Resident Stem Cell Population Research Articles

Epithelial-mesenchymal transition contrast in the amputated tail and limb of the northern house gecko, Hemidactylus flaviviridis.

The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.

Discovery of Transcription Factors Involved in the Maintenance of Resident Vascular Endothelial Stem Cell Properties.

A resident vascular endothelial stem cell (VESC) population expressing CD157 has been identified recently in mice. Herein, we identified transcription factors (TFs) regulating CD157 expression in endothelial cells (ECs) that were associated with drug resistance, angiogenesis, and EC proliferation. In the first screening, we detected 20 candidate TFs through the CD157 promoter and gene expression analyses. We found that 10 of the 20 TFs induced CD157 expression in ECs. We previously reported that 70% of CD157 VESCs were side population (SP) ECs that abundantly expressed ATP-binding cassette (ABC) transporters. Here, we found that the 10 TFs increased the expression of several ABC transporters in ECs and increased the proportion of SP ECs. Of these 10 TFs, we found that six (Atf3, Bhlhe40, Egr1, Egr2, Elf3, and Klf4) were involved in the manifestation of the SP phenotype. Furthermore, the six TFs enhanced tube formation and proliferation in ECs. Single-cell RNA sequence data in liver ECs suggested that Atf3 and Klf4 contributed to the production of CD157+ VESCs in the postnatal period. We concluded that Klf4 might be important for the development and maintenance of liver VESCs. Our work suggests that a TF network is involved in the differentiation hierarchy of VESCs.

CLEC12A sensitizes differentially responsive breast cancer cells to the anti-cancer effects of artemisinin by repressing autophagy and inflammation.

Enhanced inflammatory responses promote tumor progression by activating toll-like receptors (TLRs), which in turn are inhibited by C-type lectin like receptors (CTLRs), like CLEC12A. Although the presence of CLEC12A in acute myeloid leukemia is well established, its role in non-hematopoietic tumors is still obscure. In hematopoietic tumors, CLEC12A mostly inhibits TLRs and modulates inflammatory responses via NF-κB signaling. In this study, the fate of tumor progression was determined by modulating CLEC12A using artemisinin (ART), a FDA-approved anti-malarial drug, known for its anti-cancer and immunomodulatory properties with minimal adverse effects on normal cells. Effects of ART were primarily determined on hematological factors and primary metastatic organs, such as lungs, kidney and liver in normal and tumor-bearing BALB/c mice. Tumor-bearing mice were treated with different concentrations of ART and expressions of CLEC12A and associated downstream components were determined. CLEC12A was overexpressed in MDA-MB-231 and 4T1 cells, and the effects of ART were analyzed in the overexpressed cells. Silencing TLR4 using vivo morpholino was performed to elucidate its role in tumor progression in response to ART. Finally, CLEC12A modulation by ART was evaluated in the resident cancer stem cell (CSC) population. ART did not alter physiology of normal mice, in contrast to tumor-bearing mice, where ART led to tumor regression. In addition, ART reduced expression of CLEC12A. Expectedly, TLR4 expression increased, but surprisingly, that of NF-κB (RelA) and JNK/pJNK decreased, along with reduced inflammation, reduced autophagy and increased apoptosis. All the above observations reverted on overexpression of CLEC12A in MDA-MB-231 and 4T1 cells. Inhibition of TLR4, however, indicated no change in the expressions of CLEC12A, NF-κB, or apoptotic markers. The effect of ART showed a similar trend in the CSC population as in cancer cells. This study, for the first time, confirmed a differential role of CLEC12A in non-hematopoietic tumor and cancer stem cells in response to ART. Subsequent interaction and modulation of CLEC12A with ART induced tumor cell death and abrogation of CSCs, confirming a more comprehensive tumor therapy with reduced risk of recurrence. Therefore, ART may be repurposed as an effective drug for cancer treatment in future.

Sox11 is enriched in myogenic progenitors but dispensable for development and regeneration of the skeletal muscle

Transcription factors (TFs) play key roles in regulating differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associating domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER- or Rosa26CreER- driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remains to be elucidated.

Single-cell sequencing reveals the existence of fetal vascular endothelial stem cell-like cells in mouse liver

BackgroundA resident vascular endothelial stem cell (VESC) population expressing CD157 and CD200 has been identified recently in the adult mouse. However, the origin of this population and how it develops has not been characterized, nor has it been determined whether VESC-like cells are present during the perinatal period. Here, we investigated the presence of perinatal VESC-like cells and their relationship with the adult VESC-like cell population.MethodsWe applied single-cell RNA sequencing of endothelial cells (ECs) from embryonic day (E) 14, E18, postnatal day (P) 7, P14, and week (W) 8 liver and investigated transcriptomic changes during liver EC development. We performed flow cytometry, immunofluorescence, colony formation assays, and transplantation assays to validate the presence of and to assess the function of CD157+ and CD200+ ECs in the perinatal period.ResultsWe identified CD200− expressing VESC-like cells in the perinatal period. These cells formed colonies in vitro and had high proliferative ability. The RNA velocity tool and transplantation assay results indicated that the projected fate of this population was toward adult VESC-like cells expressing CD157 and CD200 1 week after birth.ConclusionOur study provides a comprehensive atlas of liver EC development and documents VESC-like cell lineage commitment at single-cell resolution.

Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.

Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis.

DNA Methylation Analysis Reveals Distinct Patterns in Satellite Cell-Derived Myogenic Progenitor Cells of Subjects with Spastic Cerebral Palsy.

Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient-specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC-derived myoblasts (MB) and 1774 CpG sites in differentiating SC-derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo- and hyper-methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.

RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors

Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Bone regeneration: A message from clinical medicine and basic science.

Population aging is a global phenomenon and with it, the number of bone fractures increases due to higher incidences of osteoporosis. Bone fractures in the elderly increase the risk of bedridden status and mortality. Therefore, the control of osteoporosis and bone fracture is important for healthy life expectancy, and the fundamental understanding of its pathogenesis and its application in treatment is of great social significance. To solve these clinical problems, it is necessary to integrate clinical medicine and basic research. Bone regeneration after a fracture is an essential function of the living body. The prevailing view is that a small number of resident skeletal stem cells are solely responsible for regenerative capacity. Although these cells have long been considered to be in the bone marrow, it has been shown that they are also present in the growth plate and periosteum. More recently, distinct types of cells in the bone marrow, including bone marrow stromal cells, osteoblast progenitor cells, and osteoblasts, have been shown to participate in bone regeneration. Interestingly, the cellular plasticity of differentiated cells, rather than active recruitment of resident stem cell populations, may largely account for regeneration of bone tissues; terminally differentiated cells de-differentiate into a stem cell-like state, and then re-differentiate into regenerating bone. In this review, we discuss the clinical risk and preventive therapy of bone fractures and the current concept of bone regeneration in basic mechanical insights, which may prove useful to both clinicians and researchers.

The Hunt Is On! In Pursuit of the Ideal Stem Cell Population for Cartilage Regeneration.

Cartilage injury and degeneration are hallmarks of osteoarthritis (OA), the most common joint disease. OA is a major contributor to pain, loss of function, and reduced quality of life. Over the last decade, considerable research efforts have focused on cell-based therapies, including several stem cell-derived approaches to reverse the cartilage alterations associated with OA. Although several tissue sources for deriving cell-based therapies have been identified, none of the resident stem cell populations have adequately fulfilled the promise of curing OA. Indeed, many cell products do not contain true stem cells. As well, issues with aggressive marketing efforts, combined with a lack of evidence regarding efficacy, lead the several national regulatory bodies to discontinue the use of stem cell therapy for OA until more robust evidence becomes available. A review of the evidence is timely to address the status of cell-based cartilage regeneration. The promise of stem cell therapy is not new and has been used successfully to treat non-arthritic diseases, such as hematopoietic and muscle disorders. These fields of regenerative therapy have the advantage of a considerable foundation of knowledge in the area of stem cell repair mechanisms, the role of the stem cell niche, and niche-supporting cells. This foundation is lacking in the field of cartilage repair. So, where should we look for the ideal stem cell to regenerate cartilage? It has recently been discovered that cartilage itself may contain a population of SC-like progenitors. Other potential tissues include stem cell-rich dental pulp and the adolescent growth plate, the latter of which contains chondrocyte progenitors essential for producing the cartilage scaffold needed for bone growth. In this article, we review the progress on stem cell therapies for arthritic disorders, focusing on the various stem cell populations previously used for cartilage regeneration, successful cases of stem cell therapies in muscle and hemopoietic disorders, some of the reasons why these other fields have been successful (i.e., “lessons learned” to be applied to OA stem cell therapy), and finally, novel potential sources of stem cells for regenerating damaged cartilage in vivo.

Abstract 240: Role Of Smooth Muscle-derived Vascular Progenitor Cells In Atherosclerosis

Purpose: Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis progression by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to VV expansion to drive disease progression. Methods: We generated a highly specific lineage tracing mouse model in order to track AdvSca1-SM cells in vivo even if they differentiate into other cell types. Lineage tracing mice were placed on either normal chow or Western diet for 8 or 16 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry. Results: scRNA-Seq and flow cytometry revealed that AdvSca1-SM cells in atherosclerosis primarily differentiate into mature SMCs, modulated SMCs, and myofibroblasts. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells. Additionally, despite previous evidence of SMCs gaining a macrophage-like phenotype in atherosclerosis, we identified only rare instances of AdvSca1-SM cells contributing to macrophage populations. Conclusions: As in our findings in acute vascular injury, AdvSca1-SM cells in atherosclerosis predominantly differentiate into SMCs and myofibroblasts. Future studies on advanced lesions will define the functional role of AdvSca1-SM cells in atherosclerotic plaque progression.

Assessing the Role of Ependymal and Vascular Cells as Sources of Extracellular Cues Regulating the Mouse Ventricular-Subventricular Zone Neurogenic Niche.

