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Acute respiratory distress syndrome (ARDS) is a heterogeneous clinical syndrome that is most commonly triggered by infection-related inflammation. Lung pericytes can respond to infection and act as immune and proangiogenic cells; moreover, these cells can differentiate into myofibroblasts in nonresolving ARDS and contribute to the development of pulmonary fibrosis. Here, we aimed to characterize the role of lung cells, which present characteristics of pericytes, such as peri-endothelial location and expression of a panel of specific markers. A murine model of lipopolysaccharide (LPS)-induced resolving ARDS was used to study their role in ARDS. The development of ARDS was confirmed after LPS instillation, which was resolved 14 days after onset. Immunofluorescence and flow cytometry showed early expansion of neural-glial antigen 2 + β-type platelet-derived growth factor receptor + pericytes in murine lungs with loss of CD31 + β-type platelet-derived growth factor receptor + endothelial cells. These changes were accompanied by specific changes in lung structure and loss of vascular integrity. On day 14 after ARDS onset, the composition of pericytes and endothelial cells returned to baseline values. LPS-induced ARDS activated NOTCH signaling in lung pericytes, the inhibition of which during LPS stimulation reduced the expression of its downstream target genes, pericyte markers, and angiogenic factors. Together, these data indicate that lung pericytes in response to inflammatory injury activate NOTCH signaling that supports their maintenance and in turn can contribute to recovery of the microvascular endothelium., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Heterotopic ossification (HO) means the formation of bone in muscles and soft tissues, such as ligaments or tendons. HO could have a genetic history or develop after a traumatic event, as a result of muscle injury, fractures, burns, surgery, or neurological disorders. Many lines of evidence suggest that the formation of HO is related to the pathological differentiation of stem or progenitor cells present within soft tissues or mobilized from the bone marrow. The cells responsible for the initiation and progression of HO are generally called HO precursor cells. The exact mechanisms behind the development of HO are not fully understood. However, several factors have been identified as potential contributors. For example, local tissue injury and inflammation disturb soft tissue homeostasis. Inflammatory cells release growth factors and cytokines that promote osteogenic or chondrogenic differentiation of HO precursor cells. The bone morphogenetic protein (BMP) is one of the main factors involved in the development of HO. In this study, next-generation sequencing (NGS) and RT-qPCR were performed to analyze the differences in mRNA, miRNA, and lncRNA expression profiles between muscles, control bone samples, and HO samples coming from patients who underwent total hip replacement (THR). As a result, crucial changes in the level of gene expression between HO and healthy tissues were identified. The bioinformatic analysis allowed to describe the processes most severely impacted, as well as genes which level differed the most significantly between HO and control samples. Our analysis showed that the level of transcripts involved in leukocyte migration, differentiation, and activation, as well as markers of chronic inflammatory diseases, that is, miR-148, increased in HO, as compared to muscle. Furthermore, the levels of miR-195 and miR-143, which are involved in angiogenesis, were up-regulated in HO, as compared to bone. Thus, we suggested that inflammation and angiogenesis play an important role in HO formation. Importantly, we noticed that HO is characterized by a higher level of TLR3 expression, compared to muscle and bone. Thus, we suggest that infection may also be a risk factor in HO development. Furthermore, an increased level of transcripts coding proteins involved in osteogenesis and signaling pathways, such as ALPL, SP7, BGLAP, BMP8A, BMP8B, SMPD3 was noticed in HO, as compared to muscles. Interestingly, miR-99b, miR-146, miR-204, and LINC00320 were up-regulated in HO, comparing to muscles and bone. Therefore, we suggested that these molecules could be important biomarkers of HO formation and a potential target for therapies., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Muscle satellite cells (SCs) are stem cells and the main players in skeletal muscle reconstruction. Since satellite cells are located near or in direct contact with blood vessels their niche is formed, inter alia, by endothelial cells. The cross-talk between satellite cells and endothelial cells determines quiescence or proliferation of these cells. However, little is known about the role of miRNA in these interactions. In the present study we identified miRNA that were up-regulated in SC-derived myoblasts treated with stromal derived factor-1 (SDF-1) and/or down-regulated in cells in which the expression of CXCR4 or CXCR7, that is, SDF-1 receptors, was silenced. SDF-1 is one of the important regulators of cell migration, mobilization, skeletal muscle regeneration, and angiogenesis. We hypothesized that selected miRNAs affect SC-derived myoblast fate and interactions with endothelial cells. We showed that miR-126a-3p inhibited both, myoblast migration and fusion. Moreover, the levels of Cxcl12, encoding SDF-1 and Ackr3, encoding CXCR7, were reduced by miR-126a-3p mimic. Interestingly, the miR-126a-3p mimic significantly decreased the level of numerous factors involved in myogenesis and the miR-126a-5p mimic increased the level of Vefga. Importantly, the treatment of endothelial cells with medium conditioned by miR-126-5p mimic transfected SC-derived myoblasts promoted tubulogenesis., (© 2023. Springer Nature Limited.)

