We developed a cellular model of mesenchymal stem cells (MSCs) with inducible DUX4 expression (MSC-DUX4) to investigate the potential role of MSCs in facioscapulohumeral muscular dystrophy (FSHD). DUX4 expression was successfully induced, with MSC-DUX4 maintaining the characteristic surface marker profile of MSCs. Unlike myoblasts, which rapidly undergo apoptosis upon DUX4 induction, MSC-DUX4 remained viable although they exhibited increased reactive oxygen species (ROS) accumulation. Transcriptomic analysis revealed broad changes, including strong upregulation of several myogenic genes, suggesting that DUX4 confers a partial myogenic program to MSCs. Indeed, Dox-induced MSC-DUX4 formed myotube-like structures expressing myogenic markers (myogenin, Troponin T, MF20), though fusion efficiency was markedly reduced compared to myoblasts, indicating limited and likely defective myogenic differentiation capacity. In parallel, adipogenic and osteogenic potentials were strongly impaired, as demonstrated by reduced lipid and calcium deposition, altered expression of FABP4 and leptin. Moreover, DUX4-expressing MSCs displayed pro-fibrotic features, including enhanced collagen III/IV and fibronectin, suggesting impaired extracellular matrix turnover. Together, these findings indicate that DUX4 induces a unique phenotype in MSCs, characterized by impaired differentiation, oxidative stress, partial myogenic reprogramming, and pro-fibrotic activity, contributing to muscle pathology in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of the transcription factor DUX4 in skeletal muscle, leading to progressive muscle degeneration and pathological remodelling of the muscle microenvironment. Although the effects of DUX4 in myogenic cells have been extensively investigated, its potential impact on mesenchymal stromal cells (MSCs), which contribute to tissue homeostasis and regeneration, remains poorly understood. Here, we investigated the consequences of ectopic DUX4 expression in human MSCs and assessed their differentiation potential and transcriptional phenotype. DUX4-expressing MSCs acquired selected features of the myogenic phenotype, including expression of muscle-associated markers and formation of myotube-like structures, although their myogenic differentiation and fusion capacity remained markedly lower than those of bona fide myoblasts. In parallel, DUX4 expression substantially altered MSC lineage potential, reducing adipogenic and osteogenic differentiation. Transcriptomic and functional analyses further revealed prominent activation of extracellular matrix (ECM)-associated and pro-fibrotic programmes, together with increased oxidative stress. These findings indicate that DUX4 profoundly alters the differentiation landscape of MSCs, inducing a complex cell state characterised by partial activation of muscle-associated programmes and a prominent pro-fibrotic/ECM-remodelling phenotype. Our results suggest that DUX4 may influence not only myogenic cells but also mesenchymal populations within the FSHD muscle microenvironment. Further studies are required to determine whether these altered MSC states contribute functionally to fibrosis, impaired muscle regeneration or intercellular communication in vivo.

BACKGROUND:Mesenchymal stromal cells (MSCs) are recognized for their potential in regenerative medicine, attributed to their multipotent differentiation capabilities and immunomodulatory properties. Despite this potential, the classification and detailed characterization of MSCs, especially those derived from specific tissues like the pancreas, remains challenging leading to a proliferation of terminology in the literature. This study aims to address these challenges by providing a thorough characterization of human pancreatic islets-derived mesenchymal stromal cells (hPD-MSCs). METHODS:hPD-MSCs were isolated from donor islets using enzymatic digestion, immortalized through lentiviral transduction of human telomerase reverse transcriptase (hTERT). Cells were characterized by immunostaining, flow cytometry and multilineage differentiation potential into adipogenic and osteogenic lineages. Further a transcriptomic analysis was done to compare the gene expression profiles of hPD-MSCs with other mesenchymal cells. RESULTS:We show that hPD-MSCs express the classical MSC features, including morphological characteristics, surface markers expression (CD90, CD73, CD105, CD44, and CD106) and the ability to differentiate into both adipogenic and osteogenic lineages. Furthermore, transcriptomic analysis revealed distinct gene expression profiles, showing notable similarities between hPD-MSCs and pancreatic stellate cells (PSCs). The study also identified specific genes that distinguish hPD-MSCs from MSCs of other origins, including genes associated with pancreatic function (e.g., ISL1) and neural development (e.g., NPTX1, ZNF804A). A novel gene with an unknown function (ENSG00000286190) was also discovered. CONCLUSIONS:This study enhances the understanding of hPD-MSCs, demonstrating their unique characteristics and potential applications in therapeutic strategies. The identification of specific gene expression profiles differentiates hPD-MSCs from other mesenchymal cells and opens new avenues for research into their role in pancreatic function and neural development.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease associated with ectopic expression of the DUX4 gene in skeletal muscle. Muscle degeneration in FSHD is accompanied by muscle tissue replacement with fat and connective tissue. Expression of DUX4 in myoblasts stimulates mesenchymal stem cells (MSC) migration via the CXCR4-CXCL12 axis. MSCs participate in adipose and connective tissue formation and can contribute to fibrosis. Here we studied the interaction between myoblasts and MSCs and the consequences of this interaction in the FSHD context. We used cell motility assays and coculture of MSCs with myoblasts to study their mutual effects on cell migration, differentiation, proliferation, and extracellular matrix formation. The growth medium conditioned by FSHD myoblasts stimulated MSCs migration 1.6-fold (p < 0.04) compared to nonconditioned medium. Blocking the CXCL12-CXCR4 axis with the CXCR4 inhibitor (AMD3100) or neutralizing antibodies to CXCL12 abolished this effect. FSHD myoblasts stimulated MSC proliferation 1.5-2 times (p < 0.05) compared to control myoblasts, while the presence of MSCs impaired myoblast differentiation. Under inflammatory conditions, medium conditioned by FSHD myoblasts stimulated collagen secretion by MSCs 2.2-fold as compared to the nonconditioned medium, p < 0.03. FSHD myoblasts attract MSCs via the CXCL12-CXCR4 axis, stimulate MSC proliferation and collagen secretion by MSCs. Interaction between MSCs and FSHD myoblasts accounts for several important aspects of FSHD pathophysiology. The CXCL12-CXCR4 axis may serve as a potential target to improve the state of the diseased muscles.

