Skeletal Muscle Lineage Research Articles

A 3D printed mechanical bioreactor for investigating mechanobiology and soft tissue mechanics

Mechanical loading is an important cue for directing stem cell fate and engineered tissue formation in vitro. Stem cells cultured on 2-dimensional (D) substrates and in 3D scaffolds have been shown to differentiate toward bone, tendon, cartilage, ligament, and skeletal muscle lineages depending on their exposure to mechanical stimuli. To apply this mechanical stimulus in vitro, mechanical bioreactors are needed. However, current bioreactor systems are challenged by their high cost, limited ability for customization, and lack of force measurement capabilities. We demonstrate the use of 3-dimensional printing (3DP) technology to design and fabricate a low-cost custom bioreactor system that can be used to apply controlled mechanical stimuli to cells in culture and measure the mechanical properties of small soft tissues. The results of our in vitro studies and mechanical evaluations show that 3DP technology is feasible as a platform for developing a low-cost, customizable, and multifunctional mechanical bioreactor system.• 3DP technology was used to print a multifunctional bioreactor system/tensile load frame for a fraction of the cost of commercial systems.• The system mechanically stimulated cells in culture and evaluated the mechanical properties of soft tissues.• This system is easily customizable and can be used to evaluate multiple types of soft tissues.

The Long Road to Making Muscle In Vitro.

The skeletal muscle lineage derives from the embryonic paraxial mesoderm (PM) which also gives rise to the axial skeleton, the dermis of the back, brown fat, meninges, and endothelial cells. Direct conversion was pioneered in skeletal muscle with overexpression of the transcription factor MyoD which can convert fibroblasts to a muscle fate. In contrast, directed differentiation of skeletal muscle from pluripotent cells (PC) in vitro has proven to be very difficult compared to other lineages and has only been achieved recently. Experimental strategies recapitulating myogenesis in vitro from mouse and human PC (ES/iPS) have now been reported and all rely on early activation of Wnt signaling at the epiblast stage. This leads to induction of neuromesodermal progenitors that can subsequently be induced to a PM fate and to skeletal muscle. These protocols can efficiently produce fetal muscle fibers and immature satellite cells. These new in vitro systems now open the possibility to better understand human myogenesis and to develop in vitro disease models as well as cell therapy approaches.

Dysregulated Myogenesis in Rhabdomyosarcoma.

Rhabdomyosarcoma is a mesenchymal malignancy associated with the skeletal muscle lineage and is also the most common pediatric soft tissue cancer. Between the two pediatric subtypes, embryonal and alveolar rhabdomyosarcoma, the alveolar subtype is generally more aggressive and high-risk. Despite intensive multimodal therapy, patients with high-risk rhabdomyosarcoma continue to have poor prognosis. In this chapter we address the mechanisms underlying the dysregulation of myogenesis in rhabdomyosarcoma. We specifically focus on recently identified signaling pathways that function to inhibit myogenesis and how similar functions have been shown to overlap in rhabdomyosarcoma, potentially contributing to the disease.

Insights into interplay between rexinoid signaling and myogenic regulatory factor-associated chromatin state in myogenic differentiation.

While skeletal myogenesis is tightly coordinated by myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Here, we examine the genome-wide impact of rexinoids on myogenic differentiation through integral RNA-seq and ChIP-seq analyses. We found that bexarotene promotes myoblast differentiation through the coordination of exit from the cell cycle and the activation of muscle-related genes. We uncovered a new mechanism of rexinoid action which is mediated by the nuclear receptor and largely reconciled through a direct regulation of MyoD gene expression. In addition, we determined a rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated to MyoD and myogenin. Thus, we provide novel molecular insights into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation.

Human Satellite Cell Isolation and Xenotransplantation.

