The recent rapid advances in technological pipelines are urging faster and riskier translational moves from research to practice in regenerative medicine [...]

Cellular homeostasis is maintained by three important physiological processes: proliferation, characterized by an increase in cell number; differentiation, characterized by either an increase or a decrease in cell number coincident with changes in cellular genotype and function; and cell death, which promotes the elimination of cells. Two of these alterations, proliferation and differentiation, are tightly linked to regeneration. Currently, >10 modes of programmed cell death are recognized. Among them are apoptosis, necroptosis, pyroptosis, ferroptosis, etc. Almost all of them are linked to tissue regeneration. Dead cells have a vital effect on the living cells around them. They release a wide range of stimuli (low-molecular-weight molecules, proteins, vesicles, etc.) that signal neighbors to proliferate and differentiate. Additionally, dying cells free the space and thus change the physical characteristics of tissue, arrange the elimination of dysfunctional cells, and promote regeneration. However, programmed cell death can inhibit cell proliferation and vice versa. Thus, stem cells are resistant to the induction of death and maintain their potential to affect tissue homeostasis and recovery after injury. Disturbances in the balance between cell death and regeneration lead to various pathologies. Given the importance of the interaction between cell death and regeneration, advances in this area could lay the foundation for the successful development of a rapidly growing, groundbreaking field of medicine called regenerative biomedicine, the goal of which is to support cell renewal and restore damaged organs and tissues. Here, we review these multilevel relationships to explore how cell death and regeneration balance each other.

The COVID-19 pandemic had an unprecedented impact on all aspects of human activity worldwide, frequently resulting in post-acute sequelae and affecting multiple organ systems. The underlying mechanisms driving both acute and post-acute manifestations of COVID-19 are still poorly understood, warranting further investigation for new targets. The study represents the first attempt to explore the role of T-cadherin in COVID-19 pathogenesis as well as its implications in pulmonary fibrosis and endothelial dysfunction. First, we revealed a significant decrease in T-cadherin expression in post-mortem lung samples from COVID-19 patients. This downregulated T-cadherin expression correlated with the elevated levels of VE-cadherin and reduced levels of β-catenin, suggesting a disruption in endothelial cell-cell contact integrity and function. Second, the reciprocal relation of T-cadherin and VE-cadherin expression was further confirmed using cultured human endothelial Ea.hy926 cells. T-cadherin overexpression caused a decrease in VE-cadherin mRNA expression in cultured endothelial cells providing additional evidence in favor of their interplay. Third, employing Cdh13 -/- mice, we unveiled the protective role of T-cadherin deficiency against bleomycin-induced lung fibrosis. Fourth, we demonstrated the mice lacking T-cadherin to have downregulated reactive oxygen species production and Nox2 mRNA expression in an angiotensin II-mediated endothelial dysfunction model. Our findings provide rationale for further studies into T-cadherin-mediated mechanisms in these processes.

Primary cell cultures are one of the main research objects and promising tools in regenerative biomedicine. However, their application is significantly limited by the short lifespan and rapid aging. Existing approaches to prolong the “youth” of cultured cells inevitably alter their properties, which raises questions about their applicability in regenerative biomedicine. Our review examines the main mechanisms of aging of cultured cells, existing methods used to overcome it, and safety issues associated with the produced cultures. We analyzed the data on cell immortalization and its connection with tumor transformation. Among the methods for prolonging the proliferative activity of cells are spontaneous immortalization and immortalization induced by overexpression of the catalytic subunit of telomerase (TERT), viral oncogenes (T antigens of the polyomavirus SV40, proteins E6/E7 of human papillomavirus type 16, and adenoviral proteins E1A and E1B of adenoviruses), and cellular transcription factors, such as proto-oncogenes (c-MYC, BMI1). The accumulated data suggest that increasing the expression of the gene encoding TERT is one of the relatively safe approaches to prolonging the proliferative activity of a cell line, which does not lead to the tumor transformation of cell line. Based on the analyzed data, an attempt was made to identify the “boundary” between the permissible prolongation of cell culture life and its tumor transformation.

