The cilium-centrosome axis in coupling cell cycle exit and cell fate.

The centrosome is an evolutionarily conserved, ancient organelle whose role in cell division was first described over a century ago. The structure and function of the centrosome as a microtubule-organizing center, and of its extracellular extension - the primary cilium - as a sensory antenna, have since been extensively studied, but the role of the cilium-centrosome axis in cell fate is still emerging. In this Opinion piece, we view cellular quiescence and tissue homeostasis from the vantage point of the cilium-centrosome axis. We focus on a less explored role in the choice between distinct forms of mitotic arrest - reversible quiescence and terminal differentiation, which play distinct roles in tissue homeostasis. We outline evidence implicating the centrosome-basal body switch in stem cell function, including how the cilium-centrosome complex regulates reversible versus irreversible arrest in adult skeletal muscle progenitors. We then highlight exciting new findings in other quiescent cell types that suggest signal-dependent coupling of nuclear and cytoplasmic events to the centrosome-basal body switch. Finally, we propose a framework for involvement of this axis in mitotically inactive cells and identify future avenues for understanding how the cilium-centrosome axis impacts central decisions in tissue homeostasis.

PTPRU, a quiescence-induced receptor tyrosine phosphatase negatively regulates osteogenic differentiation of human mesenchymal stem cells.

Bone marrow mesenchymal stem cells (MSCs) are heterogeneous osteo-progenitors that are mainly responsible for bone regeneration and homeostasis. In vivo, a subpopulation of bone marrow MSCs persists in a quiescent state, providing a source of new cells for repair. Previously, we reported that induction of quiescence in hMSCs in vitro skews their differentiation potential in favour of osteogenesis while suppressing adipogenesis. Herein, we uncover a new role for a protein tyrosine phosphatase, receptor type U (PTPRU) in repressing osteogenesis during quiescence. A 75 kD PTPRU protein isoform was found to be specifically induced during quiescence and down-regulated during cell cycle reactivation. Using siRNA-mediated knockdown, we report that in proliferating hMSC, PTPRU preserves self-renewal, while in quiescent hMSC, PTPRU not only maintains reversibility of cell cycle arrest but also suppresses expression of osteogenic lineage genes. Knockdown of PTPRU in proliferating or quiescent hMSC de-represses osteogenic markers, and enhances induced osteogenic differentiation. We also show that PTPRU positively regulates a ß-catenin-TCF transcriptional reporter. Taken together, our study suggests a role for a quiescence-induced 75kD PTPRU isoform in modulating bone differentiation in hMSC, potentially involving the Wnt pathway.

A transcriptionally repressed quiescence program is associated with paused RNA polymerase II and is poised for cell cycle re-entry.

Adult stem cells persist in mammalian tissues by entering a state of reversible quiescence, referred to as G0, which is associated with low levels of transcription. Using cultured myoblasts and muscle stem cells, we report that in G0, global RNA content and synthesis are substantially repressed, correlating with decreased RNA polymerase II (RNAPII) expression and activation. Integrating RNAPII occupancy and transcriptome profiling, we identify repressed networks and a role for promoter-proximal RNAPII pausing in G0. Strikingly, RNAPII shows enhanced pausing in G0 on repressed genes encoding regulators of RNA biogenesis (such as Ncl, Rps24, Ctdp1), and release of pausing is associated with increased expression of these genes in G1. Knockdown of these transcripts in proliferating cells leads to induction of G0 markers, confirming the importance of their repression in establishment of G0. A targeted screen of RNAPII regulators revealed that knockdown of Aff4 (a positive regulator of elongation) unexpectedly enhances expression of G0-stalled genes and hastens S phase; however, the negative elongation factor (NELF) complex, a regulator of pausing, appears to be dispensable. We propose that RNAPII pausing contributes to transcriptional control of a subset of G0-repressed genes to maintain quiescence and impacts the timing of the G0-G1 transition. This article has an associated First Person interview with the first authors of the paper.

SMARCA4 biology in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Inducible expression of Oct-3/4 reveals synergy with Klf4 in targeting Cyclin A2 to enhance proliferation during early reprogramming.

During reprogramming of somatic cells, heightened proliferation is one of the earliest changes observed. While other early events such as mesenchymal-to-epithelial transition have been well studied, the mechanisms by which the cell cycle switches from a slow cycling state to a faster cycling state are still incompletely understood. To investigate the role of Oct-3/4 in this early transition, we created a 4-Hydroxytamoxifen (OHT) dependent Oct-3/4 Estrogen Receptor fusion (OctER). We confirmed that OctER can substitute for Oct-3/4 to reprogram mouse embryonic fibroblasts to a pluripotent state. During the early stages of reprograming, Oct-3/4 and Klf4 individually did not affect cell proliferation but in combination hastened the cell cycle. Using OctER + Klf4, we found that proliferative enhancement is OHT dose-dependent, suggesting that OctER is the driver of this transition. We identified Cyclin A2 as a likely target of Oct-3/4 + Klf4. In mESC, Klf4 and Oct-3/4 bind ∼100bp upstream of Cyclin A2 CCRE, suggesting a potential regulatory role. Using inducible OctER, we show a dose-dependent induction of Cyclin A2 promoter-reporter activity. Taken together, our results suggest that Cyclin A2 is a key early target during reprogramming, and support the view that a rapid cell cycle assists the transition to pluripotency.

mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation.

BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation. METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells. RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta. CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Comparative nuclear matrix proteome analysis of skeletal muscle cells in different cellular states.

The nuclear matrix (NuMat) serves as the structural framework for organizing and maintaining nuclear architecture, however, the mechanisms by which this non-chromatin compartment is constructed and regulated are poorly understood. This study presents a proteomic analysis of the NuMat isolated from cultured skeletal muscle cells in three distinct cellular states- proliferating myoblasts (MBs), terminally differentiated myotubes (MTs), and mitotically quiescent (G0) myoblasts. About 40% of the proteins identified were found to be common in the NuMat proteome of these morphologically and functionally distinct cell states. These proteins, termed as the "core NuMat," define the stable, conserved, structural constituent of the nucleus, with functions such as RNA splicing, cytoskeletal organization, and chromatin modification, while the remaining NuMat proteins showed cell-state specificity, consistent with a more dynamic and potentially regulatory function. Specifically, myoblast NuMat was enriched in cell cycle, DNA replication and repair proteins, myotube NuMat in muscle differentiation and muscle function proteins, while G0 NuMat was enriched in metabolic, transcription, and transport proteins. These findings offer a new perspective for a cell-state-specific role of nuclear architecture and spatial organization, integrated with diverse cellular processes, and implicate NuMat proteins in the control of the cell cycle, lineage commitment, and differentiation.

The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts.

BACKGROUND: Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood. RESULTS: Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro, but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1. CONCLUSIONS: Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation.

Muscle differentiation is controlled by adhesion and growth factor-dependent signalling through common effectors that regulate muscle-specific transcriptional programs. Here we report that mDiaphanous1, an effector of adhesion-dependent RhoA-signalling, negatively regulates myogenesis at the level of Myogenin expression. In myotubes, over-expression of mDia1ΔN3, a RhoA-independent mutant, suppresses Myogenin promoter activity and expression. We investigated mDia1-interacting proteins that may counteract mDia1 to permit Myogenin expression and timely differentiation. Using yeast two-hybrid and mass-spectrometric analysis, we report that mDia1 has a stage-specific interactome, including Prohibitin2, MyoD, Akt2, and ß-Catenin, along with a number of proteosomal and mitochondrial components. Of these interacting partners, Prohibitin2 colocalises with mDia1 in cytoplasmic punctae in myotubes. We mapped the interacting domains of mDia1 and Phb2, and used interacting (mDia1ΔN3/Phb2 FL or mDia1ΔN3/Phb2-Carboxy) and non-interacting pairs (mDia1H + P/Phb2 FL or mDia1ΔN3/Phb2-Amino) to dissect the functional consequences of this partnership on Myogenin promoter activity. Co-expression of full-length as well as mDia1-interacting domains of Prohibitin2 reverse the anti-myogenic effects of mDia1ΔN3, while non-interacting regions do not. Our results suggest that Prohibitin2 sequesters mDia1, dampens its anti-myogenic activity and fine-tunes RhoA-mDia1 signalling to promote differentiation. Overall, we report that mDia1 is multi-functional signalling effector whose anti-myogenic activity is modulated by a differentiation-dependent interactome. The data have been deposited to the ProteomeXchange with identifier PXD012257.

Adult Muscle Stem Cells: Exploring the Links Between Systemic and Cellular Metabolism.

Emerging evidence suggests that metabolites are important regulators of skeletal muscle stem cell (MuSC) function and fate. While highly proliferative in early life, MuSCs reside in adult skeletal muscle tissue in a quiescent and metabolically depressed state, but are critical for the homeostatic maintenance and regenerative response of the tissue to damage. It is well established that metabolic activity in MuSC changes with their functional activation, but the spatiotemporal links between physiological metabolism and stem cell metabolism require explicit delineation. The quiescent MuSC is defined by a specific metabolic state, which is controlled by intrinsic and extrinsic factors during physiological and pathological tissue dynamics. However, the extent of tissue and organismal level changes driven by alteration in metabolic state of quiescent MuSC is currently not well defined. In addition to their role as biosynthetic precursors and signaling molecules, metabolites are key regulators of epigenetic mechanisms. Emerging evidence points to metabolic control of epigenetic mechanisms in MuSC and their impact on muscle regenerative capacity. In this review, we explore the links between cell-intrinsic, tissue level, and systemic metabolic state in the context of MuSC metabolic state, quiescence, and tissue homeostasis to highlight unanswered questions.

