Muscle Progenitor Research Articles

Pitx2 Differentially Regulates the Distinct Phases of Myogenic Program and Delineates Satellite Cell Lineages During Muscle Development.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors’ fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

Generation of muscle progenitors from human-induced pluripotent stem cells

BackgroundSmall molecules have a role in the differentiation of human-induced pluripotent stem cells (hiPSCs) into different cell linages. The aim of this study was to evaluate the differentiation of hiPSCs into cardiac or skeletal myogenic progenitors with a single small molecule.MethodshiPSCs were treated with three different small molecules such as Isoxazole-9, Danazol and Givinostat in serum-free medium for 7 days. Cell viability, qRT-PCR, western blots, and immunostaining were assessed after treatment of hiPSCs with small molecules.ResultsHigher hiPSC viability was observed in hiPSCs treated with Isoxazole-9 (25 µM), Danazol (25 µM) and Givinostat (150 nM) versus control (P < 0.05). Givinostat had dual effect by generating both skeletal and cardiac progenitor cells versus Isoxazole-9 and Danazol after 7 days. Givinostat treatment induced upregulation of skeletal myogenic genes and their protein expression levels on day 4 and further increased on day 8 (P < 0.05) versus control. Furthermore,positive stained cells for Pax3, Myf5, MyoD1, dystrophin, desmin, myogenin, and β-catenin at 1 month. Givinostat increased upregulation of cardiac gene expression levels versus control after day 4 (P < 0.05), with positive stained cells for Nkx2.5, GATA4, TnT, TnI, connexin 43 and α-sarcomeric actinin at 1 month.ConclusionsPretreatment of hiPSCs with Givinostat represents a viable strategy for producing both cardiac/skeletal myogenic progenitors in vitro for cell therapies against myocardial infarction and Duchenne muscular dystrophy.

Large-scale genome editing based on high-capacity adenovectors andCRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells.

Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.

Poster 247: Muscle ERRγ Overexpression Mitigates the Muscle Atrophy after ACL injury

Poster 247: Muscle ERRγ Overexpression Mitigates the Muscle Atrophy after ACL injury

Kank1 Is Essential for Myogenic Differentiation by Regulating Actin Remodeling and Cell Proliferation in C2C12 Progenitor Cells.

Actin cytoskeleton dynamics are essential regulatory processes in muscle development, growth, and regeneration due to their modulation of mechanotransduction, cell proliferation, differentiation, and morphological changes. Although the KN motif and ankyrin repeat domain-containing protein 1 (Kank1) plays a significant role in cell adhesion dynamics, actin polymerization, and cell proliferation in various cells, the functional significance of Kank1 during the myogenic differentiation of progenitor cells has not been explored. Here, we report that Kank1 acts as a critical regulator of the proliferation and differentiation of muscle progenitor cells. Kank1 was found to be expressed at a relatively high level in C2C12 myoblasts, and its expression was modulated during the differentiation. Depletion of Kank1 by siRNA (siKank1) increased the accumulation of filamentous actin (F-actin). Furthermore, it facilitated the nuclear localization of Yes-associated protein 1 (YAP1) by diminishing YAP1 phosphorylation in the cytoplasm, which activated the transcriptions of YAP1 target genes and promoted proliferation and cell cycle progression in myoblasts. Notably, depletion of Kank1 suppressed the protein expression of myogenic regulatory factors (i.e., MyoD and MyoG) and dramatically inhibited myoblast differentiation and myotube formation. Our results show that Kank1 is an essential regulator of actin dynamics, YAP1 activation, and cell proliferation and that its depletion impairs the myogenic differentiation of progenitor cells by promoting myoblast proliferation triggered by the F-actin-induced nuclear translocation of YAP1.

