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.

CXCR4/ACKR3/CXCL12 axis in the lymphatic metastasis of vulvar squamous cell carcinoma.

AIMS: Vulvar squamous cell carcinoma (VSCC) spreads early and mainly locally via direct expansion into adjacent structures, followed by lymphatic metastasis to the regional lymph nodes (LNs). In the lymphatic metastasis, cancer cells bearing CXCR4 and ACKR3 (CXCR7) receptors are recruited to the LNs that produce the CXCL12 ligand. Our study aimed to assess the role of the CXCR4/ACKR3/CXCL12 axis in VSCC progression. METHODS: Tumour and LN tissue samples were obtained from 46 patients with VSCC and 51 patients with premalignant vulvar lesions. We assessed CXCR4, ACKR3 and CXCL12 by immunohistochemistry (IHC) in the tissue samples. Additionally, CXCL12 levels were determined by ELISA in the sera of 23 patients with premalignant lesions, 37 with VSCC and 16 healthy volunteers. RESULTS: CXCR4 and ACKR3 proteins were virtually absent in vulvar precancers, while in VSCC samples the IHC staining was strong. In the LNs of patients with VSCC, 98% of metastatic cells expressed CXCR4 and 85% expressed ACKR3. Neither CXCR4 nor ACKR3 presence was correlated with tumour human papilloma virus status. Few CXCL12-positive cells were found in the analysed tissue samples, but serum CXCL12 levels were significantly increased in both patients with premalignant vulvar lesions and with VSCC compared with healthy volunteers. CONCLUSIONS: It appears that during progression and lymphatic spread of VSCC, the CXCR4/ACKR3/CXCL12 axis is activated. Moreover, our data suggest that CXCR4 antagonists merit further attention as a possible therapeutic option in patients with VSCC.

Ultrafine-Grained Ti-31Mo-Type Composites with HA and Ag, Ta2O5 or CeO2 Addition for Implant Applications.

Ultrafine-grained Ti31Mo alloy and Ti31Mo5HA, Ti31Mo5HA-Ag (or Ta2O5, CeO2) composites with a grain size of approximately 2 µm were produced by the application of mechanical alloying and powder metallurgy. Additionally, the surface of the Ti31Mo alloy was modified. In the first stage, the specimens were immersed in 5M NaOH for 24 h at 60 °C. In the second stage, hydroxyapatite (HA) was deposited on the sample surface. The cathodic deposition at -5 V vs. open circuit potential (OCP) in the electrolyte containing 0.25M CaNa2-EDTA (di-calcium ethylenediaminetetraacetic acid), 0.25M K2HPO4 in 1M NaOH at 120 °C for 2 h was applied. The bulk Ti31Mo alloy is a single ß-type phase. In the alkali-modified surface titanium oxide, Ti3O is formed. After hydrothermal treatment, the surface layer mostly consists of the Ca10(PO4)6(OH)2 (81.23%) with about 19% content of CaHPO4·2H2O. Using optical profiler, roughness 2D surface topography parameters were estimated. The in vitro cytocompatibility of synthesized materials was studied. The cell lines of normal human osteoblasts (NHost) and human periodontal ligament fibroblasts (HPdLF) was conducted in the presence of tested biomaterials. Ultrafine-grained Ti-based composites altered with HA and Ag, Ta2O5 or CeO2 have superior biocompatibility than the microcrystalline Ti metal. NHost and HPdLF cells in the contact with the synthesized biomaterial showed stable proliferation activity. Biocompatibility tests carried out indicate that the ultrafine-grained Ti31Mo5HA composites with Ag, Ta2O5, or CeO2 could be a good candidate for implant applications.

Composite and Surface Functionalization of Ultrafine-Grained Ti23Zr25Nb Alloy for Medical Applications.

In this study, the ultrafine-grained Ti23Zr25Nb-based composites with 45S5 Bioglass and Ag, Cu, or Zn additions were produced by application of the mechanical alloying technique. Additionally, the base Ti23Zr25Nb alloy was electrochemically modified in the two stages of processing: electrochemical etching in the solution of H3PO4 and HF followed by electrochemical deposition in Ca(NO3)2, (NH4)2HPO4, and HCl. The in vitro cytocompatibility studies were also done with comparison to the commercially pure titanium. The established cell lines of Normal Human Osteoblasts (NHost, CC-2538) and Human Periodontal Ligament Fibroblasts (HPdLF, CC-7049) were used. The culture was conducted among the tested materials. Ultrafine-grained titanium-based composites modified with 45S5 Bioglass and Ag, Cu, or Zn metals have higher biocompatibility than the reference material in the form of a microcrystalline Ti. Proliferation activity was at a stable level with contact with studied materials. In vitro evaluation research showed that the ultrafine-grained Ti23Zr25Nb-based composites with 45S5 Bioglass and Ag, Cu, or Zn additions, with a Young modulus below 50 GPa, can be further used in the biomedical field.

