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.

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.

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.

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.

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.

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.

Polydimethylsiloxane materials with supraphysiological elasticity enable differentiation of myogenic cells.

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.

Silencing of gelatinase expression delays myoblast differentiation in vitro.

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.

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.

The role of TGF-ß1 during skeletal muscle regeneration.

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 ß family. One of the TGF-ß family members is TGF-ß1, 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-ß1 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.

[From Gurdon to Yamanaka--a brief history of cell reprogramming]. / Od Gurdona do Yamanaki, czyli krótka historia reprogralmowania komórek.

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.

From planarians to mammals - the many faces of regeneration.

This report presents the history of the involvement of the Department of Cytology in studies of different aspects of regeneration. It can be divided into two major phases; the first focused on the regeneration of Turbellarians and the second on the regeneration of rat skeletal muscles including the differentiation of satellite cells in vitro. Regeneration of Turbellarians was investigated both at the cellular and molecular levels including the role of the protein kinase C (PKC) in this process. Studies on skeletal muscle regeneration initially focused on factors involved in regulation of signal transduction pathways. Next, we explored the influence of growth factors on the modulation of the regeneration process. Another important aspect of our studies was investigating of the distribution and function of different proteins involved in adhesion and fusion of myoblasts. Finally, we are also conducting research on the role of stem cells from other tissues in the regeneration of skeletal muscle.

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo.

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.