Nerve tissue damage is an unsolved problem in modern neurology and neurosurgery, which prompts the need to search for approaches to stimulate neuroprotection and regeneration of neural tissue. Earlier we have shown that the secretome of human mesenchymal stromal cells (MSCs) stimulates rat survival, reduces the severity of neurological deficits, and decreases the volume of brain damage in a hemorrhagic stroke model. A significant disadvantage of using the MSC secretome is the need to concentrate it (at least 5–10 fold) to achieve appreciable pharmacological activity. This increases the cost of obtaining clinically applicable amounts of secretome and slows down the clinical translation of this technology. Here, we created a number of genetically modified human MSC cultures, including immortalized MSCs and those with hyperexpression of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA) and with suppressed expression of Von Hippel–Lindau tumor suppressor (VHL), and we evaluated the pharmacological activity of their secretomes in a model of intracerebral hemorrhage (ICH) in rats. The secretome of MSCs immortalized by hyperexpression of the catalytic subunit of human telomerase (hTERT) revealed neuroprotective activity indistinguishable from that of primary MSC cultures, yet it still required 10-fold concentration to achieve neuroprotective efficacy. The secretome of MSC culture with combined hyperexpression of BDNF and uPA and suppressed expression of Von Hippel–Lindau tumor suppressor even without additional concentration reduced the severity of neurological disorders and decreased brain lesion volume in the ICH model. The secretomes of MSCs with separate overexpression of BDNF and uPA or suppression of VHL had no such effect or, on the contrary, revealed a toxic effect in the ICH model. Presumably, this may be due to an imbalance in the representation of individual growth factors in the secretome of genetically modified MSCs, which individually may lead to undesirable effects in damaged nervous tissue, such as increased permeability of the blood–brain barrier (under the influence of pro-angiogenic factors) or neural cell apoptosis (due to an excess of neurotrophic factors). The obtained data show that genetic modification of MSC cultures can enhance or alter the therapeutic activity of their secretomes, which can be used in the creation of promising sources of biopharmaceutical substances.

IntroductionImpaired function of brain morphogenic genes is considered one of the predisposing factors for the manifestation of psychiatric and cognitive disorders, such as paranoid schizophrenia (SCZ) and major depressive disorder (MDD). Identification of such genes (genes of neurotrophic factors and guidance molecules among them) and their deleterious genetic variants serves as a key to diagnosis, prevention, and possibly treatment of such disorders. In this study, we have examined the prevalence of genomic variants in brain morphogenic genes in individuals with SCZ and MDD within a Russian population.MethodsWe have performed whole-exome sequencing of 21 DNA samples: 11 from individuals with SCZ and 10 with MDD, followed by ARMS (Amplification-Refractory Mutation System) based screening of detected single nucleotide variants (SNVs) in larger groups: 102 for individuals with SCZ, 79 for those with MDD and 103 for healthy donors.ResultsWhole-exome sequencing has revealed 226 missense mutations in 79 genes (out of 140 studied), some of which occur in patients with psychiatric disorders significantly more frequently than in healthy donors. We have identified previously undescribed genomic variants in brain morphogenic genes: CDH2 (rs1944294-T and rs17445840-T), DCHS2 (rs11935573-G and rs12500437-G/T) and CDH23 (rs1227051-G/A), significantly associated with the incidence of SCZ and MDD in the Russian population. For some SNVs (rs6265-T, rs1944294-T, rs11935573-G, rs4760-G) sex-biased differences in their prevalence between SCZ/MDD patients and healthy donors was detected.DiscussionHowever, the functional significance of the SNVs identified has still to be confirmed in cellular and animal models. Once it is fulfilled, these SNVs have the potential to complement the diagnostic toolbox for assessing susceptibility to mental disorders. The data obtained indirectly confirm the importance of adequate brain structure formation for its correct functioning and preservation of mental health.

scRNA-seq is revolutionizing biomedical research by revealing tissue architecture, cellular composition, and functional interactions. However, accurate cell type annotation remains a challenge, particularly for rare cell types, with existing automated methods often falling short. Multimodal data, combining mRNA expression and protein markers, improves deep cellular analysis and make functional characterization of complex tissues more accurate. However, it is costly and complex to obtain. We present scParadise, a cutting-edge Python framework featuring three tools: scAdam for multi-level cell annotation, scEve for surface protein prediction, and scNoah for benchmarking. scAdam surpasses current methods in annotating rare cell types and ensures consistent results across diverse datasets, while scEve enhances clustering and cell type separation. With scNoah′s advanced metrics, scParadise offers a powerful, fast, and reliable solution for single-cell analysis, setting a new standard in scRNA-seq data processing. ### Competing Interest Statement The authors have declared no competing interest.

Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC’s physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17–26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8–10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate.

Mental illness and cognitive disorders represent a serious problem for the modern society. Many studies indicate that mental disorders are polygenic and that impaired brain development may lay the ground for their manifestation. Neural tissue development is a complex and multistage process that involves a large number of distant and contact molecules. In this review, we have considered the key steps of brain morphogenesis, and the major molecule families involved in these process. The review provides many indications of the important contribution of the brain development process and correct functioning of certain genes to human mental health. To our knowledge, this comprehensive review is one of the first in this field. We suppose that this review may be useful to novice researchers and clinicians wishing to navigate the field.

As regenerative medicine continues to advance as a growing field in modern biology and the healthcare industry, it attracts enormous interest from the general public and scientists [...]