Background: Skeletal muscle regeneration is a complex process regulated by many cytokines and growth factors. Among the important signaling pathways regulating the myogenic cell identity are these involving SDF-1 and NOTCH. SDF-1 participates in cell mobilization and acts as an important chemoattractant. NOTCH, on the other hand, controls cell activation and myogenic determination of satellite cells. Knowledge about the interaction between SDF-1 and NOTCH signaling is limited., Methods: We analyzed two populations of myogenic cells isolated from mouse skeletal muscle, that is, myoblasts derived from satellite cells (SCs) and muscle interstitial progenitor cells (MIPCs). First, microRNA level changes in response to SDF-1 treatment were analyzed with next-generation sequencing (NGS). Second, myogenic cells, i.e., SC-derived myoblasts and MIPCs were transfected with miRNA mimics, selected on the basis of NGS results, or their inhibitors. Transcriptional changes, as well as proliferation, migration, and differentiation abilities of SC-derived myoblasts and MIPCs, were analyzed in vitro. Naive myogenic potential was assessed in vivo, using subcutaneous engrafts and analysis of cell contribution to regeneration of the skeletal muscles., Results: SDF-1 treatment led to down-regulation of miR10a, miR151, miR425, and miR5100 in myoblasts. Interestingly, miR10a, miR425, and miR5100 regulated the expression of factors involved in the NOTCH signaling pathway, including Dll1, Jag2, and NICD. Furthermore, miR10a, miR425, and miR5100 down-regulated the expression of factors involved in cell migration: Acta1, MMP12, and FAK, myogenic differentiation: Pax7, Myf5, Myod, Mef2c, Myog, Musk, and Myh3. However, these changes did not significantly affect myogenic cell migration or fusion either in vitro or in vivo, except when miR425 was overexpressed, or miR5100 inhibitor was used. These two molecules increased the fusion of MIPCs and myoblasts, respectively. Furthermore, miR425-transfected MIPC transplantation into injured skeletal muscle resulted in more efficient regeneration, compared to control cell transplantation. However, skeletal muscles that were injected with miR10a transfected myoblasts regenerated less efficiently., Conclusions: SDF-1 down-regulates miR10a, miR425, and miR5100, what could affect NOTCH signaling, differentiation of myogenic cells, and their participation in skeletal muscle regeneration., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Heterotopic ossification (HO) is the formation of bone in non-osseous tissues, such as skeletal muscles. The HO could have a genetic or a non-genetic (acquired) background, that is, it could be caused by musculoskeletal trauma, such as burns, fractures, joint arthroplasty (traumatic HO), or cerebral or spinal insult (neurogenetic HO). HO formation is caused by the differentiation of stem or progenitor cells induced by local or systemic imbalances. The main factors described so far in HO induction are TGFβ1, BMPs, activin A, oncostatin M, substance P, neurotrophin-3, and WNT. In addition, dysregulation of noncoding RNAs, such as microRNA or long noncoding RNA, homeostasis may play an important role in the development of HO. For example, decreased expression of miRNA-630, which is responsible for the endothelial-mesenchymal transition, was observed in HO patients. The reduced level of miRNA-421 in patients with humeral fracture was shown to be associated with overexpression of BMP2 and a higher rate of HO occurrence. Down-regulation of miRNA-203 increased the expression of runt-related transcription factor 2 (RUNX2), a crucial regulator of osteoblast differentiation. Thus, understanding the various functions of noncoding RNAs can reveal potential targets for the prevention or treatment of HO., (© 2022. The Author(s).)

For many years optimal treatment for dysfunctional skeletal muscle characterized, for example, by impaired or limited regeneration, has been searched. Among the crucial factors enabling its development is finding the appropriate source of cells, which could participate in tissue reconstruction or serve as an immunomodulating agent (limiting immune response as well as fibrosis, that is, connective tissue formation), after transplantation to regenerating muscles. MSCs, including those derived from bone marrow, are considered for such applications in terms of their immunomodulatory properties, as their naive myogenic potential is rather limited. Injection of autologous (syngeneic) or allogeneic BMSCs has been or is currently being tested and compared in many potential clinical treatments. In the present study, we verified which approach, that is, the transplantation of either syngeneic or allogeneic BMSCs or the injection of BMSC-conditioned medium, would be the most beneficial for skeletal muscle regeneration. To properly assess the influence of the tested treatments on the inflammation, the experiments were carried out using immunocompetent mice, which allowed us to observe immune response. Combined analysis of muscle histology, immune cell infiltration, and levels of selected chemokines, cytokines, and growth factors important for muscle regeneration, showed that muscle injection with BMSC-conditioned medium is the most beneficial strategy, as it resulted in reduced inflammation and fibrosis development, together with enhanced new fiber formation, which may be related to, i.e., elevated level of IGF-1. In contrast, transplantation of allogeneic BMSCs to injured muscles resulted in a visible increase in the immune response, which hindered regeneration by promoting connective tissue formation. In comparison, syngeneic BMSC injection, although not detrimental to muscle regeneration, did not result in such significant improvement as CM injection.

Background: Bone marrow stromal cells (BMSCs) form a perivascular cell population in the bone marrow. These cells do not present naïve myogenic potential. However, their myogenic identity could be induced experimentally in vitro or in vivo. In vivo, after transplantation into injured muscle, BMSCs rarely fused with myofibers. However, BMSC participation in myofiber reconstruction increased if they were modified by NICD or PAX3 overexpression. Nevertheless, BMSCs paracrine function could play a positive role in skeletal muscle regeneration. Previously, we showed that SDF-1 treatment and coculture with myofibers increased BMSC ability to reconstruct myofibers. We also noticed that SDF-1 treatment changed selected miRNAs expression, including miR151 and miR5100., Methods: Mouse BMSCs were transfected with miR151 and miR5100 mimics and their proliferation, myogenic differentiation, and fusion with myoblasts were analyzed., Results: We showed that miR151 and miR5100 played an important role in the regulation of BMSC proliferation and migration. Moreover, the presence of miR151 and miR5100 transfected BMSCs in co-cultures with human myoblasts increased their fusion. This effect was achieved in an IGFBP2 dependent manner., Conclusions: Mouse BMSCs did not present naïve myogenic potential but secreted proteins could impact myogenic cell differentiation. miR151 and miR5100 transfection changed BMSC migration and IGFBP2 and MMP12 expression in BMSCs. miR151 and miR5100 transfected BMSCs increased myoblast fusion in vitro., (© 2022. The Author(s).)

miRNAs and lncRNAs do not encode proteins, but they play an important role in the regulation of gene expression. They differ in length, biogenesis, and mode of action. In this work, we focus on the selected miRNAs and lncRNAs involved in the regulation of myogenesis and muscle regeneration. We present selected miRNAs and lncRNAs that have been shown to control myogenic differentiation and show that manipulation of their levels could be used to improve myogenic differentiation of various types of stem and progenitor cells. Finally, we discuss how physical activity affects miRNA and lncRNA expression and how it affects muscle well-being.

Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2 . Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

Background: The skeletal muscle reconstruction occurs thanks to unipotent stem cells, i.e., satellite cells. The satellite cells remain quiescent and localized between myofiber sarcolemma and basal lamina. They are activated in response to muscle injury, proliferate, differentiate into myoblasts, and recreate myofibers. The stem and progenitor cells support skeletal muscle regeneration, which could be disturbed by extensive damage, sarcopenia, cachexia, or genetic diseases like dystrophy. Many lines of evidence showed that the level of oxygen regulates the course of cell proliferation and differentiation., Methods: In the present study, we analyzed hypoxia impact on human and pig bone marrow-derived mesenchymal stromal cell (MSC) and mouse myoblast proliferation, differentiation, and fusion. Moreover, the influence of the transplantation of human bone marrow-derived MSCs cultured under hypoxic conditions on skeletal muscle regeneration was studied., Results: We showed that bone marrow-derived MSCs increased VEGF expression and improved myogenesis under hypoxic conditions in vitro. Transplantation of hypoxia preconditioned bone marrow-derived MSCs into injured muscles resulted in the improved cell engraftment and formation of new vessels., Conclusions: We suggested that SDF-1 and VEGF secreted by hypoxia preconditioned bone marrow-derived MSCs played an essential role in cell engraftment and angiogenesis. Importantly, hypoxia preconditioned bone marrow-derived MSCs more efficiently engrafted injured muscles; however, they did not undergo myogenic differentiation., (© 2021. The Author(s).)