Satellite cells are mononucleated cells of the skeletal muscle lineage that exist beneath the basal lamina juxtaposed to the sarcolemma of skeletal muscle fibers. It is widely accepted that satellite cells mediate skeletal muscle regeneration. Within the satellite cell pool of adult muscle are skeletal muscle stem cells (MuSCs), also called satellite stem cells, which fulfill criteria of tissue stem cells: They proliferate and their progeny either occupies the adult MuSC niche during self-renewal or differentiates to regenerate mature muscle fibers. Here, we describe robust methods for the isolation of enriched populations of human satellite cells containing MuSCs from fresh human muscle, utilizing mechanical and enzymatic dissociation and purification by fluorescence-activated cell sorting. We also describe a process for xenotransplantation of human satellite cells into mouse muscle by injection into irradiated, immunodeficient, mouse leg muscle with concurrent notexin or bupivacaine muscle injury to increase engraftment efficiency. The engraftment of human MuSCs and the formation of human muscle can then be analyzed by histological and immunofluorescence staining, or subjected to in vivo experimentation.

Recent Insights into Notch Signaling in Embryonal Rhabdomyosarcoma.

The Notch signaling pathway is an evolutionarily conserved developmental network critical for embryonic and postnatal regulation of tissue growth, homeostasis, and repair. Signaling is initiated when transmembrane Notch ligands bind to transmembrane Notch receptors on nearby cells. Sequential proteolytic steps generate an activated Notch fragment that translocates to the nucleus, where it drives activation of canonical Notch target genes. In skeletal muscle, Notch signaling governs myogenic cell fate and stem cell maintenance. In the human soft tissue sarcoma rhabdomyosarcoma, which bears markers of skeletal muscle commitment and so is thought to be related to the skeletal muscle lineage, Notch signaling is also found to be upregulated and dysregulated. This review provides an overview of Notch signaling during normal embryonic and postnatal myogenesis, information on the recently discovered aberrant Notch signaling occurring in muscular dystrophies, the upregulation and mechanism of Notch signaling in the embryonal variant of rhabdomyosarcoma and related soft tissue sarcomas, and Notch cross-talk with other metazoan developmental pathways including Hippo, Hedgehog, Wnt, and TGF-β. The review concludes with updates on current promising efforts to target and inhibit Notch signaling in human sarcomas including rhabdomyosarcoma.

The lysine methyltransferase Ehmt2/G9a is dispensable for skeletal muscle development and regeneration.

BackgroundEuchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells.MethodsTo further investigate the importance of G9a in modulating myogenic regeneration in vivo, we crossed Ehmt2floxed mice to animals expressing Cre recombinase from the Myod locus, resulting in efficient knockout in the entire skeletal muscle lineage (Ehmt2ΔmyoD).ResultsSurprisingly, despite a dramatic drop in the global levels of H3K9me2, knockout animals did not show any developmental phenotype in muscle size and appearance. Consistent with this finding, purified Ehmt2ΔmyoD satellite cells had rates of activation and proliferation similar to wild-type controls. When induced to differentiate in vitro, Ehmt2 knockout cells differentiated with kinetics similar to those of control cells and demonstrated normal capacity to form myotubes. After acute muscle injury, knockout mice regenerated as efficiently as wildtype. To exclude possible compensatory mechanisms elicited by the loss of G9a during development, we restricted the knockout within adult satellite cells by crossing Ehmt2floxed mice to Pax7CreERT2 and also found normal muscle regeneration capacity.ConclusionsThus, Ehmt2 and H3K9me2 do not play significant roles in skeletal muscle development and regeneration in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0093-7) contains supplementary material, which is available to authorized users.

SWI/SNF-directed stem cell lineage specification: dynamic composition regulates specific stages of skeletal myogenesis.

SWI/SNF chromatin-remodeling complexes are key regulators of the epigenetic modifications that determine whether stem cells maintain pluripotency or commit toward specific lineages through development and during postnatal life. Dynamic combinatorial assembly of multiple variants of SWI/SNF subunits is emerging as the major determinant of the functional versatility of SWI/SNF. Here, we summarize the current knowledge on the structural and functional properties of the alternative SWI/SNF complexes that direct stem cell fate toward skeletal muscle lineage and control distinct stages of skeletal myogenesis. In particular, we will refer to recent evidence pointing to the essential role of two SWI/SNF components not expressed in embryonic stem cells-the catalytic subunit BRM and the structural component BAF60C-whose induction in muscle progenitors coincides with the expansion of their transcriptional repertoire.