Neurogenesis persists in selected regions of the adult mouse brain; among them, the ventricular-subventricular zone (V-SVZ) of the lateral ventricles represents a major experimental paradigm due to its conspicuous neurogenic output. Postnatal V-SVZ neurogenesis is maintained by a resident population of neural stem cells (NSCs). Although V-SVZ NSCs are largely quiescent, they can be activated to enter the cell cycle, self-renew and generate progeny that gives rise to olfactory bulb interneurons. These adult-born neurons integrate into existing circuits to modify cognitive functions in response to external stimuli, but cells shed by V-SVZ NSCs can also reach injured brain regions, suggesting a latent regenerative potential. The V-SVZ is endowed with a specialized microenvironment, which is essential to maintain the proliferative and neurogenic potential of NSCs, and to preserve the NSC pool from exhaustion by finely tuning their quiescent and active states. Intercellular communication is paramount to the stem cell niche properties of the V-SVZ, and several extracellular signals acting in the niche milieu have been identified. An important part of these signals comes from non-neural cell types, such as local vascular cells, ependymal and glial cells. Understanding the crosstalk between NSCs and other niche components may aid therapeutic approaches for neuropathological conditions, since neurodevelopmental disorders, age-related cognitive decline and neurodegenerative diseases have been associated with dysfunctional neurogenic niches. Here, we review recent advances in the study of the complex interactions between V-SVZ NSCs and their cellular niche. We focus on the extracellular cues produced by ependymal and vascular cells that regulate NSC behavior in the mouse postnatal V-SVZ, and discuss the potential implication of these molecular signals in pathological conditions.

Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell– and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFβ1). Blocking TGFβ signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell–stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BackgroundThe pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains.ObjectivesThe purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes.MethodThe literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review.ResultsMost published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence‐activated cell sorting (FACS) and magnetic‐activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta‐glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21‐days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein‐1 (DMP ‐1), the most frequent.DiscussionWhile this review highlights variability among experimental approaches, it does however identify a consensus experimental approach.ConclusionStandardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

Hindlimb Immobilization Increases IL-1β and Cdkn2a Expression in Skeletal Muscle Fibro-Adipogenic Progenitor Cells: A Link Between Senescence and Muscle Disuse Atrophy.

Loss of muscle mass and strength contributes to decreased independence and an increased risk for morbidity and mortality. A better understanding of the cellular and molecular mechanisms underlying muscle atrophy therefore has significant clinical and therapeutic implications. Fibro-adipogenic progenitors (FAPs) are a skeletal muscle resident stem cell population that have recently been shown to play vital roles in muscle regeneration and muscle hypertrophy; however, the role that these cells play in muscle disuse atrophy is not well understood. We investigated the role of FAPs in disuse atrophy in vivo utilizing a 2-week single hindlimb immobilization model. RNA-seq was performed on FAPs isolated from the immobilized and non-immobilized limb. The RNAseq data show that IL-1β is significantly upregulated in FAPs following 2 weeks of immobilization, which we confirmed using droplet-digital PCR (ddPCR). We further validated the RNA-seq and ddPCR data from muscle in situ using RNAscope technology. IL-1β is recognized as a key component of the senescence-associated secretory phenotype, or SASP. We then tested the hypothesis that FAPs from the immobilized limb would show elevated senescence measured by cyclin-dependent kinase inhibitor 2A (Cdkn2a) expression as a senescence marker. The ddPCR and RNAscope data both revealed increased Cdkn2a expression in FAPs with immobilization. These data suggest that the gene expression profile of FAPs is significantly altered with disuse, and that disuse itself may drive senescence in FAPs further contributing to muscle atrophy.

Blood vessel formation by resident endothelial stem cell population

Blood vessel formation by resident endothelial stem cell population

Tributyrin, a Butyrate Pro-Drug, Primes Satellite Cells for Differentiation by Altering the Epigenetic Landscape.

Satellite cells (SC) are a population of muscle resident stem cells that are responsible for postnatal muscle growth and repair. With investigation into the genomic regulation of SC fate, the role of the epigenome in governing SC myogenesis is becoming clearer. Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective at enhancing the myogenic program of SC, but their role in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how an HDAC inhibitor, butyrate, promotes myogenic differentiation. SC from tributyrin treated neonatal piglets showed a decrease relative to SC from control animals in the expression of enhance of zeste homologue-2 (EZH2), a chromatin modifier, ex vivo. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis of SC isolated from tributyrin treated pigs showed a global reduction of the tri-methylation of lysine 27 of histone H3 (H3K27me3) repressive chromatin mark. To determine if reductions in EZH2 was the primary mechanism through which butyrate affects SC behavior, SC were transfected with siRNA targeting EZH2, treated with 0.5 mM butyrate, or both. Treatment with butyrate reduced paired-box-7 (Pax7) and myogenic differentiation-1 (MyoD) gene expression, while siRNA caused reductions in EZH2 had no effect on their expression. EZH2 depletion did result in an increase in differentiating SC, but not in myotube hypertrophy. These results indicate that while EZH2 reduction may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance the myogenic program.

Pannexin 1 Regulates Skeletal Muscle Regeneration by Promoting Bleb-Based Myoblast Migration and Fusion Through a Novel Lipid Based Signaling Mechanism.

Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1–/– mice exhibit impaired muscle regeneration after injury. Panx1–/– myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.