99. Aging Associated Loss of Vegfa Impairs Muscle Regeneration

Purpose: Global populations, particularly in developed countries, have continued to age. Notably, aging is intimately correlated with frailty, a parameter that has been previously demonstrated to correlate significantly with mortality and concomitantly correlate with loss of muscle mass. The molecular mechanisms behind aging associated impaired skeletal muscle regeneration remain incompletely understood. Our group previously demonstrated aging-associated loss of hypoxia signaling impairs skeletal muscle myogenic potential. Given these findings, we hypothesized vascular endothelial growth factor (VEGFA), a potent downstream signal well characterized in nascent vascular ingrowth and muscle progenitor differentiation, to critically regulate regeneration in young and aged skeletal muscle. Methods: Young C57BL/6 mice (12-14 weeks old) and old C57BL/6 mice (24-25 months old) were subjected to a previously validated model of muscle cryoinjury (n=5-6/group) to induce regeneration. The tibialis anterior muscle was injured using direct contact with a metal probe cooled in dry ice. Quantifications of cross sectional area (CSA) of myofibers were performed across 10 photos/sample by blinded reviewer via ImageJ. Western blots were performed from whole TA muscle lysates for quantification of VEGFA with GAPDH for normalization. To evaluate the role of VEGF in muscle regeneration, we performed a similar and separate series of experiments using VEGFlo (Jackson Labs Stock No. 027315) mice, which exhibit a systemic, 75% decrease in VEGFA activity, and littermate controls. In addition, ML228, a hypoxia signaling activator known to increase VEGFA levels, was injected into both young and old mice, as well as VEGFlo and littermate controls, to determine whether VEGFA augmentation promotes muscle regeneration in aging. Results: Old mice demonstrated markedly impaired ability of regenerating myofiber CSA on days post injury (DPI) 5 in comparison to young mice (Young vs. Old: 526 vs. 373µm2, p<.001) and DPI 10 (Y v. O: 1250 vs. 833µm2, p<.001). VEGFA protein levels were markedly reduced in the gastrocnemius of old mice in comparison to young. Similarly, VEGFlo mice exhibited significantly smaller CSA of regenerating fibers as compared to littermate controls (WT vs. lo: 541 vs. 238µm2, p=.0011). Pharmacologic augmentation of VEGFA, using ML228, improved skeletal muscle regeneration in both old mice and VEGFlo mice. (WT vs. lo: CSA 757 vs. 662µm2, p=.387). Conclusions: Muscle regeneration declines with with aging, in a fashion correlated to loss of VEGFA levels within skeletal muscle. Systemic loss of VEGFA is further correlated with poor muscle regeneration in genetically modified mice. Furthermore, exogenous activation of hypoxic signaling/VEGFA reverses this reduced capacity for muscle regeneration in old and VEGFlo mice. Supplementation of VEGFA could represent a therapeutic target in aging patients with skeletal muscle loss.

Towards innervation of bioengineered muscle constructs: Development of a sustained neurotrophic factor delivery and release system

Surgical implantation of biomanufactured skeletal muscle constructs has recently emerged as a promising strategy to treat volumetric muscle defects. However, due to the slow rate of neural regeneration and integration, timely innervation of the implanted constructs with the host peripheral nerves remains an unresolved challenge. This study aims to develop a sustained release neurotrophic factor (NF) delivery system to accelerate peripheral nerve regeneration and innervation of three-dimensional (3D) bioprinted skeletal muscle constructs. Poly (lactic-co-glycolic acid) (PLGA) microspheres were selected as a delivery system for efficient loading and sustained release of two potent NFs: ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF). We demonstrate that the NFs can be loaded within the PLGA microspheres with a high encapsulation efficiency (75.4% ± 12.6%). The NF-loaded microspheres were incorporated into the fibrinogen-based bioink used to produce biomanufactured skeletal muscle constructs and tested for printability. The microspheres did not change the viscoelastic properties of the bioink, nor did they affect the viability of human muscle progenitor cells. The release kinetic test confirmed that the bioprinted muscle constructs with the NFs-loaded microspheres released the NFs in a sustained manner compared to the bioprinted muscle construct without microspheres. The released NFs maintained their biological activities. In an in vitro neurite outgrowth assay, the NFs released from the PLGA microspheres facilitated the neurite growth over a longer time scale than the NFs directly loaded in the hydrogel. These results demonstrate the feasibility of incorporating the microsphere-based NF delivery system for accelerating neural regeneration in future in vivo applications involving biomanufactured muscle constructs.