The transcription factor Lef1 switches partners from ß-catenin to Smad3 during muscle stem cell quiescence.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, persist in adult mammals by entering a state of quiescence (G0) during the early postnatal period. Quiescence is reversed during damage-induced regeneration and re-established after regeneration. Entry of cultured myoblasts into G0 is associated with a specific, reversible induction of Wnt target genes, thus implicating members of the Tcf and Lef1 (Tcf/Lef) transcription factor family, which mediate transcriptional responses to Wnt signaling, in the initiation of quiescence. We found that the canonical Wnt effector ß-catenin, which cooperates with Tcf/Lef, was dispensable for myoblasts to enter quiescence. Using pharmacological and genetic approaches in cultured C2C12 myoblasts and in MuSCs, we demonstrated that Tcf/Lef activity during quiescence depended not on ß-catenin but on the transforming growth factor-ß (TGF-ß) effector and transcriptional coactivator Smad3, which colocalized with Lef1 at canonical Wnt-responsive elements and directly interacted with Lef1 specifically in G0 Depletion of Smad3, but not ß-catenin, reduced Lef1 occupancy at target promoters, Tcf/Lef target gene expression, and self-renewal of myoblasts. In vivo, MuSCs underwent a switch from ß-catenin-Lef1 to Smad3-Lef1 interactions during the postnatal switch from proliferation to quiescence, with ß-catenin-Lef1 interactions recurring during damage-induced reactivation. Our findings suggest that the interplay of Wnt-Tcf/Lef and TGF-ß-Smad3 signaling activates canonical Wnt target promoters in a manner that depends on ß-catenin during myoblast proliferation but is independent of ß-catenin during MuSC quiescence.

Cycling to Meet Fate: Connecting Pluripotency to the Cell Cycle.

Pluripotent stem cells are characterized by their high proliferative rates, their ability to self-renew and their potential to differentiate to all the three germ layers. This rapid proliferation is brought about by a highly modified cell cycle that allows the cells to quickly shuttle from DNA synthesis to cell division, by reducing the time spent in the intervening gap phases. Many key regulators that define the somatic cell cycle are either absent or exhibit altered behavior, allowing the pluripotent cell to bypass cell cycle checkpoints typical of somatic cells. Experimental analysis of this modified stem cell cycle has been challenging due to the strong link between rapid proliferation and pluripotency, since perturbations to the cell cycle or pluripotency factors result in differentiation. Despite these hurdles, our understanding of this unique cell cycle has greatly improved over the past decade, in part because of the availability of new technologies that permit the analysis of single cells in heterogeneous populations. This review aims to highlight some of the recent discoveries in this area with a special emphasis on different states of pluripotency. We also discuss the highly interlinked network that connects pluripotency factors and key cell cycle genes and review evidence for how this interdependency may promote the rapid cell cycle. This issue gains translational importance since disruptions in stem cell proliferation and differentiation can impact disorders at opposite ends of a spectrum, from cancer to degenerative disease.

Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics.

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.

Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells' morphology and proliferation.

The effect of substrate stiffness on the cellular morphology, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) has been extensively researched and well established. However, the majority of these studies are done with a low seeding density where cell to cell interactions do not play a significant role. While these conditions permit an analysis of cell-substratum interactions at the single cell level, such a model system fails to capture a critical aspect of the cellular micro-environment in vivo, i.e. the cell-cell interaction via matrix deformation (i.e., strain). To address this question, we seeded hMSCs on soft poly-acrylamide (PAA) gels, at a seeding density that permits cells to be mechanically interacting via the underlying substrate. We found that as the intercellular distance decreases with the increasing seeding density, cellular sensitivity towards the substrate rigidity becomes significantly diminished. With the increasing seeding density, the cell spread area increased on a soft substrate (500 Pa) but reduced on an even slightly stiffer substrate (2 kPa) as well as on glass making them indistinguishable at a high seeding density. Not only in terms of cell spread area but also at a high seeding density, cells formed mature focal adhesions and prominent stress fibres on a soft substrate similar to that of the cells being cultured on a stiff substrate. The decreased intercellular distance also influenced the proliferation rate of the cells: higher seeding density on the soft substrate showed cell cycle progression similar to that of the cells on glass substrates. In summary, this paper demonstrates how the effect of substrate rigidity on the cell morphology and fate is a function of inter-cellular distance when seeded on a soft substrate. Our AFM data suggest that such changes happen due to local strain stiffening of the soft PAA gel, an effect that has been rarely reported in the literature so far.

Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis.

Regenerative potential in adult stem cells is closely associated with the establishment of-and exit from-a temporary state of quiescence. Emerging evidence not only provides a rationale for the link between lineage determination programs and cell cycle regulation but also highlights the understanding of quiescence as an actively maintained cellular program, encompassing networks and mechanisms beyond mitotic inactivity or metabolic restriction. Interrogating the quiescent genome and transcriptome using deep-sequencing technologies offers an unprecedented view of the global mechanisms governing this reversibly arrested cellular state and its importance for cell identity. While many efforts have identified and isolated pure target stem cell populations from a variety of adult tissues, there is a growing appreciation that their isolation from the stem cell niche in vivo leads to activation and loss of hallmarks of quiescence. Thus, in vitro models that recapitulate the dynamic reversibly arrested stem cell state in culture and lend themselves to comparison with the activated or differentiated state are useful templates for genome-wide analysis of the quiescence network.In this chapter, we describe the methods that can be adopted for whole genome epigenomic and transcriptomic analysis of cells derived from one such established culture model where mouse myoblasts are triggered to enter or exit quiescence as homogeneous populations. The ability to synchronize myoblasts in G0 permits insights into the genome in "deep quiescence." The culture methods for generating large populations of quiescent myoblasts in either 2D or 3D culture formats are described in detail in a previous chapter in this series (Arora et al. Methods Mol Biol 1556:283-302, 2017). Among the attractive features of this model are that genes isolated from quiescent myoblasts in culture mark satellite cells in vivo (Sachidanandan et al., J Cell Sci 115:2701-2712, 2002) providing a validation of its approximation of the molecular state of true stem cells. Here, we provide our working protocols for ChIP-seq and RNA-seq analysis, focusing on those experimental elements that require standardization for optimal analysis of chromatin and RNA from quiescent myoblasts, and permitting useful and revealing comparisons with proliferating myoblasts or differentiated myotubes.

C-State: an interactive web app for simultaneous multi-gene visualization and comparative epigenetic pattern search.

BACKGROUND: Comparative epigenomic analysis across multiple genes presents a bottleneck for bench biologists working with NGS data. Despite the development of standardized peak analysis algorithms, the identification of novel epigenetic patterns and their visualization across gene subsets remains a challenge. RESULTS: We developed a fast and interactive web app, C-State (Chromatin-State), to query and plot chromatin landscapes across multiple loci and cell types. C-State has an interactive, JavaScript-based graphical user interface and runs locally in modern web browsers that are pre-installed on all computers, thus eliminating the need for cumbersome data transfer, pre-processing and prior programming knowledge. CONCLUSIONS: C-State is unique in its ability to extract and analyze multi-gene epigenetic information. It allows for powerful GUI-based pattern searching and visualization. We include a case study to demonstrate its potential for identifying user-defined epigenetic trends in context of gene expression profiles.

Mimicking Muscle Stem Cell Quiescence in Culture: Methods for Synchronization in Reversible Arrest.

Growing evidence supports the view that in adult stem cells, the defining stem cell features of potency and self-renewal are associated with the quiescent state. Thus, uncovering the molecular logic of this reversibly arrested state underlies not only a fundamental understanding of adult tissue dynamics but also hopes for therapeutic regeneration and rejuvenation of damaged or aging tissue. A key question concerns how adult stem cells use quiescence to establish or reinforce the property of self-renewal. Since self-renewal is largely studied by assays that measure proliferation, the concept of self-renewal programs imposed during non-proliferating conditions is counterintuitive. However, there is increasing evidence generated by deconstructing the quiescent state that highlights how programs characteristic of this particular cell cycle exit may enhance stem cell capabilities, through both cell-intrinsic and extrinsic programs.Toward this end, culture models that recapitulate key aspects of stem cell quiescence are useful for molecular analysis to explore attributes and regulation of the quiescent state. In this chapter, we review the different methods used to generate homogeneous populations of quiescent muscle cells, largely by manipulating culture conditions that feed into core signaling programs that regulate the cell cycle. We also provide detailed protocols developed or refined in our lab over the past two decades.

Exosomes: mobile platforms for targeted and synergistic signaling across cell boundaries.

Intercellular communications play a vital role during tissue patterning, tissue repair, and immune reactions, in homeostasis as well as in disease. Exosomes are cell-derived secreted vesicles, extensively studied for their role in intercellular communication. Exosomes have the intrinsic ability to package multiple classes of proteins and nucleic acids within their lumens and on their membranes. Here, we explore the hypothesis that exosomal targeting may represent a cellular strategy that has evolved to deliver specific combinations of signals to specific target cells and influence normal or pathological processes. This review aims to evaluate the available evidence for this hypothesis and to identify open questions whose answers will illuminate our understanding and applications of exosome biology.

Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest.

OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts. MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFß, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and ß-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.