Implication of retinoic acid receptor selective signaling in myogenic differentiation.

Signaling molecules are important for committing individual cells into tissue-specific lineages during early vertebrate development. Retinoic acid (RA) is an important vertebrate morphogen, in that its concentration gradient is essential for correct patterning of the vertebrate embryo. RA signaling is mediated through the activation of retinoic acid receptors (RARs), which function as ligand-dependent transcription factors. In this study, we examined the molecular mechanisms of RAR-selective signaling in myogenic differentiation. We found that just like natural ligand RA, a RAR-selective ligand is an effective enhancer in the commitment of skeletal muscle lineage at the early stage of myogenic differentiation. Interestingly, the kinetics and molecular basis of the RAR-selective ligand in myogenic differentiation are similar to that of natural ligand RA. Also similar to natural ligand RA, the RAR-selective ligand enhances myogenic differentiation through β-catenin signaling pathway while inhibiting cardiac differentiation. Furthermore, while low concentrations of natural ligand RA or RAR-selective ligand regulate myogenic differentiation through RAR function and coactivator recruitment, high concentrations are critical to the expression of a model RA-responsive gene. Thus our data suggests that RAR-mediated gene regulation may be highly context-dependent, affected by locus-specific interaction or local chromatin environment.

Retinoid X Receptor-selective Signaling in the Regulation of Akt/Protein Kinase B Isoform-specific Expression

The differentiation and fusion of myoblasts into mature myotubes are complex processes responding to multiple signaling pathways. The function of Akt/PKB is critical for myogenesis, but less is clear as to the regulation of its isoform-specific expression. Bexarotene is a drug already used clinically to treat cancer, and it has the ability to enhance the commitment of embryonic stem cells into skeletal muscle lineage. Whereas bexarotene regulates fundamental biological processes through retinoid X receptor (RXR)-mediated gene expression, molecular pathways underlying its positive effects on myogenesis remain unclear. In this study, we have examined the signaling pathways that transmit bexarotene action in the context of myoblast differentiation. We show that bexarotene promotes myoblast differentiation and fusion through the activation of RXR and the regulation of Akt/PKB isoform-specific expression. Interestingly, bexarotene signaling appears to correlate with residue-specific histone acetylation and is able to counteract the detrimental effects of cachectic factors on myogenic differentiation. We also signify an isoform-specific role for Akt/PKB in RXR-selective signaling to promote and to retain myoblast differentiation. Taken together, our findings establish the viability of applying bexarotene in the prevention and treatment of muscle-wasting disorders, particularly given the lack of drugs that promote myogenic differentiation available for potential clinical applications. Furthermore, the model of bexarotene-enhanced myogenic differentiation will provide an important avenue to identify additional genetic targets and specific molecular interactions that we can study and apply for the development of potential therapeutics in muscle regeneration and repair.

Distinct Activities of Myf5 and MyoD Indicate Separate Roles in Skeletal Muscle Lineage Specification and Differentiation

Most transcription factor families contain highly related paralogs generated by gene duplication, and functional divergence is generally accomplished by activation of distinct sets of genes by each member. Here we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find that MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits Pol II, and robustly activates gene transcription. Therefore, the initial specification of the muscle lineage by Myf5 occurs without significant induction of gene transcription. Transcription of the skeletal muscle program is then achieved by the subsequent expression of MyoD, which binds to the same sites as Myf5, indicating that each factor regulates distinct steps in gene initiation and transcription at a shared set of binding sites.

Defective myogenesis in the absence of the muscle-specific lysine methyltransferase SMYD1

The SMYD (SET and MYND domain) family of lysine methyltransferases harbor a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein–protein interaction MYND domain. SMYD proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Smyd1 is critical for development of the right ventricle in mice. In zebrafish, Smyd1 is necessary for sarcomerogenesis in fast-twitch muscles. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. To determine the role of Smyd1 in mammalian myogenesis, we conditionally eliminated Smyd1 from the skeletal muscle lineage at the myoblast stage using Myf5cre. Deletion of Smyd1 impaired myoblast differentiation, resulted in fewer myofibers and decreased expression of muscle-specific genes. Muscular defects were temporally restricted to the second wave of myogenesis. Thus, in addition to the previously described functions for Smyd1 in heart development and skeletal muscle sarcomerogenesis, these results point to a novel role for Smyd1 in myoblast differentiation.