Engineered meatballs via scalable skeletal muscle cell expansion and modular micro-tissue assembly using porous gelatin micro-carriers

The emerging field of cultured meat faces several technical hurdles, including the scale-up production of quality muscle and adipose progenitor cells, and the differentiation and bioengineering of these cellular materials into large, meat-like tissue. Here, we present edible, 3D porous gelatin micro-carriers (PoGelat-MCs), as efficient cell expansion scaffolds, as well as modular tissue-engineering building blocks for lab-grown meat. PoGelat-MC culture in spinner flasks, not only facilitated the scalable expansion of porcine skeletal muscle satellite cells and murine myoblasts, but also triggered their spontaneous myogenesis, in the absence of myogenic reagents. Using 3D-printed mold and transglutaminase, we bio-assembled pork muscle micro-tissues into centimeter-scale meatballs, which exhibited similar mechanical property and higher protein content compared to conventional ground pork meatballs. PoGelat-MCs also supported the expansion and differentiation of 3T3L1 murine pre-adipocytes into mature adipose micro-tissues, which could be used as modular assembly unit for engineered fat-containing meat products. Together, our results highlight PoGelat-MCs, in combination with dynamic bioreactors, as a scalable culture system to produce large quantity of highly-viable muscle and fat micro-tissues, which could be further bio-assembled into ground meat analogues.

Impaired Mitochondrial Folate Metabolism Reduces Mitochondrial Respiration in Mouse Muscle Progenitor Cells

ObjectivesMitochondrial dysfunction is a central contributor to aging. Mitochondrial DNA mutations increase with aging and are associated with low serum folate. Furthermore, both increased age and low serum folate are suggested to increase skeletal muscle fiber loss and impair muscle function. However, no research has focused on the metabolic pathway linking the causal relationship. Data from our lab indicate that folate-mediated one-carbon metabolism may play a role, as loss of the mitochondrial specific isozyme (serine hydroxymethyltransferase 2, SHMT2) and folate depletion both result in mitochondrial DNA instability and impaired mitochondrial function. Furthermore, serum folate and SHMT2 gene expression levels decrease with aging, potentially providing a causal mechanism for age-associated mitochondrial DNA mutations and skeletal muscle impairments. To date, little is known about the relationship between loss of SHMT2 enzyme expression and folate status in skeletal muscles.MethodsTibialis anterior muscles were harvested from 10-week-old wild-type (Shmt2+/+) and heterozygous (Shmt2+/−) mice that were weaned onto either a folate-sufficient diet or a modified diet lacking folic acid. Skeletal muscles used to isolate primary muscle progenitor cells (MPCs) were isolated from 20-week-old Shmt2+/+ and Shmt2+/− mouse hindlimbs. MPCs were cultured to 80–90% confluency and then differentiated in low-serum medium for 7–10 days. Skeletal muscles were examined for mitochondrial DNA content and MPCs were examined for mitochondrial function.ResultsHeterozygous loss of Shmt2 resulted in an 80% reduction in SHMT2 protein levels in mouse tibialis anterior muscle. Mice fed a folate-depleted diet for 7 weeks exhibited a 25% reduction in mitochondrial DNA content compared to the folate-sufficient group. In MPCs, reduced Shmt2 expression impaired basal and maximal respiration. Furthermore, protein levels of mitochondrial NADH dehydrogenase 3 were reduced and protein levels of PGC1α and mitofusin 2 were elevated with Shmt2 heterozygosity in MPCs.ConclusionsOur results demonstrate that folate depletion reduced mitochondrial DNA content in tibialis anterior skeletal muscle and loss of SHMT2 impairs MPCs mitochondrial respiration and may influence mitochondrial biogenesis and dynamics.Funding SourcesPresident's Council of Cornell Women Award.