Background: The skeletal muscle regeneration relays on the satellite cells which are stem cells located between basal lamina and plasmalemma of muscle fiber. In the injured muscles, the satellite cells become activated, start to proliferate, and then differentiate into myoblasts, which fuse to form myotubes and finally myofibers. The satellite cells play the crucial role in the regeneration; however, other cells present in the muscle could also support this process. In the present study, we focused on one population of such cells, i.e., muscle interstitial progenitor cells., Methods: We used the CD146 marker to identify the population of mouse muscle interstitial cells. We analyzed the expression of selected markers, as well as clonogenic, myogenic, adipogenic, and chondrogenic potential in vitro. Simultaneously, we analyzed satellite cell-derived myoblasts and bone marrow-derived mesenchymal stem/stromal cells that allowed us to pinpoint the differences between these cell populations. Moreover, we isolated CD146+ cells and performed heterotopic transplantations to follow their in vivo differentiation., Results: Mouse muscle CD146+ interstitial progenitor cells expressed nestin and NG2 but not PAX7. These cells presented clonogenic and myogenic potential both in vitro and in vivo. CD146+ cells fused also with myoblasts in co-cultures in vitro. However, they were not able to differentiate to chondro- or adipocytes in vitro. Moreover, CD146+ cells followed myogenic differentiation in vivo after heterotopic transplantation., Conclusion: Mouse CD146+ cells represent the population of mouse muscle interstitial progenitors that differ from satellite cell-derived myoblasts and have clonogenic and myogenic properties.

The proper functioning of tissues and organs depends on their ability to self-renew and repair. Some of the tissues, like epithelia, renew almost constantly while in the others this process is induced by injury or diseases. The stem or progenitor cells responsible for tissue homeostasis have been identified in many organs. Some of them, such as hematopoietic or intestinal epithelium stem cells, are multipotent and can differentiate into various cell types. Others are unipotent. The skeletal muscle tissue does not self-renew spontaneously, however, it presents unique ability to regenerate in response to the injury or disease. Its repair almost exclusively relies on unipotent satellite cells. However, multiple lines of evidence document that some progenitor cells present in the muscle can be supportive for skeletal muscle regeneration. Here, we summarize the current knowledge on the complicated landscape of stem and progenitor cells that exist in skeletal muscle and support its regeneration. We compare the cells from two model organisms, i.e., mouse and human, documenting their similarities and differences and indicating methods to test their ability to undergo myogenic differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Pluripotent stem cells present the ability to self-renew and undergo differentiation into any cell type building an organism. Importantly, a lot of evidence on embryonic stem cell (ESC) differentiation comes from in vitro studies. However, ESCs cultured in vitro do not necessarily behave as cells differentiating in vivo. For this reason, we used teratomas to study early and advanced stages of in vivo ESC myogenic differentiation and the role of Pax7 in this process. Pax7 transcription factor plays a crucial role in the formation and differentiation of skeletal muscle precursor cells during embryonic development. It controls the expression of other myogenic regulators and also acts as an anti-apoptotic factor. It is also involved in the formation and maintenance of satellite cell population., Methods: In vivo approach we used involved generation and analysis of pluripotent stem cell-derived teratomas. Such model allows to analyze early and also terminal stages of tissue differentiation, for example, terminal stages of myogenesis, including the formation of innervated and vascularized mature myofibers., Results: We determined how the lack of Pax7 function affects the generation of different myofiber types. In Pax7-/- teratomas, the skeletal muscle tissue occupied significantly smaller area, as compared to Pax7+/+ ones. The proportion of myofibers expressing Myh3 and Myh2b did not differ between Pax7+/+ and Pax7-/- teratomas. However, the area of Myh7 and Myh2a myofibers was significantly lower in Pax7-/- ones. Molecular characteristic of skeletal muscles revealed that the levels of mRNAs coding Myh isoforms were significantly lower in Pax7-/- teratomas. The level of mRNAs encoding Pax3 was significantly higher, while the expression of Nfix, Eno3, Mck, Mef2a, and Itga7 was significantly lower in Pax7-/- teratomas, as compared to Pax7+/+ ones. We proved that the number of satellite cells in Pax7-/- teratomas was significantly reduced. Finally, analysis of neuromuscular junction localization in samples prepared with the iDISCO method confirmed that the organization of neuromuscular junctions in Pax7-/- teratomas was impaired., Conclusions: Pax7-/- ESCs differentiate in vivo to embryonic myoblasts more readily than Pax7+/+ cells. In the absence of functional Pax7, initiation of myogenic differentiation is facilitated, and as a result, the expression of mesoderm embryonic myoblast markers is upregulated. However, in the absence of functional Pax7 neuromuscular junctions, formation is abnormal, what results in lower differentiation potential of Pax7-/- ESCs during advanced stages of myogenesis.

Under physiological conditions skeletal muscle regeneration depends on the satellite cells. After injury these cells become activated, proliferate, and differentiate into myofibers reconstructing damaged tissue. Under pathological conditions satellite cells are not sufficient to support regeneration. For this reason, other cells are sought to be used in cell therapies, and different factors are tested as a tool to improve the regenerative potential of such cells. Many studies are conducted using animal cells, omitting the necessity to learn about human cells and compare them to animal ones. Here, we analyze and compare the impact of IL-4 and SDF-1, factors chosen by us on the basis of their ability to support myogenic differentiation and cell migration, at mouse and human adipose tissue-derived stromal cells (ADSCs). Importantly, we documented that mouse and human ADSCs differ in certain reactions to IL-4 and SDF-1. In general, the selected factors impacted transcriptome of ADSCs and improved migration and fusion ability of cells in vitro. In vivo, after transplantation into injured muscles, mouse ADSCs more eagerly participated in new myofiber formation than the human ones. However, regardless of the origin, ADSCs alleviated immune response and supported muscle reconstruction, and cytokine treatment enhanced these effects. Thus, we documented that the presence of ADSCs improves skeletal muscle regeneration and this influence could be increased by cell pretreatment with IL-4 and SDF-1.