The work is based on obtaining a living skin equivalent based on a three-dimensional scaffold consisting of type I collagen and other components of the extracellular matrix. The composition of the equivalent includes dermal and epidermal cells, it significantly accelerates and normalizes the healing process of chronic wounds. The living equivalent of the skin is a start for the further development of tissue-engineered constructs-analogs of skin and equivalents of other human tissues: at present, in particular, work is underway to study the stromal fractions of the dermis to control epithelial-mesenchymal interactions in vitro.

This study was focused on the problem of regulation of the proinflammatory activity of mast cells through a specific class of protease-activated receptors, PAR1. The study demonstrated for the first time the regulation of the activity of RBL-2H3 cells, mast cell analogues, by a new PAR1 agonist, peptide NPND-KYEPF-amide. It has been shown that the PAR1 agonist peptide, like activated protein C (APC), exhibited anti-inflammatory and cytoprotective effects on RBL-2H3 cells under activation by pro-inflammatory factors. Incubation of mast cells with lipopolysaccharide (LPS) caused a transient increase in the concentration of intracellular free calcium, increased the level of histamine secretion by the cells, and reduced their proliferation activity. Pre-incubation of the cells with both the peptide and APC prevented the effect of endotoxin on the RBL-2H3 cells. Application of thrombin and calcium ionophore led to actin reorganization, which might indicate cell activation and initiation of secretion. Pretreatment of the cells with both the peptide and APC in the presence of activators led to the ordering of actin in the submembrane region of the cells, which was typical for the control group. Thus, the newly discovered anti-inflammatory and protective properties of the PAR1 agonist peptide open the possibility of searching for new approaches to the treatment of inflammatory processes based on drugs of a peptide nature through modulating the activity of the PAR family receptors.

Background. The search for protein (these include c-fos, ERK1/2, MAP2, NOTCH1) expression that provide neuroplasticity mechanisms of the cerebral cortex after ischemic stroke (IS) patterns is an urgent task. Aims to reveal c-fos, ERK1/2, MAP2, NOTCH1 proteins expression patterns in human cerebral cortex neurons after IS. Materials and methods. We studied 9 left middle cerebral artery (LMCA) IS patients cerebral cortex samples from 3 zones: 1 the zone adjacent to the necrotic tissue focus; 2 zone remote from the previous one by 47 cm; 3 zone of the contralateral hemisphere, symmetric to the IS focus. Control samples were obtained from 3 accident died people. Identification of targeted proteins NSE, c-fos, ERK1/2, MAP2, NOTCH1 was performed by indirect immunoperoxidase immunohistochemical method. Results. Moving away from the ischemic focus, there is an increase in the density of neurons and a decrease in the damaged neurons proportion, the largest share of c-fos protein positive neurons in zone 2, NOTCH1 positive neurons in zone 1, smaller fractions of ERK1/2 and MAP2 positive neurons compared to the control only in samples of zone 1. Conclusions. With the IS development, the contralateral hemisphere is intact tissue increased activation zone, while the zones 1 and 2 have pathological activation signs. In zone 1 of the range, the adaptive response of the tissue decreases, and in zone 2 it expands. Therefore, a key target for therapeutic intervention is zone 2.