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

Continuous Production of Acoustically Patterned Cells Within Hydrogel Fibers for Musculoskeletal Tissue Engineering

AbstractMany mammalian tissues have a specific cellular arrangement that enables their unique function. For example, parallel alignment of myofibers enables uniaxial muscle contraction. To engineer structured tissues ex vivo, it is critical to recapitulate this cellular arrangement. Conventional 3D encapsulation often fails to recapitulate this complexity, motivating the need for advanced patterning approaches. In this work, an acoustofluidic device to continuously pattern mammalian cells within hydrogel fibers is engineered. Contactless acoustofluidic forces are used to control the spacing between parallel lines of cells. To enable continuous extrusion of cell‐laden hydrogel fibers, a low friction Teflon tube is integrated into the device. A photopolymerizable hydrogel allows triggering gelation externally with light once the cells are under the influence of the acoustic field, setting the patterned cells within the hydrogel fiber. Using this device, the muscle progenitor cells (myoblasts) within the hydrogel are patterned in parallel lines to mimic the structure of skeletal muscle. The increased formation of myotubes and spontaneous twitching of the myotubes in patterned samples are observed. This approach combining continuous fabrication with the tunability of acoustofluidics can create complex 3D tissues to engineer skeletal muscles as well as tendons, ligaments, vascular networks, or combinations thereof in the future.

Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro

Thyroid hormone (TH) signaling controls muscle progenitor cells differentiation. However, inflammation can alter muscle TH signaling by modulating the expression of TH transporters (Slc16a2), receptors (Thra1), and deiodinase enzymes (Dio2 and Dio3). Thus, a proinflammatory environment could affect myogenesis. The role of a low-grade inflammatory milieu in TH signaling during myogenesis needs further investigation. Herein, we aimed to study the impact of the bacterial lipopolysaccharide (LPS)-induced inflammatory stimulus on the TH signaling during myogenesis. C2C12 myoblasts differentiation was induced without (CTR) or with 10 ng/mL LPS presence. The myoblasts under LPS stimulus release the proinflammatory cytokines (IL-6 and IL-1β) and chemokines (CCL2 and CXCL-1). LPS decreases Myod1 expression by 28% during the initial myogenesis, thus reducing the myogenic stimulus. At the same time, LPS reduced the expression of Dio2 by 41% but doubled the D2 enzymatic activity. The late differentiation was not affected by inflammatory milieu, which only increased the Slc16a2 gene expression by 38%. LPS altered the intracellular metabolism of TH and reduced the initial myogenic stimulus. However, it did not affect late differentiation. Increased intracellular TH activation may be the compensatory pathway involved in the recovery of myogenic differentiation under a low-grade inflammatory milieu.

Characterization of the Three Dimensional Genome in Human Skeletal Muscle Progenitor Cells

Duchenne Muscular Dystrophy (DMD) is a devastating disease with no cure affecting approximately 1 in 3,500‐5,000 boys. Stem cell treatments using skeletal muscle stem cells (or satellite cells, SCs) provide great potential for regenerating new muscle and we have developed directed differentiation strategies to generate skeletal muscle cells from human induced pluripotent stem cells (hiPSCs). Our work has shown that hiPSCs generate PAX7+ skeletal muscle progenitor cells (SMPCs) resembling early myogenic cells that align closer to week 7‐12 in human development and are not equivalent to adult SCs. We are interested in understanding the key molecular and functional differences that control SMPC versus SC cell states. Recently, there has been an intense interest in the three dimensional (3D) organization of the genome and its involvement in cell specific gene regulation. This has led to the discovery of chromatin loops between gene enhancers and promoters as well as self‐interacting domains termed topologically associating domains (TADs). Recently published data support a role for the 3D genome in cellular differentiation, however, the differences between the 3D genome in human SMPCs compared to adult SCs is unknown. High throughput chromosome conformation capture (Hi‐C) was used to characterize the 3D genome of SMPCs. We found genome‐wide 3D configurations were different between hPSCs and SMPCs as was the number of TADs. Interestingly, TAD size was also different between cell types, suggesting dynamic control of TADs during differentiation. When focusing on the PAX7 locus, TAD boundaries differ between cell types further highlighting the dynamic nature of TADs with respect to cell specific gene expression. Not all muscle specific loci were different between cell types, however, suggesting that some TADs may be pre‐established early in the differentiation process while others are established de‐novo. With respect to chromatin looping at the PAX7 locus, we found SMPC specific looping between the PAX7 promoter and downstream sequences that were unique in SMPCs. Considering that PAX7 enhancer sequences have yet to be determined, these sequences may serve as candidate enhancers for PAX7. These data, for the first time, characterize the 3D genome of human SMPCs using Hi‐C. Moreover, these data provide candidate enhancer sequences for that could provide unique candidates for support of PAX7 SMPCs.