Heterotopic ossification (HO) manifests as bone development in the skeletal muscles and surrounding soft tissues. It can be caused by injury, surgery, or may have a genetic background. In each case, its development might differ, and depending on the age, sex, and patient's conditions, it could lead to a more or a less severe outcome. In the case of the injury or surgery provoked ossification development, it could be, to some extent, prevented by treatments. As far as genetic disorders are concerned, such prevention approaches are highly limited. Many lines of evidence point to the inflammatory process and abnormalities in the bone morphogenetic factor signaling pathway as the molecular and cellular backgrounds for HO development. However, the clear targets allowing the design of treatments preventing or lowering HO have not been identified yet. In this review, we summarize current knowledge on HO types, its symptoms, and possible ways of prevention and treatment. We also describe the molecules and cells in which abnormal function could lead to HO development. We emphasize the studies involving animal models of HO as being of great importance for understanding and future designing of the tools to counteract this pathology., Competing Interests: The authors declare no conflict of interest.

Skeletal muscle regeneration depends on the satellite cells, which, in response to injury, activate, proliferate, and reconstruct damaged tissue. However, under certain conditions, such as large injuries or myopathies, these cells might not sufficiently support repair. Thus, other cell populations, among them adipose tissue-derived stromal cells (ADSCs), are tested as a tool to improve regeneration. Importantly, the pro-regenerative action of such cells could be improved by various factors. In the current study, we tested whether IL-4 and SDF-1 could improve the ability of ADSCs to support the regeneration of rat skeletal muscles. We compared their effect at properly regenerating fast-twitch EDL and poorly regenerating slow-twitch soleus. To this end, ADSCs subjected to IL-4 and SDF-1 were analyzed in vitro and also in vivo after their transplantation into injured muscles. We tested their proliferation rate, migration, expression of stem cell markers and myogenic factors, their ability to fuse with myoblasts, as well as their impact on the mass, structure and function of regenerating muscles. As a result, we showed that cytokine-pretreated ADSCs had a beneficial effect in the regeneration process. Their presence resulted in improved muscle structure and function, as well as decreased fibrosis development and a modulated immune response.

Interplay between CXCR7 and other CXC receptors, namely CXCR4 or CXCR3, binding such ligands as SDF-1 or ITAC, was shown to regulate multiple cellular processes. The developmental role of signaling pathways mediated by these receptors was proven by the phenotypes of mice lacking either functional CXCR4, or CXCR7, or SDF-1, showing that formation of certain lineages relies on these factors. In this study, using in vitro differentiating mouse embryonic stem cells that lacked the function of CXCR7, we asked the question about the role of CXCR mediated signaling during early steps of differentiation. Our analysis showed that interaction of SDF-1 or ITAC with CXC receptors is necessary for the regulation of crucial developmental regulators expression and that CXCR7 is involved in the control of ESC pluripotency and differentiation into mesodermal lineages., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Myogenic differentiation during muscle regeneration is guided by various physical and biochemical factors. Recently, substratum elasticity has gained attention as a physical signal that influences both cell differentiation and tissue regeneration. In this work, we investigated the influence of substratum elasticity on proliferation and differentiation of myogenic cells, mouse myoblasts of the C2C12 cell line and mouse primary myoblasts derived from satellite cells-muscle stem cells playing key role in muscle regeneration. Materials with different elastic moduli within the MPa scale based on polydimethylsiloxane (PDMS) were used as cell substratum and characterized for surface roughness, wettability, and micromechanical characteristics. We found that surface properties of PDMS substrates are alter nonlinearly with the increase of the material's elastic modulus. Using this system we provide an evidence that materials with elastic modulus higher than that of physiological skeletal muscle tissue do not perturb myogenic differentiation of both types of myoblasts; thus, can be used as biomaterials for muscle tissue engineering. PDMS materials with elasticity within the range of 2.5-4 MPa may transiently limit the proliferation of myoblasts, but not the efficiency of their differentiation. Direct correlation between substratum elasticity and myogenic differentiation efficiency was not observed but the other surface properties of the PDMS materials such as nanoroughness and wettability were also diverse., (© 2019 Wiley Periodicals, Inc.)

Background: Satellite cells, a population of unipotent stem cells attached to muscle fibers, determine the excellent regenerative capability of injured skeletal muscles. Myogenic potential is also exhibited by other cell populations, which exist in the skeletal muscles or come from other niches. Mesenchymal stromal/stem cells inhabiting the bone marrow do not spontaneously differentiate into muscle cells, but there is some evidence that they are capable to follow the myogenic program and/or fuse with myoblasts., Methods: In the present study we analyzed whether IGF-1, IL-4, IL-6, and SDF-1 could impact human and porcine bone marrow-derived mesenchymal stromal/stem cells (hBM-MSCs and pBM-MSCs) and induce expression of myogenic regulatory factors, skeletal muscle-specific structural, and adhesion proteins. Moreover, we investigated whether these factors could induce both types of BM-MSCs to fuse with myoblasts. IGF-1, IL-4, IL-6, and SDF-1 were selected on the basis of their role in embryonic myogenesis as well as skeletal muscle regeneration., Results: We found that hBM-MSCs and pBM-MSCs cultured in vitro in the presence of IGF-1, IL-4, IL-6, or SDF-1 did not upregulate myogenic regulatory factors. Consequently, we confirmed the lack of their naïve myogenic potential. However, we noticed that IL-4 and IL-6 impacted proliferation and IL-4, IL-6, and SDF-1 improved migration of hBM-MSCs. IL-4 treatment resulted in the significant increase in the level of mRNA encoding CD9, NCAM, VCAM, and m-cadherin, i.e., proteins engaged in cell fusion during myotube formation. Additionally, the CD9 expression level was also driven by IGF-1 treatment. Furthermore, the pre-treatment of hBM-MSCs either with IGF-1, IL-4, or SDF-1 and treatment of pBM-MSCs either with IGF-1 or IL-4 increased the efficacy of hybrid myotube formation between these cells and C2C12 myoblasts., Conclusions: To conclude, our study revealed that treatment with IGF-1, IL-4, IL-6, or SDF-1 affects BM-MSC interaction with myoblasts; however, it does not directly promote myogenic differentiation of these cells.