Обоснование. Поиск новых направлений патогенетически обоснованной терапии и реабилитации пациентов после ишемического инсульта является актуальной задачей. Для ее решения необходимы новые знания о закономерностях экспрессии после ишемического инсульта в нейронах коры головного мозга белков, обеспечивающих механизмы нейропластичности. К ним относят с-fos, ERK1/2, MAP2, NOTCH1. Цель исследования — выявить закономерности экспрессии белков с-fos, ERK1/2, MAP2, NOTCH1 в нейронах коры головного мозга человека после ишемического инсульта. Методы. Анализировали парафиновые срезы образцов коры головного мозга 9 пациентов, умерших в срок от 2 до 6 сут после развития ишемического инсульта в бассейне левой средней мозговой артерии (ЛСМА) из трех зон: 1 — зоны, прилежащей непосредственно к очагу некротической ткани; 2 — зоны, отдаленной от предыдущей на 4–7 см; 3 — зоны контралатерального полушария, симметричной очагу ишемического инсульта. Контрольные образцы получены от погибших в результате несчастного случая (3 человека). Оценку экспрессии белков NSE, с-fos, ERK1/2, MAP2, NOTCH1 нейронами проводили непрямым иммунопероксидазным иммуногистохимическим методом. Результаты. Выявлены увеличение плотности и уменьшение доли поврежденных нейронов при удалении от ишемического очага, наибольшая доля с-fos протеин-позитивных нейронов в зоне 2, NOTCH1- позитивных нейронов — в зоне 1, меньшие доли ERK1/2- и MAP2-позитивных нейронов по сравнению с контрольными только в образцах зоны 1. Заключение. При развитии ишемического инсульта контралатеральное очагу полушарие является зоной повышенной активации интактной ткани, тогда как участки коры, прилежащие непосредственно к очагу и отдаленные от него, имеют признаки патологической активации. При этом для зоны 1 характерно снижение диапазона адаптационного ответа ткани, а для зоны 2 — его расширение. Поэтому ключевой мишенью для терапевтического воздействия является зона 2.

Protease-activated receptors (PARs) are involved not only in hemostasis but also in the development of ischemic brain injury. In the present work, we examined in vivo effects of a new peptide (AP9) composing Asn47-Phen55 of PAR1 "tethered ligand" generated by activated protein C. We chose a mouse model of photothrombosis (PT)-induced ischemia to assess AP9 effects in vivo. To reveal the molecular mechanism of AP9 action, mice lacking β-arrestin-2 were used. AP9 was injected intravenously once 10 min before PT at doses of 0.2, 2, or 20 mg/kg, or twice, that is, 10 min before and 1 h after PT at a dose of 20 mg/kg. Lesion volume was measured by magnetic resonance imaging and staining of brain sections with tetrazolium salt. Neurologic deficit was estimated using the cylinder and the grid-walk tests. Blood-brain barrier (BBB) disruption was assessed by Evans blue dye extraction. Eosin-hematoxylin staining and immunohistochemical staining were applied to evaluate the number of undamaged neurons and activated glial cells in the penumbra. A single administration of AP9 (20 mg/kg), as well as its two injections (20 mg/kg), decreased brain lesion volume. A double administration of AP9 also reduced BBB disruption and neurological deficit in mice. We did not observe the protective effect of AP9 in mice lacking β-arrestin-2 after PT. Thus, we demonstrated for the first time protective properties of a PAR1 agonist peptide, AP9, in vivo. β-Arrestin-2 was required for the protective action of AP9 in PT-induced brain ischemia.

Thrombin is a multifunctional serine protease that attracts the attention of many researchers in particular in connection with a wide range of its effects in the nervous tissue. It is known that the main thrombin receptor is a protease-activated type 1 receptor, the functional activity of which is associated with adapter protein β-arrestin-2. Here we assessed potential involvement of β-arrestin-2 in the thrombin toxicity both in vitro and in vivo using gene knockout mice. It was found that thrombin induced dose-dependent cell death of cultured β-arrestin-2–/– astrocytes 48 h after the exposure. In contrast, thrombin did not exert any effects on the survival of astrocytes from wild-type animals. The in vivo study showed that β-arrestin-2 gene knockout did not alter the severity of the focal photoinduced cerebral ischemia aftereffects, which could involve additional cell types and molecular mechanisms in nervous tissue damage. Our findings demonstrate for the first time the necessity of β-arrestin-2 for the survival of mouse astrocytes under the toxic action of thrombin. At the CNS level, however, further studies are required to determine the key targets of this protease in each cell types of the nervous tissue and to clarify the role of β-arrestin signaling in neuroprotection.