Changes in muscle force recovery and myofiber satellite cell incorporation by modulating nitric oxide signaling in vivo during muscle repair after lengthening contractions

During muscle repair, nitric oxide (NO) has an important role in the activation and proliferation of muscle satellite cells (MuSC). Our group has shown that increasing intracellular S‐nitrosothiol signaling in vitro by the inhibition of S‐nitrosoglutathione reductase (GSNOR), potentiates the proliferation of muscle progenitor cells. However, it is not known whether S‐nitrosothiols interfere with muscle regeneration following injury. We hypothesize that inhibiting GSNOR activity during the proliferative phase of MuSC accelerates force recovery after lengthening contractions. We investigated whether providing excess NO (with ISDN) or inhibiting inducible NO synthase (with L‐NIL) or inhibiting GSNOR (with SPL‐334) during the first 3 days of recovery from lengthening contractions (MuSC proliferation phase) in vivo would affect muscle re‐gain of strength and MuSC fusion to damaged myofibers. Procedures were approved by UCSD‐IACUC (protocol# S00250) and followed APS guidelines for animal research. Experiments were performed in 5‐6 months old Pax7CreERTdTomato mice (n=20 mice, 10 male and 10 female). Mice were treated tamoxifen (2 mg/mouse/day, i.p., for 5 days), and 7 days later the right leg was subjected to a lengthening contraction protocol (LCP). Contralateral leg was used as a non‐injured control. LCP consisted of contracting the anterior crural muscles via electrical stimulation of the peroneal nerve for 200 ms (0.1 ms pulses, 150 Hz pulse‐frequency), implementing active plantar flexion in the last 60 ms of stimulation (1,000º.s‐1), every 12 s for 30 min (150 contractions). After LCP, mice were separated in 5 groups of 4 mice each and in the first 3 days of recovery from LCP, animals were treated with either DMSO (50 µL/day), or ISDN (20 mg/Kg/day in DMSO), or SPL‐334 ([GSNORi] 1mg.Kg/day in DMSO) or L‐NIL (0.6 mg/mL in drinking water) or untreated. After 5 days of recovery from LCP, extensor digitorum longus (EDL) muscles from both legs were isolated for contractility (300 ms trains, 0.5 ms pulses, 1‐200 Hz, 95% O2, 5% CO2, 22ºC), tibialis anterior (TA) muscles were fixed for immunohistology, and serum was collected for NO byproducts (nitrate + nitrite) measurements. The LCP resulted in a decrease of the initial torque by ~72% which was not different between the groups of mice. Maximum force of the EDL from LCP‐subjected legs was significantly smaller vs control legs for untreated (20±5%), DMSO (7±11%), ISDN (47±10%), and L‐NIL (27±7%) (P&lt;0.05 vs. control, two‐way ANOVA), but not for GSNORi (‐7±5%; P&gt;0.05). TA muscles were fixed, frozen in OCT compound, sliced, then labeled with anti‐laminin antibody and DAPI. Satellite cell incorporation into myofibers was determined by the appearance of endogenous TdTomato fluorescence in myofibers. TA and EDL muscles from LCP legs showed a higher quantity of immunofluorescent myofibers compared to the control legs. Plasma NO byproducts were not different between groups. The data suggest that either excessive NO (ISDN) or low NO (L‐NIL) impair recovery of force after injury but blocking GSNOR may accelerate the re‐gain of strength after a physiologically relevant model of muscle injury.