Adolescent idiopathic scoliosis (AIS) is a relatively frequent disease within a range 0.5%-5.0% of population, with higher frequency in females. While a resultant spinal deformity is usually medically benign condition, it produces far going psychosocial consequences, which warrant attention. The etiology of AIS is unknown and current therapeutic approaches are symptomatic only, and frequently inconvenient or invasive. Muscular contribution to AIS is widely recognized, although it did not translate to clinical routine as yet. Muscle asymmetry has been documented by pathological examinations as well as systemic muscle disorders frequently leading to scoliosis. It has been also reported numerous genetic, metabolic and radiological alterations in patients with AIS, which are linked to muscular and neuromuscular aspects. Therefore, muscles might be considered an attractive and still insufficiently exploited therapeutic target for AIS. Stem cell-based regenerative medicine is rapidly gaining momentum based on the tremendous progress in understanding of developmental biology. It comes also with a toolbox of various stem cells such as satellite cells or mesenchymal stem cells, which could be transplanted; also, the knowledge acquired in research on regenerative medicine can be applied to manipulation of endogenous stem cells to obtain desired therapeutic goals. Importantly, paravertebral muscles are located relatively superficially; therefore, they can be an easy target for minimally invasive approaches to treatment of AIS. It comes in pair with a fast progress in image guidance, which allows for precise delivery of therapeutic agents, including stem cells to various organs such as brain, muscles, and others. Summing up, it seems that there is a link between AIS, muscles, and stem cells, which might be worth of further investigations with a long-term goal of setting foundations for eventual bench-to-bedside translation.

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging-i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting-and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1 , Pax3 , and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

In case of large injuries of skeletal muscles the pool of endogenous stem cells, i.e., satellite cells, might be not sufficient to secure proper regeneration. Such failure in reconstruction is often associated with loss of muscle mass and excessive formation of connective tissue. Therapies aiming to improve skeletal muscle regeneration and prevent fibrosis may rely on the transplantation of different types of stem cell. Among such cells are adipose tissue-derived stromal cells (ADSCs) which are relatively easy to isolate, culture, and manipulate. Our study aimed to verify applicability of ADSCs in the therapies of severely injured skeletal muscles. We tested whether 3D structures obtained from Matrigel populated with ADSCs and transplanted to regenerating mouse gastrocnemius muscles could improve the regeneration. In addition, ADSCs used in this study were pretreated with myoblasts-conditioned medium or anti-TGFβ antibody, i.e., the factors modifying their ability to proliferate, migrate, or differentiate. Analyses performed one week after injury allowed us to show the impact of 3D cultured control and pretreated ADSCs at muscle mass and structure, as well as fibrosis development immune response of the injured muscle.

Resolution of old problems with new tools seems to be a new way of challenging and finding the keys to innovation. A holistic view of different branches of science and industry, including services for society, is critically important for the development of modern science. The International Congress Eurobiotech 2017 was a special opportunity for such a universal view. In this paper, we discuss the application of different small RNAs, stem cells, epigenetics and sequencing, as well as art and bioeconomy. Our most significant message is not a new one but is very universal: biotechnology is vital for society., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Skeletal muscle regeneration is possible thanks to unipotent stem cells, which are satellite cells connected to the myofibers. Populations of stem cells other than muscle-specific satellite cells are considered as sources of cells able to support skeletal muscle reconstruction. Among these are bone marrow-derived mesenchymal stem cells (BM-MSCs), which are multipotent, self-renewing stem cells present in the bone marrow stroma. Available data documenting the ability of BM-MSCs to undergo myogenic differentiation are not definitive. In the current work, we aimed to check if the satellite cell niche could impact the ability of bone marrow-derived cells to follow a myogenic program., Methods: We established a new in-vitro method for the coculture of bone marrow-derived cells (BMCs) that express CXCR4 (CXCR4 + BMCs; the stromal-derived factor-1 (Sdf-1) receptor) with myofibers. Using various tests, we analyzed the myogenic identity of BMCs and their ability to fuse with myoblasts in vitro and in vivo., Results: We showed that Sdf-1 treatment increased the number of CXCR4 + BMCs able to bind the myofiber and occupy the satellite cell niche. Moreover, interaction with myofibers induced the expression of myogenic regulatory factors (MRFs) in CXCR4 + BMCs. CXCR4 + BMCs, pretreated by the coculture with myofibers and Sdf-1, participated in myotube formation in vitro and also myofiber reconstruction in vivo. We also showed that Sdf-1 overexpression in vivo (in injured and regenerating muscles) supported the participation of CXCR4 + BMCs in new myofiber formation., Conclusion: We showed that CXCR4 + BMC interaction with myofibers (that is, within the satellite cell niche) induced CXCR4 + BMC myogenic commitment. CXCR4 + BMCs, pretreated using such a method of culture, were able to participate in skeletal muscle regeneration.

MicroRNAs (miRNAs) are known regulators of various cellular processes, including pluripotency and differentiation of embryonic stem cells (ESCs). We analyzed differentiation of two ESC lines-D3 and B8, and observed significant differences in the expression of miRNAs and genes involved in pluripotency and differentiation. We also examined if transient miRNA overexpression could serve as a sufficient impulse modulating differentiation of mouse ESCs. ESCs were transfected with miRNA Mimics and differentiated in embryoid bodies and embryoid body outgrowths. miRNAs involved in differentiation of mesodermal lineages, such as miR145 and miR181, as well as miRNAs regulating myogenesis (MyomiRs)-miR1, miR133a, miR133b, and miR206 were tested. Using such approach, we proved that transient overexpression of molecules selected by us modulated differentiation of mouse ESCs. Increase in miR145 levels upregulated Pax3, Pax7, Myod1, Myog, and MyHC2, while miR181 triggered the expression of such crucial myogenic factors as Myf5 and MyHC2. As a result, the ability of ESCs to initiate myogenic differentiation and form myotubes was enhanced. Premature expression of MyomiRs had, however, an adverse effect on myogenic differentiation of ESCs. Stem Cells 2018;36:655-670., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Skeletal muscle growth and regeneration relies on the activation of muscle specific stem cells, that is, satellite cells. The activation and differentiation of satellite cells into myoblasts, as well as their migration, proliferation, and fusion of mononuclear myoblasts into a functional multi-nucleated muscle fiber, are associated with extracellular matrix (ECM) protein synthesis and degradation. The extracellular environment is dynamically adapting to the changes accompanying skeletal muscle growth or repair. Enzymes engaged in many biological processes that involve ECM remodeling are matrix metalloproteinases (MMPs). Among metalloproteinases crucial for skeletal muscles are two gelatinases-MMP-9 and MMP-2. In the current study we test the effect of silencing the MMP-9 and MMP-2 expression on the proliferation and differentiation of in vitro cultured skeletal muscle myoblasts. We show that downregulating gelatinase MMP-9 expression results in a delayed myoblast differentiation., (© 2017 International Federation for Cell Biology.)

The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous studies we showed that Sdf-1 (stromal derived factor -1) increased migration of stem cells and their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1 receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the cells of different migration and myogenic potential. We showed that Sdf-1 altered actin organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the transcription profile of genes encoding factors engaged in cells adhesion and migration. As the result, cells such as primary myoblasts or embryonic stem cells, became characterized by more effective migration when transplanted into regenerating muscle.

The injury of adult skeletal muscle initiates series of well-coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF-β family members is TGF-β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF-β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function., (© 2017 The Authors. Cell Biology International Published by Wiley Periodicals, Inc.)

Introduction: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so-called fast-twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow-twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation., Methods: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury., Results: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles., Conclusions: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow- and fast-twitch muscles. Muscle Nerve 55: 400-409, 2017., (© 2016 Wiley Periodicals, Inc.)

Professor Andrzej Krzysztof Tarkowski passed away last September (2016) at the age of 83. His findings, have become indispensable tools for immunological, genetic, and oncological studies, as well as for generating transgenic animals which are instrumental for studying gene function in living animals. His work and discoveries provided a tremendous input to the contemporary developmental biology of mammals.

Regeneration of skeletal muscle relies on the presence of satellite cells. Satellite cells deficiency accompanying some degenerative diseases is the reason for the search for the "replacement cells" that can be used in the muscle therapies. Due to their unique properties embryonic stem cells (ESCs), as well as myogenic cells derived from them, are considered as a promising source of therapeutic cells. Among the factors crucial for the specification of myogenic precursor cells is Pax7 that sustains proper function of satellite cells. In our previous studies we showed that ESCs lacking functional Pax7 are able to form myoblasts in vitro when differentiated within embryoid bodies and their outgrowths. In the current study we showed that ESCs lacking functional Pax7, cultured in vitro in monolayer in the medium supplemented with horse serum and 5azaC, expressed higher levels of factors associated with myogenesis, such as Pdgfra, Pax3, Myf5, and MyoD. Importantly, skeletal myosin immunolocalization confirmed that myogenic differentiation of ESCs was more effective in case of cells lacking Pax7. Our in vivo studies showed that ESCs transplanted into regenerating skeletal muscles were detectable at day 7 of regeneration and the number of Pax7-/- ESCs detected was significantly higher than of control cells. Our results support the concept that lack of functional Pax7 promotes proliferation of differentiating ESCs and for this reason more of them can turn into myogenic lineage., (Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.

Background: The skeletal muscle has the ability to regenerate after injury. This process is mediated mainly by the muscle specific stem cells, that is, satellite cells. In case of extensive damage or under pathological conditions, such as muscular dystrophy, the process of muscle reconstruction does not occur properly. The aim of our study was to test whether mobilized stem cells, other than satellite cells, could participate in skeletal muscle reconstruction., Methods: Experiments were performed on wild-type mice and mice lacking the functional Pax7 gene, that is, characterized by the very limited satellite cell population. Gastrocnemius mice muscles were injured by cardiotoxin injection, and then the animals were treated by stromal derived factor-1 (Sdf-1) with or without granulocyte-colony stimulating factor (G-CSF) for 4 days. The muscles were subjected to thorough assessment of the tissue regeneration process using histological and in vitro methods, as well as evaluation of myogenic factors' expression at the transcript and protein levels., Results: Stromal derived factor-1 alone and Sdf-1 in combination with G-CSF significantly improved the regeneration of Pax7-/- skeletal muscles. The Sdf-1 and G-CSF treatment caused an increase in the number of mononucleated cells associated with muscle fibres. Further analysis showed that Sdf-1 and G-CSF treatment led to the rise in the number of CD34+ and Cxcr4+ cells and expression of Cxcr7., Conclusions: Stromal derived factor-1 and G-CSF stimulated regeneration of the skeletal muscles deficient in satellite cells. We suggest that mobilized CD34+, Cxcr4+, and Cxcr7+ cells can efficiently participate in the skeletal muscle reconstruction and compensate for the lack of satellite cells.

The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs.

Pluripotent stem cells (PSCs), such as embryonic stem cells or induced pluripotent stem cells are a promising source of cells for regenerative medicine as they can differentiate into all cell types building a mammalian body. However, protocols leading to efficient and safe in vitro generation of desired cell types must be perfected before PSCs can be used in cell therapies or tissue engineering. In vivo, i.e. in developing mouse embryo or teratoma, PSCs can differentiate into skeletal muscle, but in vitro their spontaneous differentiation into myogenic cells is inefficient. Numerous attempts have been undertaken to enhance this process. Many of them involved mimicking the interactions occurring during embryonic myogenesis. The key regulators of embryonic myogenesis, such as Wnts proteins, fibroblast growth factor 2, and retinoic acid, have been tested to improve the frequency of in vitro myogenic differentiation of PSCs. This review summarizes the current state of the art, comparing spontaneous and directed myogenic differentiation of PSCs as well as the protocols developed this far to facilitate this process.

The skeletal muscle injury triggers the inflammatory response which is crucial for damaged muscle fiber degradation and satellite cell activation. Immunodeficient mice are often used as a model to study the myogenic potential of transplanted human stem cells. Therefore, it is crucial to elucidate whether such model truly reflects processes occurring under physiological conditions. To answer this question we compared skeletal muscle regeneration of BALB/c, i.e. animals producing all types of inflammatory cells, and SCID mice. Results of our study documented that initial stages of muscles regeneration in both strains of mice were comparable. However, lower number of mononucleated cells was noticed in regenerating SCID mouse muscles. Significant differences in the number of CD14-/CD45+ and CD14+/CD45+ cells between BALB/c and SCID muscles were also observed. In addition, we found important differences in M1 and M2 macrophage levels of BALB/c and SCID mouse muscles identified by CD68 and CD163 markers. Thus, our data show that differences in inflammatory response during muscle regeneration, were not translated into significant modifications in muscle regeneration.

Introduction: Understanding the mechanism of stem cell mobilization into injured skeletal muscles is a prerequisite step for the development of muscle disease therapies. Many of the currently studied stem cell types present myogenic potential; however, when introduced either into the blood stream or directly into the tissue, they are not able to efficiently engraft injured muscle. For this reason their use in therapy is still limited. Previously, we have shown that stromal-derived factor-1 (Sdf-1) caused the mobilization of endogenous (not transplanted) stem cells into injured skeletal muscle improving regeneration. Here, we demonstrate that the beneficial effect of Sdf-1 relies on the upregulation of the tetraspanin CD9 expression in stem cells., Methods: The expression pattern of adhesion proteins, including CD9, was analysed after Sdf-1 treatment during regeneration of rat skeletal muscles and mouse Pax7-/- skeletal muscles, that are characterized by the decreased number of satellite cells. Next, we examined the changes in CD9 level in satellite cells-derived myoblasts, bone marrow-derived mesenchymal stem cells, and embryonic stem cells after Sdf-1 treatment or silencing expression of CXCR4 and CXCR7. Finally, we examined the potential of stem cells to fuse with myoblasts after Sdf-1 treatment., Results: In vivo analyses of Pax7-/- mice strongly suggest that Sdf-1-mediates increase in CD9 levels also in mobilized stem cells. In the absence of CXCR4 receptor the effect of Sdf-1 on CD9 expression is blocked. Next, in vitro studies show that Sdf-1 increases the level of CD9 not only in satellite cell-derived myoblasts but also in bone marrow derived mesenchymal stem cells, as well as embryonic stem cells. Importantly, the Sdf-1 treated cells migrate and fuse with myoblasts more effectively., Conclusions: We suggest that Sdf-1 binding CXCR4 receptor improves skeletal muscle regeneration by upregulating expression of CD9 and thus, impacting at stem cells mobilization to the injured muscles.

Duchenne muscular dystrophy (DMD), the most common and most severe form of all muscular dystrophies, leads to progressive muscle fiber necrosis, fibroblast proliferation, and growth of fibrous tissue and fat. The most common cause of death in DMD patients is cardiac and respiratory failure. Current pharmacological and other treatment methods do not lead to full recovery. For this reason, new alternatives for skeletal muscle regeneration are being investigated. Transplantation of myoblasts from healthy donors is one studied approach to muscle treatment in DMD patients. However, the results of intramuscular injection of in vitro cultured myoblasts are still not satisfactory. The use of autologous stem cells is also proposed. Despite many ongoing studies, this therapy is still in preliminary testing and requires more experiments.

Pluripotent stem cells are a potential source of various cell types for use in regenerative medicine. Despite accumulating knowledge, there is currently no efficient and reproducible protocol that does not require genetic manipulation for generation of myogenic cells from pluripotent stem cells. Here, we examined whether mouse embryonic stem (ES) cells are able to undergo myogenic differentiation and fusion in response to signals released by differentiating myoblasts. Using ES cells expressing the histone 2B-green fluorescent fusion protein, we were able to detect hybrid myotubes formed by ES cells and differentiating myoblasts. ES cells that fused with myoblasts downregulated the expression of pluripotency markers and induced the expression of myogenic markers, while unfused ES cells did not exhibit this expression pattern. Thus, the signals released by myoblasts were not sufficient to induce myogenic differentiation of ES cells. Although ES cells synthesize many proteins involved in myoblast adhesion and fusion, we did not observe any myotubes formed exclusively by ES cells. We found that ES cells lacked M-cadherin and vascular cell adhesion molecule-1, which may account for the low frequency of hybrid myotube formation in ES cell-myoblast co-cultures and the inability of ES cells alone to form myotubes.

Adherent A431, BHK-21, and C2C12 cells were cultured on a flexible interface formed between two immiscible liquid phases: (i) hydrophobic perfluorodecalin (PFD) and (ii) aqueous culture medium (DMEM). BHK-21 cells formed multicellular aggregates characterized by irregular shapes. A431, as well as C2C12 cells, grew as tight multicellular sheets of 3-D cells. Enhanced mass transfer and facilitated access of the cells to the O2 dissolved in PFD/DMEM by approx. 250 % and thereby increased the density of BHK-21 cells. Thus the liquid/liquid system is a simple, ready-to-use, and fully scalable (independent of vessel shapes); consequently it is a method for 3-D cultures of adherent animal cells in which the growth of anchorage-dependent cells is not limited by confluence effect.

Background Information: Matrix metalloproteinases (MMPs) are the key enzymes responsible for the remodelling of extracellular matrix. Two of them, namely MMP-2 and MMP-9 (gelatinases A and B, respectively), are expressed in skeletal muscles and are involved in their regeneration after the injury. Although MMPs are primarily known to act extracellularly, recent studies have shown that some of them are also found within the cell. In this study, we examine intracellular localisation of gelatinases during myoblasts differentiation in vitro, focussing the impact of MMPs inhibition on the myoblasts proliferation and function., Results: We show that MMP-9 localises within the S-phase nuclei of in vitro differentiating myoblasts. The inhibition of MMPs activity achieved by either doxycycline (a non-competitive inhibitor of collagenases), TIMP-1 (tissue inhibitor of metalloproteinases 1) or neutralising anti-MMP-9 antibody affects nuclear localisation of this gelatinase, and impacts at myoblasts proliferation., Conclusions: During myoblasts differentiation, MMP-9 that is localised in nuclei might be involved in the processes regulating cell cycle progression., (© 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Formation of mammalian skeletal muscle myofibers, that takes place during embryogenesis, muscle growth or regeneration, requires precise regulation of myoblast adhesion and fusion. There are few evidences showing that adhesion proteins play important role in both processes. To follow the function of these molecules in myoblast differentiation we analysed integrin alpha3, integrin beta1, ADAM12, CD9, CD81, M-cadherin, and VCAM-1 during muscle regeneration. We showed that increase in the expression of these proteins accompanies myoblast fusion and myotube formation in vivo. We also showed that during myoblast fusion in vitro integrin alpha3 associates with integrin beta1 and ADAM12, and also CD9 and CD81, but not with M-cadherin or VCAM-1. Moreover, we documented that experimental modification in the expression of integrin alpha3 lead to the modification of myoblast fusion in vitro. Underexpression of integrin alpha3 decreased myoblasts' ability to fuse. This phenomenon was not related to the modifications in the expression of other adhesion proteins, i.e. integrin beta1, CD9, CD81, ADAM12, M-cadherin, or VCAM-1. Apparently, aberrant expression only of one partner of multiprotein adhesion complexes necessary for myoblast fusion, in this case integrin alpha3, prevents its proper function. Summarizing, we demonstrated the importance of analysed adhesion proteins in myoblast fusion both in vivo and in vitro.

