Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.
In the mature central nervous system (CNS) regulated secretion of ATP from astrocytes is thought to play a significant role in cell signaling. Whether such a mechanism is also operative in the developing nervous system and, if so, during which stage of development, has not been investigated. We have tackled this question using cells derived from reconstituted neurospheres, as well as brain explants of embryonic mice. Here, we show that in both models of neural cell development, astrocyte progenitors are competent for the regulated secretion of ATP-containing vesicles. We further document that this secretion is dependent on cytosolic Ca 2+ and the v-SNARE system, and takes place by exocytosis. Interference with ATP secretion alters spontaneous Ca 2+ oscillations and migration of neural progenitors. These data indicate that astrocyte progenitors acquire early in development the competence for regulated secretion of ATP, and that this event is implicated in the regulation of at least two cell functions, which are critical for the proper morphogenesis and functional maturation of the CNS.
Accruing evidence indicates that gap junctions are involved in neuronal survival after brain injury. The present study was aimed at clarifying the contribution of the neuronal gap-junction protein connexin36 (Cx36) to secondary cell loss after injury in the mouse retina. A focal retinal lesion was induced by infrared laser photocoagulation. Remarkably, this model allowed spatial and temporal definition of the lesion with high reproducibility. Moreover, Cx36 is abundantly expressed in the retina and plays an essential role in the visual transmission process. Taking advantage of these features, cell death was assessed using TUNEL assay and light and electron microscopy, and the extent of Cx36 expression was studied by immunohistochemistry, Western blot, in situ hybridization and real-time RT-PCR. Secondary cell loss was most prominent between 24 and 48 h after lesioning. This peak was accompanied by an increase in Cx36 expression. When cultured explanted retinas were subjected to gap-junction blockers a significant increase in the extent of secondary cell loss after laser photocoagulation became evident. Using the same experimental paradigm we compared the incidence of cell death in wild-type and Cx36(-/-) mice. A significant increase in total number of TUNEL-positive cells occurred in the Cx36(-/-) mice compared to controls. From these data we conclude that Cx36 contributes to the survival and resistance against damage of retinal cells and thus constitutes a protective factor after traumatic injury of the retina.
Connexin43 (Cx43) is widely expressed in embryonic brain, and its expression becomes restricted mainly to astrocytes as the central nervous system matures. Recent studies have indicated that Cx43 plays important, nonchannel, roles during central nervous system development by affecting neuronal cell migration. Here, we evaluated the effects of Cx43 on neuronal differentiation. For that we used an in vitro model of neural cell development (neurospheres) to evaluate, through immunocytochemistry, electrophysiology, and molecular biology, the degree of neuronal maturation from neurospheres derived from wild-type (WT) and Cx43-null mice. Our results indicate that Cx43 is a negative modulator of neuronal differentiation. The percent neurospheres containing differentiated neurons and the number of cells displaying inward currents were significantly higher in Cx43-null than in WT littermate neurospheres. Knockdown of Cx43 with small interfering RNA increased the number of WT neurospheres generating differentiated neurons. Blockade of gap junctional communication with carbenoxolone did not induce neuronal differentiation in WT neurospheres. Transfection of Cx43-null neurospheres with Cx43 mutants revealed that Cx43 carboxyl terminus prevents neuronal maturation. In agreement with these in vitro data, in situ analysis of embryonic day 16 brains revealed increased -III-tubulin expression in germinal zones of Cx43-null compared with that of WT littermates. These results indicate that Cx43, and specifically its carboxyl terminus, is crucial for signaling mechanisms preventing premature neuronal differentiation during embryonic brain development.Multiple distinct mechanisms contribute to neocortical development. Diffusible and nondiffusible molecules have been implicated in neural cell proliferation, migration, and differentiation during brain development (1-4). Connexins (Cx), 2 the proteins forming gap junctions, are prominent in neural progenitor cells and play important roles in neural cell proliferation, migration, and differentiation (5-9). Among the central nervous system gap junction proteins, Cx43 and Cx26 are the two most abundantly expressed in neural stem cells, although transcripts for other connexins (Cx29, Cx30, Cx30.2, Cx32, Cx36, and Cx47) are also present in embryonic states (10 -12). The expression pattern of connexin proteins dramatically changes as the central nervous system matures, becoming restricted with respect to cell type. For instance, following postnatal development, Cx43 together with Cx30 are mainly found in astrocytes, whereas Cx29, Cx32, and Cx47 form the oligodendrocyte group of gap junction proteins, and Cx36, Cx45, and Cx30.2 are expressed in neurons (for review, see Ref. 13).The dramatic decrease in Cx43 expression during central nervous system development, specifically during neuroblast maturation, has raised the possibility that concurrent loss of gap junctional communication may play an important role during neuronal differentiation. Indeed, initial studies (14, 15) indicating an inver...
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