Secreted Frizzled-Related Protein 3 (SFRP3) Is Required for Tumorigenesis of PAX3-FOXO1-Positive Alveolar Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma associated with the skeletal muscle lineage. Of the two predominant subtypes, known as embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a five-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3-FOXO1. As PAX3-FOXO1 has proven chemically intractable, this study aims to identify targetable proteins that are downstream from or cooperate with PAX3-FOXO1 to support tumorigenesis. Microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PAX3-FOXO1 revealed alteration of several Wnt pathway gene members, including secreted frizzled related protein 3 (SFRP3), a secreted Wnt pathway inhibitor. Loss-of-function using shRNAs against SFRP3 was used to interrogate the role of SFRP3 in human aRMS cell lines in vitro and conditional murine xenograft systems in vivo. The combination of SFRP3 genetic suppression and the chemotherapeutic agent vincristine was also examined. In vitro, suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. In vivo, doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold, in addition to increasing myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.

Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells

Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies.

Arsenic and Its Methylated Metabolites Inhibit the Differentiation of Neural Plate Border Specifier Cells.

Exposure to arsenic in food and drinking water has been correlated with adverse developmental outcomes, such as reductions in birth weight and neurological deficits. Additionally, studies have shown that arsenic suppresses sensory neuron formation and skeletal muscle myogenesis, although the reason why arsenic targets both of these cell types in unclear. Thus, P19 mouse embryonic stem cells were used to investigate the mechanisms by which arsenic could inhibit cellular differentiation. P19 cells were exposed to 0, 0.1, or 0.5 μM sodium arsenite and induced to form embryoid bodies over a period of 5 days. The expression of transcription factors necessary to form neural plate border specifier (NPBS) cells, neural crest cells and their progenitors, and myocytes and their progenitors were examined. Early during differentiation, arsenic significantly reduced the transcript and protein expression of Msx1 and Pax3, both needed for NPBS cell formation. Arsenic also significantly reduced the protein expression of Sox 10, needed for neural crest progenitor cell production, by 31-50%, and downregulated the protein and mRNA levels of NeuroD1, needed for neural crest cell differentiation, in a time- and dose-dependent manner. While the overall protein expression of transcription factors in the skeletal muscle lineage was not changed, arsenic did alter their nuclear localization. MyoD nuclear translocation was significantly reduced on days 2-5 between 15 and 70%. At a 10-fold lower concentration, monomethylarsonous acid (MMA III) appeared to be just as potent as inorganic arsenic at reducing the mRNA levels Pax3 (79% vs84%), Sox10 (49% vs 65%), and Msx1 (56% vs 56%). Dimethylarsinous acid (DMA III) also reduced protein and transcript expression, but the changes were less dramatic than those with MMA or arsenite. All three arsenic species reduced the nuclear localization of MyoD and NeuroD1 in a similar manner. The early changes in the differentiation of neural plate border specifier cells may provide a mechanism for arsenic to suppress both neurogenesis and myogenesis.

Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes.

