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.
Age-related loss of muscle mass and function negatively impacts healthspan and lifespan. Satellite cells function as muscle stem cells in muscle maintenance and regeneration by self-renewal, ...activation, proliferation and differentiation. These processes are perturbed in aging at the stem cell population level, contributing to muscle loss. However, how representation of subpopulations within the human satellite cell pool change during aging remains poorly understood. We previously reported a comprehensive baseline of human satellite cell (Hu-MuSCs) transcriptional activity in muscle homeostasis describing functional heterogenous human satellite cell subpopulations such as CAV1+ Hu-MUSCs. Here, we sequenced additional satellite cells from new healthy donors and performed extended transcriptomic analyses with regard to aging. We found an age-related loss of global transcriptomic heterogeneity and identified new markers (CAV1, CXCL14, GPX3) along with previously described ones (FN1, ITGB1, SPRY1) that are altered during aging in human satellite cells. These findings describe new transcriptomic changes that occur during aging in human satellite cells and provide a foundation for understanding functional impact.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Regeneration and repair of skeletal muscle is driven by tissue-specific progenitor cells called satellite cells, which occupy a minority of the cells in the muscle. This protocol provides researchers ...with techniques to efficiently isolate and purify functional satellite cells from human muscle tissue. The proven techniques described here enable the preparation of purified and minimally altered satellite cells for in vitro and in vivo experimentation and for potential clinical applications.
For complete details on the use and execution of this protocol, please refer to Barruet et al. (2020) and Garcia et al. (2018).
Display omitted
•Techniques are described for efficient isolation of human satellite cells•Quiescent and activated satellite cells are purified using surface markers•Purification involves minimal alteration compared to culture or activation•Purified satellite cells faithfully represent the natural state for applications
Regeneration and repair of skeletal muscle is driven by tissue-specific progenitor cells called satellite cells, which occupy a minority of the cells in the muscle. This protocol provides researchers with techniques to efficiently isolate and purify functional satellite cells from human muscle tissue. The proven techniques described here enable the preparation of purified and minimally altered satellite cells for in vitro and in vivo experimentation and for potential clinical applications.
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 β-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.
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.
Stem cell behavior is tightly regulated by spatiotemporal signaling from the niche, which is a four-dimensional microenvironment that can instruct stem cells to remain quiescent, self-renew, ...proliferate, or differentiate. In this review, we discuss recent advances in understanding the signaling cues provided by the stem cell niche in two contrasting adult tissues, the rapidly cycling intestinal epithelium and the slowly renewing skeletal muscle. Drawing comparisons between these two systems, we discuss the effects of niche-derived growth factors and signaling molecules, metabolic cues, the extracellular matrix and biomechanical cues, and immune signals on stem cells. We also discuss the influence of the niche in defining stem cell identity and function in both normal and pathophysiologic states.
Irreversible denervation atrophy remains an unsolved clinical problem, and the role of skeletal muscle stem cell (MuSC, satellite cell) depletion in this process is unclear. We investigated the ...ability of MuSCs to regenerate muscle in the context of denervation. Three to 12 months following sciatic denervation in mice, MuSC number, size, EdU uptake, rate of division, and mitochondrial activity were increased. Following acute myotoxin injury, denervated muscles formed new muscle fibers in situ. MuSCs isolated via flow cytometry from denervated mouse muscle, or from atrophic denervated gluteus maximus muscles of humans with complete spinal cord injuries two decades prior, formed new muscle fibers and reoccupied the anatomic niche after transplantation into uninjured muscle. Our results show unequivocally that, even after prolonged denervation, MuSCs retain intrinsic regenerative potential similar to that of uninjured MuSCs. Treatment of denervation atrophy will require elucidating the non‐MuSC environmental changes in muscle that prevent functional regeneration.
Human satellite cells isolated from gluteus maximus muscle denervated >20 years retain the ability to engraft and form new muscle fiber when transplanted into NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, and also reoccupy the anatomic niche. Left: NSG muscle showing human dystrophin (green) and laminin (red). Right: NSG muscle showing costaining of human lamin A/C (red) Pax7 (green). Laminin (grey), 4′,6‐diamidino‐2‐phenylindole (DAPI, blue) counterstain.
Spontaneous calcium activity of neural progenitors is largely dependent on a paracrine signaling mechanism involving release of ATP and activation of purinergic receptors. Although it is well ...documented that, in mature astrocytes, cytokines modulate the expression levels of certain purinergic receptors, nothing is known about their impact during early stages of development. Here we provide evidence that conditioned medium from activated microglia and interleukin-1beta, but not tumor necrosis factor-alpha, decrease the frequency of calcium oscillations and reduce the rate of in vitro migration of astrocyte progenitors. Such alterations were due to changes in activity of two purinergic P2 receptors, and not to the amount of released ATP. These results indicate that interleukin-1beta plays an important role during early stages of CNS development, modulating calcium signaling and cell migration.
Abstract Spontaneous calcium activity of neural progenitors is largely dependent on a paracrine signaling mechanism involving release of ATP and activation of purinergic receptors. Although it is ...well documented that, in mature astrocytes, cytokines modulate the expression levels of certain purinergic receptors, nothing is known about their impact during early stages of development. Here we provide evidence that conditioned medium from activated microglia and interleukin-1β, but not tumor necrosis factor-α, decrease the frequency of calcium oscillations and reduce the rate of in vitro migration of astrocyte progenitors. Such alterations were due to changes in activity of two purinergic P2 receptors, and not to the amount of released ATP. These results indicate that interleukin-1β plays an important role during early stages of CNS development, modulating calcium signaling and cell migration.
PDF
