Bifeo3 Harmonic Nanoparticle (Bfo-Hnps) Use for the Stem Cell Tracking: Labeling Investigation by Non Linear Microscopy and X-Ray Fluorescence

Duchenne Muscular Dystrophy is the most common form of degenerative muscle disease; currently, there is no effective treatment. In 2011, our group showed that an adult stem cell population (MuStem) isolated from healthy dog skeletal muscle induces long-term muscle repair and striking clinical efficacy after its systemic delivery in clinically relevant dystrophic dog. During last years, our group isolated the human counterparts (hMuStem) [1]. To achieve the full therapeutic potential of the hMuStem cells, their homing process, survival and engraftment post-transplantation must be clearly understood. BiFeO3 harmonic nanoparticles (BFO-HNPs) were used as probes for the hMuStem cell tracking [2]. We demonstrate the possibility of identifying <100 nm BFO-HNPs in depth of muscle tissue at more than 1 mm from the surface by multiphoton microscopy. Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of the hMuStem cells upon exposure to BFO-HNPs revealing their high biocompatibility. To complete these studies, the stability of BFO-HNPs was followed in the labeled hMuStem cells by investigation of Bi and Fe X-ray fluorescence mapping on both Nanoscopium (Soleil, Gif-sur-Yvette, France) and ID16B (ESRF, Grenoble, France) beamlines. In this work, correlation between non-linear microscopy and X-Ray fluorescence was done. Bi and Fe X-Ray fluorescence allowed us to localize with high resolution the BFO-HNPs in the labeled hMuStem cells and the variation of Bi/Fe ratio was analyzed to detect possible dissociation of the nanoparticles in the labeled cells

Molecular Signature, Metabolic Profile, and Therapeutic Potential of Human Muscle Reserve Cells

Muscle stem cell therapy is being studied for the treatment of skeletal muscle pathologies, such as severe muscle injury or muscular dystrophies, to enhance muscle regeneration. In vivo, the pool of skeletal muscle stem cells (MuSCs) is the main cell population that allows skeletal muscle regeneration. Therefore, MuSCs are considered as a good cell source for cell therapy. However, it is known that MuSCs lose some of their regenerative potential when expanded in vitro. To date, there has been no identification of the ideal cell product. In the laboratory, it has been demonstrated that human muscle reserve cells (MuRCs) generated in vitro are highly comparable to human MuSCs. Human MuRCs are quiescent Pax7+/MyoD- cells with enhanced survival and a higher potency to generate Pax7+ cells in vivo compared to human myoblasts. Furthermore, it is possible to generate human MuRCs in vitro in large numbers, making them suitable for future therapeutic applications. Based on these promising results obtained earlier in the laboratory, I then proposed to evaluate the molecular signature, metabolic profile, and therapeutic potential of human MuRCs. In a first study, I assessed the expression of Pax7 in human MuRCs, a known marker of MuSCs. I showed by flow cytometry that human MuRCs are heterogeneous for Pax7 expression with Pax7High and Pax7Low subpopulations. Using bulk-RNA sequencing, we showed that Pax7High cells exhibited a deeper quiescent state and a stronger shift towards fatty acid oxidation (FAO) compared to Pax7Low -MuRCs. However, it is not possible to obtain viable cells to study their functional properties in vitro or in vivo with the procedure (fixation and permeabilization) used to isolate human Pax7High and Pax7Low MuRCs populations. In a second study, I investigated whether autofluorescence could be used as a tool to isolate viable and functional human MuRCs. I showed that compared to human MB, human MuRCs are highly autofluorescent (AF) cells. We can distinguish two subpopulations of MuRCs based on their autofluorescence AFHigh and AFLow. After sorting, these cells were shown to be viable and functional in vitro. In addition, AFHigh and AFLow were shown to be functional cells in vivo as they participate in muscle regeneration and in MuSCs pool formation after their intramuscular injection in immunodeficient mice. Overall, my PhD project has the potential to improve our knowledge concerning the molecular signature, metabolic profile and therapeutic potential of human MuRCs. Taken together, these results suggest that human MuRCs AFHigh/Pax7High may be a suitable stem cell source for potential therapeutic applications in skeletal muscle diseases.</p

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

Abstract Background The capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies. Methods In the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile. Results We observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake. Conclusions Overall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases

Additional file 1 of In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

Additional file 1. Figure S1. Notch signaling is activated and required for the generation of human MuRC in vitro. Total protein extracts were isolated from human myoblasts in growth medium (MB) or after 24h in DM. MuRC and myotubes fractions were separated after 48h, 72h or 96h in DM. Western blot analysis for Pax7, MyoD and the notch intracellular domain (NICD). Figure S2. Human MuRC overexpressed active AMPKa1. Western blot analysis of AMPKa1, acetyl CoA carboxylase (ACC) and phosphoACC (pACC) in human myoblasts (MB), human 48h-MuRC and 96h-MuRC

Bifeo3 Harmonic Nanoparticle (Bfo-Hnps) Use for the Stem Cell Tracking: Labeling Investigation by Non Linear Microscopy and X-Ray Fluorescence

Duchenne Muscular Dystrophy is the most common form of degenerative muscle disease; currently, there is no effective treatment. In 2011, our group showed that an adult stem cell population (MuStem) isolated from healthy dog skeletal muscle induces long-term muscle repair and striking clinical efficacy after its systemic delivery in clinically relevant dystrophic dog. During last years, our group isolated the human counterparts (hMuStem) [1]. To achieve the full therapeutic potential of the hMuStem cells, their homing process, survival and engraftment post-transplantation must be clearly understood. BiFeO3 harmonic nanoparticles (BFO-HNPs) were used as probes for the hMuStem cell tracking [2]. We demonstrate the possibility of identifying <100 nm BFO-HNPs in depth of muscle tissue at more than 1 mm from the surface by multiphoton microscopy. Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of the hMuStem cells upon exposure to BFO-HNPs revealing their high biocompatibility. To complete these studies, the stability of BFO-HNPs was followed in the labeled hMuStem cells by investigation of Bi and Fe X-ray fluorescence mapping on both Nanoscopium (Soleil, Gif-sur-Yvette, France) and ID16B (ESRF, Grenoble, France) beamlines. In this work, correlation between non-linear microscopy and X-Ray fluorescence was done. Bi and Fe X-Ray fluorescence allowed us to localize with high resolution the BFO-HNPs in the labeled hMuStem cells and the variation of Bi/Fe ratio was analyzed to detect possible dissociation of the nanoparticles in the labeled cells

Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis

Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis