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. To study their role in ARDS, we used a murine model of lipopolysaccharide (LPS)-induced resolving ARDS. We confirmed the development of ARDS after LPS instillation, which was resolved 14 days after onset. Using immunofluorescence and flow cytometry, we observed 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, 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.

Coding and noncoding RNA profile of human heterotopic ossifications - Risk factors and biomarkers.

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.

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.

The role of miRNA and lncRNA in heterotopic ossification pathogenesis.

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 TGFß1, 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.

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.

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.

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 Survey of Cells Responsible for Heterotopic Ossification Development in Skeletal Muscles-Human and Mouse Models.

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.

Human and mouse skeletal muscle stem and progenitor cells in health and disease.

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.

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.

The role of CXC receptors signaling in early stages of mouse embryonic stem cell differentiation.

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.

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.

Muscular Contribution to Adolescent Idiopathic Scoliosis from the Perspective of Stem Cell-Based Regenerative Medicine.

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.

Induction of bone marrow-derived cells myogenic identity by their interactions with the satellite cell niche.

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.

Aspects of pericytes and their potential therapeutic use.

Pericytes, which are multi-potential stem cells, co-create the walls of the microvessels: capillaries, terminal arterioles and postcapillary venules. These cells are localized under the basement membrane, tightly encircling the endothelium. The most frequently mentioned molecular markers of pericytes include NG2 (neural-glial antigen 2), ß-type platelet-derived growth factor receptor (PDGFRß), smooth muscle α-actin (α-SMA), regulator of G protein signalling 5 (RGS5), the adhesion protein CD146 and nestin. Different functions in physiological processes are assigned to pericytes such as maintaining the integrity and senescence of endothelial cells, transregulation of vascular tone or the potential to differentiate into other cells. Probably they are also involved in pathological processes such as tissues fibrosis. In this review, we focus on the participation of pericytes in the process of blood vessel formation, the regeneration of skeletal muscle tissue and fibrosis. Strong evidence for pericytes' participation in endothelial homeostasis, as well as in pathological conditions such as fibrosis, reveals a broad potential for the therapeutic use of these cells. Targeted pharmacological modulation of pericytes, leading to blocking signalling pathways responsible for the differentiation of pericytes into myofibroblasts, seems to be a promising strategy for the treatment of fibrosis in the early stages.

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.