This paper describes the genesis of discoveries that have allowed cell reprogramming and derivation of induced pluripotent stem cells. This achievement has been distinguished by the 2012 Nobel Prize in Physiology or Medicine awarded to John B. Gurdon and Shinya Yamanaka. The verdict of the Nobel Committee was as follows: "for the discovery that mature cells can be reprogrammed to become pluripotent". The basis for the discovery was done by Gurdon in the 60s of the twentieth century, although he was not a pioneer in his field of research. The last word was pronounced, however, by Yamanaka at the beginning of the twenty-first century. The Japanese was born fifty years ago, that is exactly the year when Gurdon made his most important discoveries. Despite such a large difference in age of the two scientists their studies complement each other perfectly and promise numerous applications in regenerative medicine.

Different sources of stem cells are considered as a potential source of precursor cells that could improve skeletal muscle regeneration. Under physiological conditions muscle regeneration is based on the satellite cells, i.e. adult muscle precursor cells that are localized between muscle fiber and surrounding basal lamina. These cells remain quiescent but after skeletal muscle injury activate, proliferate, differentiate, and fuse either to form new muscle fibers or reconstruct the damaged ones. As it was shown in many studies few populations of stem cells other than satellite cells are able to support skeletal muscle regeneration. Among them are mesenchymal stem cells (MSCs) that are present in many niches within adult organism and also in fetal tissues, such as human umbilical cord blood (HUCB) or umbilical cord connective tissue, i.e. Wharton's jelly. Thus, MSCs are intensively tested to prove that they are able to differentiate into various cell types, including skeletal myoblasts, and therefore could be useful in regenerative medicine. In our previous study we showed that MSCs isolated from Wharton's jelly expressed pluripotency as well as myogenic markers and were able to undergo myogenic differentiation both in vitro and in vivo. We also analyzed the potential of HUCB cells population which contains not only MSCs but also hematopoietic precursors. Our analyses of whole population of HUCB cells showed that these cells express myogenic regulatory factors, i.e. MyoD, and are able to contribute to skeletal muscle regeneration. In the present study we document that adherent fraction of HUCB cells, i.e. the cells that constitute the subpopulation enriched in MSCs, expresses pluripotency and myogenic markers, and have a positive impact at the regeneration of injured mouse skeletal muscle.

Background Information: The regeneration of skeletal muscles involves satellite cells, which are muscle-specific precursor cells. In muscles, injured either mechanically or as a consequence of a disease, such as muscular dystrophy, local release of the growth factors and cytokines leads to satellite cells activation, proliferation and differentiation of the resulting myoblasts, followed by the formation of new myofibres. Various cell types, such as stem and progenitor cells, originating from other tissues different than the muscle, are also able to follow a myogenic program. Participation of these cells in the repair process depends on their precise mobilisation to the site of the injury., Results: In this study, we showed that stromal-derived factor-1 (Sdf-1) impacts on the mobilisation of CXC chemokine receptor (Cxcr)4-positive cells and improves skeletal muscle regeneration. Analysis of isolated and in vitro cultured satellite cells showed that Sdf-1 did not influence myoblasts proliferation and expression of myogenic regulatory transcription factors but induced migration of the myoblasts in Cxcr4-dependent ways. This phenomenon was also associated with the increased activity of crucial extracellular matrix modifiers, i.e. metalloproteases Mmp-2 and Mmp-9., Conclusions: Thus, positive impact of Sdf-1 on muscle regeneration is related to the mobilisation of endogenous cells, that is satellite cells and myoblasts, as well as non-muscle stem cells, expressing Cxcr4 and CD34., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)

The regeneration of skeletal muscles relies on the function of satellite cells that are quiescent myogenic precursors associated with adult skeletal muscle fibers. Upon injury, the satellite cells are activated, divide extensively, and differentiate into new myofibers. These events are accompanied by the remodeling of the surrounding extracellular matrix, which is mediated by variety of factors, including matrix metalloproteinases (MMPs). Regeneration of certain type of muscles, such as Soleus slow twitch muscle, is often inefficient and hindered by the development of fibrosis. Here, we studied the effect of inhibition of MMP-9 and MMP-2 activity on the Soleus muscle regeneration in vivo and on the in vitro differentiation of myoblasts derived from this muscle. Using in situ and in-gel zymography, we tested the activity of these two MMPs in vivo, during regeneration of the muscle, and in vitro, during differentiation of the myoblasts. We also analyzed the histology of regenerating muscles and morphology of differentiating myoblasts. All these analyses showed that treatment with doxycycline and anti-MMP-9, but not MMP-2 antibody, significantly improved Soleus muscle regeneration and ameliorated development of excessive fibrosis, as well as delayed myoblast proliferation and differentiation in vitro.

The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are an important tool in the studies focusing at the differentiation of various cell types, including skeletal myoblasts. They are also considered as a source of the cells that due to their pluripotent character and availability could be turned into any required tissue and then used in future in regenerative medicine. However, the methods of the derivation of some of cell types from pluripotent cells still need to be perfected. This chapter summarizes the history and current advancements in the derivation and testing of pluripotent stem cells-derived skeletal myoblasts. It focuses at the in vitro methods allowing the differentiation of stem cells grown in monolayer or propagated as embryoid bodies, and also at in vivo tests allowing the verification of the functionality of obtained skeletal myoblasts.

Nonhematopoietic cord blood cells and mesenchymal cells of umbilical cord Wharton's jelly have been shown to be able to differentiate into various cell types. Thus, as they are readily available and do not raise any ethical issues, these cells are considered to be a potential source of material that can be used in regenerative medicine. In our previous study, we tested the potential of whole mononucleated fraction of human umbilical cord blood cells and showed that they are able to participate in the regeneration of injured mouse skeletal muscle. In the current study, we focused at the umbilical cord mesenchymal stromal cells isolated from Wharton's jelly. We documented that limited fraction of these cells express markers of pluripotent and myogenic cells. Moreover, they are able to undergo myogenic differentiation in vitro, as proved by coculture with C2C12 myoblasts. They also colonize injured skeletal muscle and, with low frequency, participate in the formation of new muscle fibers. Pretreatment of Wharton's jelly mesenchymal stromal cells with SDF-1 has no impact on their incorporation into regenerating muscle fibers but significantly increased muscle mass. As a result, transplantation of mesenchymal stromal cells enhances the skeletal muscle regeneration.