The fundamental question about the functionality of in vitro derived human primordial germ cell-like cells remains unanswered, despite ongoing research in this area. Attempts have been made to imitate the differentiation of human primordial germ cells (hPGCs) and meiocytes in vitro from human pluripotent stem cells (hPSCs). A defined system for developing human haploid cells in vitro is the challenge that scientists face to advance the knowledge of human germ cell development. To develop human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) that are capable of giving rise to haploid cells, we applied a sequential induction protocol via the early mesodermal push of female human embryonic and induced pluripotent stem cells. BMP4-induced early mesoderm-like cells showed significant alterations in their expression profiles toward early (PRDM1 and NANOS3) and late (VASA and DAZL) germ cell markers. Furthermore, using retinoic acid (RA), we induced hPGCLCs in embryoid bodies and identified positive staining for the meiotic initiation marker STRA8. Efforts to find the cells exhibiting progression to meiosis were unsuccessful. The validation by the expression of SCP3 did not correspond to the natural pattern. Regarding the 20-day meiotic induction, the derived hPGCLCs containing two X-chromosomes were unable to complete the meiotic division. We observed the expression of the oocyte marker PIWIL1 and PIWIL4. RNAseq analysis and cluster dendrogram showed a similar clustering of hPGCLC groups and meiotic like cell groups as compared to previously published data. This reproducible in vitro model for deriving hPGCLCs provides opportunities for studying the molecular mechanisms involved in the specification of hPGCs. Moreover, our results will support a further elucidation of gametogenesis and meiosis of female hPGCs.

F1,6BP also activates Ras and its downstream targets ERK and MEK [5]. In turn, small GTPases of the Ras family directly bind to the catalytic subunit p110a of PI3K, activating PI3K-AKT signaling [6]. As a result, a vicious cycle is created between F1,6P and major oncogenic drivers (Ras and PI3K-AKT) acting with HIF-1a to promote the Warburg effect [7]. Moreover, PFKFB and dimeric PKM2 translocate into nucleus, where F2,6BP represses p27Kip1, a strong inhibitor of cell cycle progression [2], while dimeric PKM2 promotes c-Myc expression, favoring the Warburg effect and cell cycle progression [8].

DUX4, a double homeobox transcription factor, has been mostly studied in facioscapulohumeral dystrophy (FSHD), a pathology linked to a deletion of subtelomeric repeats on chromosome 4q. More recently, however, the gene has been associated with various sarcomas and haematological malignancies. Drugs developed for FSHD could be tested on cancer cells to develop efficient treatment strategies for both pathologies.

Background: Photothrombosis is a minimally invasive method for induction of cortical ischemia. However, different ways of applying some methods to assess photothrombosis-induced damage need to be developed. New Methods: We applied the tongue protrusion test and H&E staining of brain sections to detect ischemic damage after photothrombosis. Evaluation of the local status of the BBB using Evans blue dye was proposed. We also assessed the sensitivity of the grid-walk test. Moreover, we examined the interchangeability of MRI and TTC staining to measure lesion volume. Results: We evaluated ischemic outcomes at 24 h after photothrombosis in mice. The tongue protrusion test did not reveal impairments of the neurological status whereas the grid-walk test showed the high sensitivity. Using histological techniques, we determined the reduction in the number of neurons with normal morphology in the penumbra. 3D reconstruction of the brain, which reflected Evans blue dye distribution in the nervous tissue, revealed BBB disruption in areas remote from the ischemic core. We also showed the strong correlation between damage volumes assessed by MRI and TTC staining. Comparison with Existing Methods: The present work demonstrates the efficacy of the classical histological approach and TTC staining that are more affordable than MRI and immunohistochemical methods. Detection of 3D distribution of Evans blue dye in the brain in contrast to its total extraction reveals BBB damage in details. Conclusions: We proposed the simple methods for describing the severity of brain ischemia at the cellular and whole organism levels without significant labor and financial expenditures.

This paper presents a literature review considering the role and mechanism of apoptosis in the pathogenesis of ischemic stroke (IS). The authors introduce a new concept: the functional request of the patient as a set of external (the nature and intensity of rehabilitation measures, characteristics of everyday life, diet, etc.) and internal (genetic factors, internal picture of the disease, availability of rental and other psychological facilities and etc.) attributes. This concept allows a new angle in understanding the pathogenesis of IS and creates fundamental and clinical potential for more successful approaches to therapy and rehabilitation after IS.