RESUMEN
Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.
Asunto(s)
Envejecimiento , Genes Reporteros , Factor 5 Regulador Miogénico , Factor de Transcripción PAX7 , Regeneración , Animales , Factor de Transcripción PAX7/metabolismo , Factor de Transcripción PAX7/genética , Factor 5 Regulador Miogénico/metabolismo , Factor 5 Regulador Miogénico/genética , Ratones , Envejecimiento/metabolismo , Células Madre/metabolismo , Células Madre/citología , Proliferación Celular , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Diferenciación Celular , Ratones Transgénicos , Autorrenovación de las CélulasRESUMEN
Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Animales , Diferenciación Celular , Células Endoteliales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Desarrollo de Músculos , Fosfatidilinositol 3-Quinasas/metabolismo , Nicho de Células MadreRESUMEN
We developed and validated a reliable, robust, and easy-to-implement quantitative method for multielemental analysis of low-volume samples. Our ICP-MS-based method comprises the analysis of 20 elements (Mg, P, S, K, Ca, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Br, Rb, Sr, Mo, I, Cs, and Ba) in 10 µL of serum and 12 elements (Mg, S, Mn, Fe, Co, Cu, Zn Se, Br, Rb, Mo, and Cs) in less than 250â¯000 cells. As a proof-of-concept, we analyzed the elemental profiles of serum and sorted immune T cells derived from naiÌve and tumor-bearing mice. The results indicate a tumor systemic effect on the elemental profiles of both serum and T cells. Our approach highlights promising applications of multielemental analysis in precious samples such as rare cell populations or limited volumes of biofluids that could provide a deeper understanding of the essential role of elements as cofactors in biological and pathological processes.
Asunto(s)
Compuestos Inorgánicos/análisis , Espectrometría de Masas/métodos , Neoplasias/química , Animales , Línea Celular Tumoral , Cobre/análisis , Cobre/sangre , Compuestos Inorgánicos/sangre , Límite de Detección , Magnesio/análisis , Magnesio/sangre , Ratones , Ratones Endogámicos C57BL , Neoplasias/patología , Linfocitos T/química , Linfocitos T/citología , Linfocitos T/metabolismo , Trasplante Homólogo , Zinc/análisis , Zinc/sangreRESUMEN
Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis.
Asunto(s)
Adipocitos/metabolismo , Envejecimiento/metabolismo , Proteínas CCN de Señalización Intercelular/metabolismo , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/metabolismo , Adipocitos/citología , Adipogénesis , Animales , Proteínas CCN de Señalización Intercelular/deficiencia , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/citología , Proteínas Proto-Oncogénicas/deficiencia , Células Madre/citologíaRESUMEN
Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body Pparg null mice generated by epiblast-specific Cre/lox deletion (PpargΔ/Δ). We demonstrate that deletion of PPARγ completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in PpargΔ/Δ mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in PpargΔ/Δ mice and suggests that PPARγ signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPARγ-dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPARγ is required for MuSC function and efficient muscle repair.