RESUMO
Myogenic differentiation results in different cell type cooperation, but the molecules involved in the myogenic cell activation remain elusive. Here, we show that muscle-resident pre-adipocytes promote myogenic differentiation through the secretion of factors. Using proteomic and transcriptomic analyses, we identified that proliferative adipogenic lineage cells produce and secrete a key factor of the innate immune system, the complement C3. Cell culture experiments revealed that C3 promotes the differentiation of myogenic progenitors following internalisation of the immune molecule. These data demonstrate that the third component of the complement system, which is a pivotal factor in the immune response to pathogens, is also involved in the differentiation of myogenic progenitor cells.
Assuntos
Complemento C3/genética , Complemento C3/metabolismo , Desenvolvimento Muscular , Células-Tronco/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Células Musculares/citologia , Células Musculares/metabolismo , Proteômica , Células-Tronco/metabolismoRESUMO
We studied the behavior of myogenic progenitors from donor desmin(+/-) LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of beta-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.
Assuntos
Desmina/genética , Embrião de Mamíferos/citologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Desmina/análise , Embrião de Mamíferos/metabolismo , Feminino , Imuno-Histoquímica/métodos , Masculino , Camundongos , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Neurônios/citologia , Neurônios/fisiologia , Notocorda/metabolismo , Notocorda/fisiologia , Fatores de Tempo , Transgenes , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
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.
Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem da Célula , Feto/citologia , Desenvolvimento Muscular , Músculo Esquelético/citologia , Neovascularização Fisiológica , Adipogenia , Animais , Citometria de Fluxo , Imuno-Histoquímica , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.
Assuntos
Antígenos CD34/metabolismo , Células Musculares/transplante , Distrofia Muscular Animal/terapia , Animais , Fusão Celular , Desmina/metabolismo , Distrofina/metabolismo , Fadiga/induzido quimicamente , Feminino , Proteínas de Fluorescência Verde/genética , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Células Musculares/fisiologia , Músculos/embriologia , Músculos/efeitos da radiaçãoRESUMO
The staging of murine cardiomyocyte specification and determination was investigated in cultures of tissue explants from pre- and postgastrulation embryos and after transplantation of cardiac or cardiogenic tissues from mouse embryos into chick embryos. The development of cultured and transplanted cells in cardiomyocytes was evaluated by testing the expression of several cardiac transcription factor genes (Nkx 2.5, eHAND, dHAND, GATA 4), alpha cardiac actin, and beta myosin heavy chain protein. In vitro analyses showed that cells with the potential to form cardiac muscle were present prior to gastrulation in 6.5-day postconception (dpc) epiblasts. Although, as shown by in vivo experiments, neurectodermal derived structures did not influence cardiogenesis in epiblast transplants, these transplants did not exhibit full cardiogenic cell differentiation in the chicken environment. In in vitro culture, the neurectoderm also had no effect on murine cardiomyogenesis. In contrast, the presence of endoderm in explants between mid- and late streak stages stimulated emerging mesodermal cells to adopt a myocardial pathway. Mesoderm from late streak explants (7.5 dpc) was capable of differentiating into a cardiac phenotype in the avian heterotopic environment, indicating that the specification of cardiac precursors became irreversible around the late streak stage in mouse embryo.
Assuntos
Coração/embriologia , Animais , Desenvolvimento Embrionário e Fetal , Mamíferos , Camundongos , Morfogênese , Células Musculares/citologia , Miocárdio/citologia , Técnicas de Cultura de Órgãos , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.