The Role of Pericytes in Lipopolysaccharide-Induced Murine Acute Respiratory Distress Syndrome.

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+ ß-type platelet-derived growth factor receptor+ pericytes in murine lungs with loss of CD31+ ß-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.

miRNA-126a plays important role in myoblast and endothelial cell interaction.

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.

SDF-1 and NOTCH signaling in myogenic cell differentiation: the role of miRNA10a, 425, and 5100.

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.

Comparison of Muscle Regeneration after BMSC-Conditioned Medium, Syngeneic, or Allogeneic BMSC Injection.

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.

The miR151 and miR5100 Transfected Bone Marrow Stromal Cells Increase Myoblast Fusion in IGFBP2 Dependent Manner.

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.

Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions.

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.

Hypoxia preconditioned bone marrow-derived mesenchymal stromal/stem cells enhance myoblast fusion and skeletal muscle regeneration.

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.

Mouse CD146+ muscle interstitial progenitor cells differ from satellite cells and present myogenic potential.

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.

Beneficial Effect of IL-4 and SDF-1 on Myogenic Potential of Mouse and Human Adipose Tissue-Derived Stromal Cells.

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.

Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas.

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.

IL-4 and SDF-1 Increase Adipose Tissue-Derived Stromal Cell Ability to Improve Rat Skeletal Muscle Regeneration.

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.

The factors present in regenerating muscles impact bone marrow-derived mesenchymal stromal/stem cell fusion with myoblasts.

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.

Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion.

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.

Adipose Tissue-Derived Stromal Cells in Matrigel Impacts the Regeneration of Severely Damaged Skeletal Muscles.

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-TGFß 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.

Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle.

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.

Inflammatory response during slow- and fast-twitch muscle regeneration.

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.

Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/Cxcr7 axis.

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.

Stromal derived factor-1 and granulocyte-colony stimulating factor treatment improves regeneration of Pax7-/- mice skeletal muscles.

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.

Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene.

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.

Progression of inflammation during immunodeficient mouse skeletal muscle regeneration.

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.