Mitochondria from Skeletal Muscle Cells Derived from Old Donors Show Reduced Energy Production and Respiratory Capacity

With age, skeletal muscle mitochondria lose their oxidative capacity and their ability to respond to energy demand. These phenomena can lead to a reduction in skeletal muscle oxygen consumption, atrophy, and increased risk of developing age‐related diseases such as sarcopenia. Whether age‐derived changes in mitochondrial function correspond to morphological changes in the mitochondrial reticulum remains unknown. Our objective is to investigate age‐related changes in mitochondrial morphology and function using primary skeletal muscle cells derived from healthy young and old men. Primary skeletal muscle progenitor cells (SkM) derived from the Rectus abdominis muscle of healthy active 18‐19‐year‐old men (Young), and 66‐69‐year‐old men (Old) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Mitochondrial morphology was analyzed in live cells using confocal microscopy. Oxygen Consumption Rate (OCR) was measured in intact cells using the extracellular flux assay and a Seahorse analyzer (Agilent Technologies; Santa Clara, CA). Seahorse Cell Mito Stress Test of primary cells derived from Young revealed a higher Basal and Maximal OCR compared to Old (Basal: 23.20 ± 1.49 vs. 11.34 ± 2.31; Maximal: 41.50 ± 4.72 vs. 18.36 ± 1.65 pmol/min/protein). Young also had higher ATP production and Spare Respiratory Capacity (ATP: 19.08 ± 1.21 vs. 9.19 ± 2.10; SRC: 18.30 ± 3.47 vs. 7.03 ± 1.29 pmol/min/protein). No differences were revealed in morphology (Individuals: 37.80 ± 23.18 vs. 46.67 ± 22.43 counts; Networks: 10.20 ± 2.48 vs. 15.33 ± 5.75 counts; Mean Branches per Network: 15.52 ± 6.41 vs. 11.27 ± 3.67 counts). These preliminary results show primary skeletal muscle progenitor cells derived from old donors have lower respiration, produce less ATP, and have a reduced capacity to adapt to energy demands when compared to SkM derived from young donors. Further analyses can give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial dysfunction in vitro helps us understand the effects of aging on skeletal muscle mitochondria and whole‐body declines of resting and exercise metabolic rates. Studies on aging individuals will be required to determine if predicted decrements in energy coupling efficiency occur with aging in vivo.

Extracellular Vesicles from iPSC derived Muscle Progenitor Cells Rejuvenate the Dysfunctional Muscle Stem Cells during Aging

