression of Laminin and Integrin Isoforms in Muscles Regenerating after Crush Injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin α2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and “preactivated” myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin α1 was not detectable in skeletal muscle. Laminin α2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin α2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin α chains in early myogenic differentiation, such as laminin α4 and α5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin β1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin α3 was not expressed in uninjured or regenerating muscle, while integrin α6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin α6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin α6 occurred on some newly formed myotubes. Integrin α7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin α7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin α7 negative. No marked difference was observed in integrin α7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin α4 and integrin α6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin α4 and integrin α6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin α2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.

The compound 5-azacytidine has been previously shown to convert cells of the rat embryonic fibroblastic cell line, C3H/10T1/2, into myoblasts, adipocytes, and chondrocytes. Rare, resident cells of bone marrow and periosteum, referred to as mesenchymal stem cells, have been shown to differentiate into a number of mesenchymal phenotypes including bone, cartilage, and adipocytes. Rat bone marrow-derived mesenchymal stem cells were exposed to 5-azacytidine beginning 24 h after seeding twice-passaged cells into culture dishes. After an exposure of 24 h, long, multinucleated myotubes were observed in some of the dishes 7-11 days later. Cells containing Sudan black-positive droplets in their cytoplasm were also observed. Thus, culture-propagated rat bone marrow mesenchymal stem cells appear to have the capacity to be induced to differentiate in vitro into myogenic and adipocytic phenotypes, although nonmesenchymal cells (rat brain fibroblasts) cannot be so induced. Taken together, these observations provide support for the suggestion that mesenchymal stem cells in the bone marrow of postnatal organisms may provide a source for myoprogenitor cells which could function in clinically relevant myogenic regeneration.

Repair mechanisms involved in muscle regeneration following partial excision of the rat gastrocnemius muscle.

The sequential cytological events of the regeneration process, after partial excision of the gastrocnemius muscle in the rat, were followed by light and electron microscopy. During the first 2 days after injury leukocytes and macrophages infiltrate into the traumatized area. Myogenic regeneration is then characterized by mainly two repair mechanisms. Mononucleated cells, that populate the excised area, most probably fuse together to give rise to newly formed multinucleated myotubes that further develop to striated myofibers. Another mechanism involves the repair of injured muscle fibers by the possible fusion of mononucleated cells with their necrotic cut ends. Consequently, by addition of nuclei and new muscular material, sarcoplasmic outgrowths from the injured fibers are formed. It is concluded that mainly two repair mechanisms are involved in the regeneration process following partial excision of a muscle: addition of new muscle fibers in a process similar to that of embryonic myogenesis and also meristic growth from the injured fibers.

Enhancement of human muscle growth in diffusion chambers by bone marrow cells.

Samples of minced human muscle were cultured in millipore diffusion chambers incubated in the peritoneal cavities of mice. In about half the chambers the minced muscle samples were mixed with autogenous bone marrow cells which lead to improved myogenic growth. A similar but less marked effect was produced by mononuclear cells from the patients' blood. No growth enhancement occurred when the muscle and marrow cells were separated by a filter in double chambers. In addition to accelerated myogenesis, the chambers with added bone marrow cells had a much lower incidence of infection. This work may have practical clinical implications for the treatment of muscle injuries. Local implantation of autogenous marrow cells (+/- minced muscle) may prove useful in improving myogenic regeneration.