Myogenic regulatory factor (MRF) genes, MYOD1, MYOG, MYF6 and MYF5, are critical for the skeletal muscle lineage. Here, we used various epigenome profiles from human myoblasts (Mb), myotubes (Mt), muscle and diverse non-muscle samples to elucidate the involvement of multigene neighborhoods in the regulation of MRF genes. We found more far-distal enhancer chromatin associated with MRF genes in Mb and Mt than previously reported from studies in mice. For the MYF5/MYF6 gene-pair, regions of Mb-associated enhancer chromatin were located throughout the adjacent 236-kb PTPRQ gene even though Mb expressed negligible amounts of PTPRQ mRNA. Some enhancer chromatin regions inside PTPRQ in Mb were also seen in PTPRQ mRNA-expressing non-myogenic cells. This suggests dual-purpose PTPRQ enhancers that upregulate expression of PTPRQ in non-myogenic cells and MYF5/MYF6 in myogenic cells. In contrast, the myogenic enhancer chromatin regions distal to MYOD1 were intergenic and up to 19 kb long. Two of them contain small, known MYOD1 enhancers, and one displayed an unusually high level of 5-hydroxymethylcytosine in a quantitative DNA hydroxymethylation assay. Unexpectedly, three regions of MYOD1-distal enhancer chromatin in Mb and Mt overlapped enhancer chromatin in umbilical vein endothelial cells, which might upregulate a distant gene (PIK3C2A). Lastly, genes surrounding MYOG were preferentially transcribed in Mt, like MYOG itself, and exhibited nearby myogenic enhancer chromatin. These neighboring chromatin regions may be enhancers acting in concert to regulate myogenic expression of multiple adjacent genes. Our findings reveal the very different and complex organization of gene neighborhoods containing closely related transcription factor genes.

Deconstruction of DNA methylation patterns during myogenesis reveals specific epigenetic events in the establishment of the skeletal muscle lineage.

The progressive restriction of differentiation potential from pluripotent embryonic stem cells (ESCs) to tissue-specific stem cells involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Skeletal muscle stem cells are required for the growth, maintenance, and regeneration of skeletal muscle. To investigate the contribution of DNA methylation to the establishment of the myogenic program, we analyzed ESCs, skeletal muscle stem cells in proliferating (myoblasts) and differentiating conditions (myotubes), and mature myofibers. About 1.000 differentially methylated regions were identified during muscle-lineage determination and terminal differentiation, mainly located in gene bodies and intergenic regions. As a whole, myogenic stem cells showed a gain of DNA methylation, while muscle differentiation was accompanied by loss of DNA methylation in CpG-poor regions. Notably, the hypomethylated regions in myogenic stem cells were neighbored by enhancer-type chromatin, suggesting the involvement of DNA methylation in the regulation of cell-type specific enhancers. Interestingly, we demonstrated the hypomethylation of the muscle cell-identity Myf5 super-enhancer only in muscle cells. Furthermore, we observed that upstream stimulatory factor 1 binding to Myf5 super-enhancer occurs upon DNA demethylation in myogenic stem cells. Taken altogether, we characterized the unique DNA methylation signature of skeletal muscle stem cells and highlighted the importance of DNA methylation-mediated regulation of cell identity Myf5 super-enhancer during cellular differentiation.

MP21-08 SKELETAL MYOGENIC DIFFERENTIATION OF HUMAN URINE-DERIVED CELLS AS A POTENTIAL SOURCE OF CELL THERAPY FOR URETHRAL SPHINCTER MUSCLE DYSFUNCTION

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2015MP21-08 SKELETAL MYOGENIC DIFFERENTIATION OF HUMAN URINE-DERIVED CELLS AS A POTENTIAL SOURCE OF CELL THERAPY FOR URETHRAL SPHINCTER MUSCLE DYSFUNCTION Wei Chen, M Xie, Bin Yang, S Bharadwaj, Song Li, Guihua Liu, Anthony Atala, and Yuanyuan Zhang Wei ChenWei Chen More articles by this author , M XieM Xie More articles by this author , Bin YangBin Yang More articles by this author , S BharadwajS Bharadwaj More articles by this author , Song LiSong Li More articles by this author , Guihua LiuGuihua Liu More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Yuanyuan ZhangYuanyuan Zhang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.960AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adult stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration in treatment of urethral sphincter dysfunction. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle lineage cells, and potentially be used for skeletal muscle regeneration when treating urinary incontinence due to urethral sphincter dysfunction. METHODS USCs were harvested from six healthy individuals aged 25–55 years old. Expression profiles of cell-surface markers were assessed by flow cytometry. Growth factors and cytokines secreted from USCs and human bone marrow stroma cells (BMSC, as a control) culture supernatant were measure by specific ELISAs. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. RESULTS Human USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. A series of growth factors and cytokines (such as IGF1, HGF, FGF, VEGF, and PDGF) were identified at higher levels in the culture supernatant of USCs than in BMSCs. After myogenic differentiation, morphology of USCs changed from ‘rice-grain’-like cells to spindle-shaped cells and then to myotube-like structures. The USCs expressed specific skeletal muscle transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed skeletal muscle markers stably in vivo. CONCLUSIONS Our findings suggest that USCs can differentiate into a skeletal muscle lineage cells in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy to induce urethral sphincter skeletal muscle regeneration and thereby treat urinary incontinence. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e235 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei Chen More articles by this author M Xie More articles by this author Bin Yang More articles by this author S Bharadwaj More articles by this author Song Li More articles by this author Guihua Liu More articles by this author Anthony Atala More articles by this author Yuanyuan Zhang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Radioelectric Asymmetric Conveyed Fields and Human Adipose-Derived Stem Cells Obtained with a Nonenzymatic Method and Device: A Novel Approach to Multipotency

