transplantation-induced-pluripotent-stem-cell-derived-mesoangioblast

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 ...

University College London

San Raffaele Hospital

Skeletal muscle damaged by injury or by degenerative diseases such as muscular dystrophy is able to regenerate new muscle fibers. Regeneration mainly depends upon satellite cells, myogenic progenitors localized between the basal lamina and the muscle fiber membrane. However, other cell types outside the basal lamina, such as pericytes, also have myogenic potency. Here, we discuss the main properties of satellite cells and other myogenic progenitors as well as recent efforts to obtain myogenic cells from pluripotent stem cells for patient-tailored cell therapy. Clinical trials utilizing these cells to treat muscular dystrophies, heart failure, and stress urinary incontinence are also briefly outlined.

Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells.

Mesoangioblasts are progenitor endowed with multipotent mesoderm differentiation ability. Despite the promising results obtained with mesoangioblast transplantation in muscle dystrophy, an improvement of their efficient engrafting and survival within damaged muscles, as well as their ex vivo activation/expansion and commitment toward myogenic lineage, is highly needed and should greatly increase their therapeutic potential. We show that globular adiponectin, an adipokine endowed with metabolic and differentiating functions for muscles, regulates vital cues of mesoangioblast cell biology. The adipokine drives mesoangioblasts to entry cell cycle and strongly counteracts the apoptotic process triggered by growth factor withdrawal, thereby serving as an activating and prosurvival stem cell factor. In addition, adiponectin provides a specific protection against anoikis, the apoptotic death due to lack of anchorage to extracellular matrix, suggesting a key protective role for these nonresident stem cells after systemic injection. Finally, adiponectin behaves as a chemoattractive factor toward mature myotubes and stimulates their differentiation toward the skeletal muscle lineage, serving as a positive regulator in mesoangioblast homing to injured or diseased muscles. We conclude that adiponectin exerts several advantageous effects on mesoangioblasts, potentially valuable to improve their efficacy in cell based therapies of diseased muscles.

Globular adiponectin as a complete mesoangioblast regulator: role in proliferation, survival, motility, and skeletal muscle differentiation.

Complementation of pluripotency genes may improve adult stem cell functions.

Sox2 transduction enhances cardiovascular repair capacity of blood-derived mesoangioblasts.

In contrast to conventional gene therapy vectors, human artificial chromosomes (HACs) are episomal vectors that can carry large regions of the genome containing regulatory elements. So far, HACs have not been used as vectors in gene therapy for treating genetic disorders. Here, we report the amelioration of the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD) using a combination of HAC-mediated gene replacement and transplantation with blood vessel-associated stem cells (mesoangioblasts). We first genetically corrected mesoangioblasts from dystrophic mdx mice with a HAC vector containing the entire (2.4 Mb) human dystrophin genetic locus. Genetically corrected mesoangioblasts engrafted robustly and gave rise to many dystrophin-positive muscle fibers and muscle satellite cells in dystrophic mice, leading to morphological and functional amelioration of the phenotype that lasted for up to 8 months after transplantation. Thus, HAC-mediated gene transfer shows efficacy in a preclinical model of DMD and offers potential for future clinical translation.

Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy.

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo.

Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy.

Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration.

Muscular dystrophies are genetic disorders characterized by skeletal muscle wasting and weakness. Although there is no effective therapy, a number of experimental strategies have been developed over recent years and some of them are undergoing clinical investigation. In this review, we highlight recent developments and key challenges for strategies based upon gene replacement and gene/expression repair, including exon-skipping, vector-mediated gene therapy and cell therapy. Therapeutic strategies for different forms of muscular dystrophy are discussed, with an emphasis on Duchenne muscular dystrophy, given the severity and the relatively advanced status of clinical studies for this disease.

Repair or replace? Exploiting novel gene and cell therapy strategies for muscular dystrophies.

Pericytes are endothelial-associated cells that contribute to vessel wall. Here, we report that pericytes may derive from direct conversion of committed skeletal myoblasts. When exposed to Dll4 and PDGF-BB, but not Dll1, skeletal myoblasts downregulate myogenic genes, except Myf5, and upregulate pericyte markers, whereas inhibition of Notch signaling restores myogenesis. Moreover, when cocultured with endothelial cells, skeletal myoblasts, previously treated with Dll4 and PDGF-BB, adopt a perithelial position stabilizing newly formed vessel-like networks in vitro and in vivo. In a transgenic mouse model in which cells expressing MyoD activate Notch, skeletal myogenesis is abolished and pericyte genes are activated. Even if overexpressed, Myf5 does not trigger myogenesis because Notch induces Id3, partially sequestering Myf5 and inhibiting MEF2 expression. Myf5-expressing cells adopt a perithelial position, as occasionally also observed in wild-type (WT) embryos. These data indicate that endothelium, via Dll4 and PDGF-BB, induces a fate switch in adjacent skeletal myoblasts.

Dll4 and PDGF-BB convert committed skeletal myoblasts to pericytes without erasing their myogenic memory.

Adult muscle stem cells, or satellite cells have essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here, we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1(-/-) mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1(-/-) muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases.

Collagen VI regulates satellite cell self-renewal and muscle regeneration.

Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

Francesco Saverio Tedesco

Department of Cell and Developmental Biology

University College London

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

Francesco Saverio Tedesco

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Developmental Biology

University College London

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

Department of Cell and Developmental Biology