Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD.

Mesenchymal stem cells derived from human induced pluripotent stem cells improve the engraftment of myogenic cells by secreting urokinase-type plasminogen activator receptor (uPAR)

Background Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by mutations in the dystrophin gene. Transplantation of myogenic stem cells holds great promise for treating muscular dystrophies. However, poor engraftment of myogenic stem cells limits the therapeutic effects of cell therapy. Mesenchymal stem cells (MSCs) have been reported to secrete soluble factors necessary for skeletal muscle growth and regeneration. Methods We induced MSC-like cells (iMSCs) from induced pluripotent stem cells (iPSCs) and examined the effects of iMSCs on the proliferation and differentiation of human myogenic cells and on the engraftment of human myogenic cells in the tibialis anterior (TA) muscle of NSG-mdx4Cv mice, an immunodeficient dystrophin-deficient DMD model. We also examined the cytokines secreted by iMSCs and tested their effects on the engraftment of human myogenic cells. Results iMSCs promoted the proliferation and differentiation of human myogenic cells to the same extent as bone marrow-derived (BM)-MSCs in coculture experiments. In cell transplantation experiments, iMSCs significantly improved the engraftment of human myogenic cells injected into the TA muscle of NSG-mdx4Cv mice. Cytokine array analysis revealed that iMSCs produced insulin-like growth factor-binding protein 2 (IGFBP2), urokinase-type plasminogen activator receptor (uPAR), and brain-derived neurotrophic factor (BDNF) at higher levels than did BM-MSCs. We further found that uPAR stimulates the migration of human myogenic cells in vitro and promotes their engraftment into the TA muscles of immunodeficient NOD/Scid mice. Conclusions Our results indicate that iMSCs are a new tool to improve the engraftment of myogenic progenitors in dystrophic muscle.

pNiPAM-Nanoparticle-Based Antiapoptotic Approach for Pro-Regenerative Capacity of Skeletal Myogenic Cells

The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells.

Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets

Muscle growth of low birth weight (LBW) piglets may be improved with adapted nutrition. This study elucidated effects of glutamine (Gln) supplementation on the cellular muscle development of LBW and normal birth weight (NBW) piglets. Male piglets (n = 144) were either supplemented with 1 g Gln/kg body weight or an isonitrogeneous amount of alanine (Ala) between postnatal day 1 and 12 (dpn). Twelve piglets per group were slaughtered at 5, 12 and 26 dpn, one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Muscle samples were collected and myogenic cells were isolated and cultivated. Expression of muscle growth related genes was quantified with qPCR. Proliferating, BrdU-positive cells in muscle sections were detected with immunohistochemistry indicating different cell types and decreasing proliferation with age. More proliferation was observed in muscle tissue of LBW-GLN than LBW-ALA piglets at 5 dpn, but there was no clear effect of supplementation on related gene expression. Cell culture experiments indicated that Gln could promote cell proliferation in a dose dependent manner, but expression of myogenesis regulatory genes was not altered. Overall, Gln supplementation stimulated cell proliferation in muscle tissue and in vitro in myogenic cell culture, whereas muscle growth regulatory genes were barely altered.

Mesenchymal Stem Cells Derived From Human Induced Pluripotent Stem Cells Improve the Engraftment of Myogenic Cells by Secreting Urokinase-type Plasminogen Activator Receptor (uPAR)

Background: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by mutations in the dystrophin gene. Transplantation of myogenic stem cells holds great promise for treating muscular dystrophies. However, poor engraftment of myogenic stem cells limits the therapeutic effects of cell therapy. Mesenchymal stem cells (MSCs) have been reported to secrete soluble factors necessary for skeletal muscle growth and regeneration. Methods: we induced MSC-like cells (iMSCs) from induced pluripotent stem cells (iPSCs) and examined the effects of iMSCs on the proliferation and differentiation of human myogenic cells and on the engraftment of human myogenic cells in the tibialis anterior (TA) muscle of NSG-mdx4Cv mice, an immunodeficient dystrophin-deficient DMD model. We also examined the cytokines secreted by iMSCs and tested their effects on the engraftment of human myogenic cells.Results: iMSCs promoted the proliferation and differentiation of human myogenic cells to the same extent as bone marrow-derived (BM)-MSCs in coculture experiments. In cell transplantation experiments, iMSCs significantly improved the engraftment of human myogenic cells injected into the TA muscle of NSG-mdx4Cv mice. Cytokine array analysis revealed that iMSCs produced insulin-like growth factor-binding protein 2 (IGFBP2), urokinase-type plasminogen activator receptor (uPAR), and brain-derived neurotrophic factor (BDNF) at higher levels than did BM-MSCs. We further found that uPAR stimulates the migration of human myogenic cells in vitro and promotes their engraftment into the TA muscles of immunodeficient NOD/Scid mice. Conclusions: Our results indicate that iMSCs are new tools to improve the engraftment of myogenic progenitors in dystrophic muscle.

Uhrf1 governs the proliferation and differentiation of muscle satellite cells

DNA methylation is an essential form of epigenetic regulation responsible for cellular identity. In muscle stem cells, termed satellite cells, DNA methylation patterns are tightly regulated during differentiation. However, it is unclear how these DNA methylation patterns are maintained. We demonstrate that a key epigenetic regulator, ubiquitin like with PHD and RING finger domains 1 (Uhrf1), is activated in proliferating myogenic cells but not expressed in quiescent or differentiated myogenic cells in mice. Ablation of Uhrf1 in mouse satellite cells impairs their proliferation and differentiation, leading to failed muscle regeneration. Loss of Uhrf1 in satellite cells alters transcriptional programs, leading to DNA hypomethylation with activation of Cdkn1a and Notch signaling. Although down-regulation of Cdkn1a rescued proliferation but not differentiation, inhibition of Notch signaling rescued impaired differentiation of Uhrf1-deficient satellite cells. These findings point to Uhrf1 as a regulator of self-renewal and differentiation of satellite cells via genome-wide DNA methylation patterning.