Response of inflammatory cells to biodegradable ultra-fine grained Mg-based composites.

Ultra-fine grained biodegradable Mg-based Mg1Zn1Mn0.3 Zr - HA and Mg4Y5.5Dy0.5 Zr - 45S5 Bioglass composites have shown great medical potential. Two types of these Mg-based biomaterials subjected to different treatments were tested and as shown earlier they are biocompatible. The aim of the study is to determine how much culture media incubated with these ultra-fine trained Mg-based composites can cause inflammatory reactions and /or periodontal cell death. The incubation of composites in the medium releases metal ions into the solution. It can be assumed that this process is permanent and also occurs in the human body. The results have shown that the effect of proinflammatory IL-6 and TNF- cytokines results in the strongest production of the acute phase proteins in the first day on the Mg1Zn1Mn0.3 Zr-5 wt.% HA-1 wt. % Ag HF-treated biocomposite after immersion for 2 h in 40 % HF and then the fastest decrease in these processes on the third day. In turn, the inflammatory process induced on the Mg1Zn1Mn0.3 Zr-5 wt.% HA-1 wt. % Ag biomaterial, in BAX / BCL ratio assessment, is the strongest on the third day and maintains a significantly high level on the following day, which, at the same time, confirms its persistence and development. In addition, these results confirm the successively generated necrotic processes. Ions can induce inflammatory reactions, which in the case of the implant may take a long time, which results in the loss of the implant. Even if the material is biocompatible in rapid in-vitro tests, it can induce inflammation in the body after some time due to the release of ions. Not every treatment improves the material's properties in terms of subsequent safety.

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.

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.

Somatic mutation profiling of vulvar cancer: Exploring therapeutic targets.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) constitutes over 90% of vulvar cancer. Its pathogenesis can follow two different pathways; high risk human papillomavirus (hrHPV)-dependent and HPV-independent. Due to the rarity of VSCC, molecular mechanisms underlying VSCC development remain largely unknown. The study aimed to identify pathogenic mutations implicated in the two pathways of VSCC development. METHODS: Using next generation sequencing, 81 VSCC tumors, 52 hrHPV(+) and 29 hrHPV(-), were screened for hotspot mutations in 50 genes covered by the Ion AmpliSeq Cancer Hotspot Panel v2 Kit (Thermo Fisher Scientific). RESULTS: Mutations of TP53 (46% and 41%, of hrHPV(+) and hrHPV(-) cases respectively) and CDKN2A (p16) (25% and 21%, of hrHPV(+) and hrHPV(-) cases respectively) were the most common genetic alterations identified in VSCC tumors. Further mutations were identified in PIK3CA, FBXW7, HRAS, FGFR3, STK11, AKT1, SMAD4, FLT3, JAK3, GNAQ, and PTEN, albeit at low frequencies. Some of the identified mutations may activate the PI3K/AKT/mTOR pathway. The activation of mTOR was confirmed in the vast majority of VSCC samples by immunohistochemical staining. CONCLUSIONS: Detecting pathogenic mutations in 13/50 genes examined at comparable frequencies in hrHPV(+) and hrHPV(-) tumors suggest that genetic mechanisms of the two routes of VSCC pathogenesis may be similar, despite being initiated from different premalignant lesions. Importantly, our data provide a rationale for new anti-VSCC therapies targeting the PI3K/AKT/mTOR pathway.

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.

Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged 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.

[Are satellite cells stem cells?]. / Czy komórki satelitowe sa macierzyste?

Satellite cells, localized in the niche between the membrane of muscle fiber and basal lamina that surrounds it, serve as a source of myoblasts that are necessary for both growth and regeneration of skeletal muscle. Apart from their ability to convert into myoblasts, satellite cells are also able to self-renew, thus, they meet requirements for tissue specific, unipotent stem cells. Recently conducted research revealed that population of satellite cells is heterogeneous. The article summarizes current information on biology and characteristics of satellite cells, and also describes models concerning mechanisms of self-renewal and differentiation of satellite cells. Experiments regarding in vitro differentiation of satellite cells into other cell types are also discussed. Moreover, other population of stem cells localized in the muscle are described in this review.

Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells.

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.