Human adipose-derived stem cells (hASCs) have been recently proposed as a suitable tool for regenerative therapies for their simple isolation procedure and high proliferative capability in culture. Although hASCs can be committed into different lineages in vitro, the differentiation is a low-yield and often incomplete process. We have recently developed a novel nonenzymatic method and device, named Lipogems, to obtain a fat tissue derivative highly enriched in pericytes/mesenchymal stem cells by mild mechanical forces from human lipoaspirates. When compared to enzymatically dissociated cells, Lipogems-derived hASCs exhibited enhanced transcription of vasculogenic genes in response to provasculogenic molecules, suggesting that these cells may be amenable for further optimization of their multipotency. Here we exposed Lipogems-derived hASCs to a radioelectric asymmetric conveyer (REAC), an innovative device asymmetrically conveying radioelectric fields, affording both enhanced differentiating profiles in mouse embryonic stem cells and efficient direct multilineage reprogramming in human skin fibroblasts. We show that specific REAC exposure remarkably enhanced the transcription of prodynorphin, GATA-4, Nkx-2.5, VEGF, HGF, vWF, neurogenin-1, and myoD, indicating the commitment toward cardiac, vascular, neuronal, and skeletal muscle lineages, as inferred by the overexpression of a program of targeted marker proteins. REAC exposure also finely tuned the expression of stemness-related genes, including NANOG, SOX-2, and OCT-4. Noteworthy, the REAC-induced responses were fashioned at a significantly higher extent in Lipogems-derived than in enzymatically dissociated hASCs. Therefore, REAC-mediated interplay between radioelectric asymmetrically conveyed fields and Lipogems-derived hASCs appears to involve the generation of an ideal "milieu" to optimize multipotency expression from human adult stem cells in view of potential improvement of future cell therapy efforts.

Concise review: mesoangioblast and mesenchymal stem cell therapy for muscular dystrophy: progress, challenges, and future directions.

Mesenchymal stem cells (MSCs) and mesoangioblasts (MABs) are multipotent cells that differentiate into specialized cells of mesodermal origin, including skeletal muscle cells. Because of their potential to differentiate into the skeletal muscle lineage, these multipotent cells have been tested for their capacity to participate in regeneration of damaged skeletal muscle in animal models of muscular dystrophy. MSCs and MABs infiltrate dystrophic muscle from the circulation, engraft into host fibers, and bring with them proteins that replace the functions of those missing or truncated. The potential for systemic delivery of these cells increases the feasibility of stem cell therapy for the large numbers of affected skeletal muscles in patients with muscular dystrophy. The present review focused on the results of preclinical studies with MSCs and MABs in animal models of muscular dystrophy. The goals of the present report were to (a) summarize recent results, (b) compare the efficacy of MSCs and MABs derived from different tissues in restoration of protein expression and/or improvement in muscle function, and (c) discuss future directions for translating these discoveries to the clinic. In addition, although systemic delivery of MABs and MSCs is of great importance for reaching dystrophic muscles, the potential concerns related to this method of stem cell transplantation are discussed.