Background and ObjectiveSarcopenia is defined as the loss of skeletal muscle mass and function due to age, which is a major concern for life long health and wellbeing in elderly population. Age‐associated decline in the performance of muscle stem cells (MuSC) contributes to the deterioration of skeletal muscle during aging. Using small molecule Givinostat and CHIR99021 we previously demonstrated the generation of muscle progenitor cells (Givi‐MPC) of human Induced pluripotent stem cells (iPSC) from multiple cell lines. We found that extracellular vesicles (EVs) from these MPC have a specific cargo compared with human myoblasts. Here, we investigated whether these EVs can rejuvenate the aging dysfunctional muscle stem cells.Methods and ResultsUsing size exclusion chromatography EVs were isolated and purified from iPSC derived Givi‐MPC and primary human myoblasts. The EVs were visualized by transmission electron microscopy. Next, we isolated primary MuSC from 25 months old aged C57/B6 mice, which were confirmed by the expression of myogenic transcription factor, Pax7. We found the aged MuSC pretreated with EVs from MPC promoted clonal expansion and restored myogenic potential in vitro with more myotubes formed after 72h differentiation in medium with 2% horse serum compared with PBS pretreated MuSC (MuSC (Fusion index: 40.3±5.2% vs.15.0± 2.1%, n=3).). To test the regenerative potential of the EVs on aged MuSC, 8 weeks old mdx/scid mice (dystrophin deficient mice) were irradiated to destroy the host MuSC and then injured their tibialis anterior (TA) muscle with cardiotoxin (CTX). 24 hours after injury, we transplanted the aging MuSC (5000 cells) either with EV or PBS pretreatment into TA muscles, respectively. 5 days after transplantation, the EVs pretreated MuSC increased muscle regeneration with muscle fibers positive for embryonic myosin heavy chain compared to PBS pretreated MuSC. One month later, we found transplantation of EVs pretreated MuSC restored dystrophin expression in these mdx mice. We further tested the effects of EVs pretreatment on MuSC in 25 months old mice following CTX injury. Consistent with the results in mdx mice, pretreatment of mice with EVs from Givi‐MPC showed enhanced regeneration (6‐fold increase in number of new muscle cells with central nuclei) in CTX injured aged muscle accompanied by an increase of Pax7 positive MuSC compared with PBS treated mice or treatment with EVs from human myoblasts (number of Pax7 positive MuSC: EVs‐Givi‐MPC 10±5/mm2; PBS 2±1/mm2; EVs‐human myoblast 6±2/mm2, n=3).ConclusionThe regenerative potential of aging MuSC is restored with the treatment of EVs from Givi‐MPC allowing stem cell renewal and differentiation upon activation.

Single-cell RNA-seq reveals heterogeneity in hiPSC-derived muscle progenitors and E2F family as a key regulator of proliferation.

Human pluripotent stem cell-derived muscle progenitor cells (hiPSC-MuPCs) resemble fetal-stage muscle progenitor cells and possess in vivo regeneration capacity. However, the heterogeneity of hiPSC-MuPCs is unknown, which could impact the regenerative potential of these cells. Here, we established an hiPSC-MuPC atlas by performing single-cell RNA sequencing of hiPSC-MuPC cultures. Bioinformatic analysis revealed four cell clusters for hiPSC-MuPCs: myocytes, committed, cycling, and noncycling progenitors Using FGFR4 as a marker for noncycling progenitors and cycling cells and CD36 as a marker for committed and myocyte cells, we found that FGFR4+ cells possess a higher regenerative capacity than CD36+ cells. We also identified the family of E2F transcription factors are key regulators of hiPSC-MuPC proliferation. Our study provides insights on the purification of hiPSC-MuPCs with higher regenerative potential and increases the understanding of the transcriptional regulation of hiPSC-MuPCs.

IL-17A-producing γδT cells promote muscle regeneration in a microbiota-dependent manner.

Subsequent to acute injury, skeletal muscle undergoes a stereotypic regenerative process that reestablishes homeostasis. Various types of innate and adaptive immunocytes exert positive or negative influences at specific stages along the course of muscle regeneration. We describe an unanticipated role for γδT cells in promoting healthy tissue recovery after injection of cardiotoxin into murine hindlimb muscle. Within a few days of injury, IL-17A-producing γδT cells displaying primarily Vγ6+ antigen receptors accumulated at the wound site. Punctual ablation experiments showed that these cells boosted early inflammatory events, notably recruitment of neutrophils; fostered the proliferation of muscle stem and progenitor cells; and thereby promoted tissue regeneration. Supplementation of mice harboring low numbers of IL-17A+ γδT cells with recombinant IL-17A largely reversed their inflammatory and reparative defects. Unexpectedly, the accumulation and influences of γδT cells in this experimental context were microbiota dependent, unveiling an orthogonal perspective on the treatment of skeletal muscle pathologies such as catastrophic wounds, wasting, muscular dystrophies, and myositides.

Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

In vivo transcriptomic profiling using cell encapsulation identifies effector pathways of systemic aging.

Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here, we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence, and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.