Pulmonary fibrosis, a debilitating lung disorder characterised by excessive fibrous tissue accumulation in lung parenchyma, compromises respiratory function leading to a life-threatening respiratory failure. While its origins are multifaceted and poorly understood, the urokinase system, including urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plays a significant role in regulating fibrotic response, extracellular matrix remodelling, and tissue repair. Mesenchymal stem/stromal cells (MSCs) hold promise in regenerative medicine for treating pulmonary fibrosis. Our study aimed to investigate the potential of MSCs to inhibit pulmonary fibrosis as well as the contribution of uPAR expression to this effect. We found that intravenous MSC administration significantly reduced lung fibrosis in the bleomycin-induced pulmonary fibrosis model in mice as revealed by MRI and histological evaluations. Notably, administering the MSCs isolated from adipose tissue of uPAR knockout mice (Plaur-/- MSCs) attenuated lung fibrosis to a lesser extent as compared to WT MSCs. Collagen deposition, a hallmark of fibrosis, was markedly reduced in lungs treated with WT MSCs versus Plaur-/- MSCs. Along with that, endogenous uPA levels were affected differently; after Plaur-/- MSCs were administered, the uPA content was specifically decreased within the blood vessels. Our findings support the potential of MSC treatment in attenuating pulmonary fibrosis. We provide evidence that the observed anti-fibrotic effect depends on uPAR expression in MSCs, suggesting that uPAR might counteract the uPA accumulation in lungs.

Recent research indicates that some types of mental illnesses (schizophrenia, autism, depressive disorders) may be associated with impaired functioning of a number of genes, including those involved in brain morphogenesis. To assess the possible contribution of brain morphogenesis genes in the formation of predisposition to depressive disorders in Russian population, we performed whole-exome sequencing of genomic DNA of such patients. We identified 166 missense genomic variants in 66 genes (out of 140 studied) involved in the formation of brain tissue. The prevalence of some of them was estimated by allele-specific PCR. For the first time, a significantly higher frequency of occurrence of genomic variants rs17445840‑T (CDH2 gene), rs12923655‑C (CDH3 gene), rs1227051‑G/A (CDH23 gene), and rs12500437‑G/T (DCHS2 gene) was shown in a group of patients suffering from endogenous depressive disorder, and an association of some of the identified genomic variants with gender was established. The data obtained confirm the previously stated assumption that genes of brain tissue morphogenesis may be associated with a predisposition to the development of mental and cognitive disorders. The functional significance of the identified genetic variants remains to be established. The identification of pathogenic genomic variants with the confirmation of their functional significance allows better understanding of the pathogenesis of mental disorders and opens prospects for the development of approaches to objective diagnosis of such diseases, their early prevention, and pathogenetic therapy.

IntroductionT-cadherin, a non-canonical member of the cadherin superfamily, was initially identified for its involvement in homophilic recognition within the nervous and vascular systems. Apart from its adhesive function, T-cadherin acts as a receptor for two ligands: LDL, contributing to atherogenic processes, and HMW adiponectin, a hormone with well-known cardiovascular protective properties. However, the precise role of T-cadherin in adipose tissue remains elusive. Previously, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene, which encodes the T-cadherin protein, and characterized their phenotype.MethodsUsing wild-type (WT) and T-cadherin-deficient mice (Cdh13ΔExon3), we isolated and cultured mesenchymal stem cells to explore the role of T-cadherin in adipogenic differentiation. The experimental approaches employed include culturing cells under standard or adipogenic conditions, performing Oil Red O and Nile Red staining followed by quantitative analysis, conducting rescue experiments to reintroduce T-cadherin using lentiviral constructs in T-cadherin-deficient cells combined with automated adipocyte differentiation quantification via a neural network. Additionally, Western blotting, ELISA assays, and statistical analysis were utilized to verify the results.ResultsIn this study, we demonstrate for the first time that T-cadherin influences the adipogenic differentiation of MSCs. The presence of T-cadherin dictates distinct morphological characteristics in MSCs. Lack of T-cadherin leads to spontaneous differentiation into adipocytes with the formation of large lipid droplets. T-cadherin-deficient cells (T−/− MSCs) exhibit an enhanced adipogenic potential upon induction with differentiating factors. Western Blot, ELISA assays, and rescue experiments collectively corroborate the conclusion that T−/− MSCs are predisposed toward adipogenic differentiation. We carried out an original comparative analysis to explore the effects of T-cadherin ligands on lipid droplet accumulation. LDL stimulate adipogenic differentiation, while T-cadherin expression mitigates the impact of LDL on lipid droplet accumulation. We also examined the effects of both low molecular weight (LMW) and high molecular weight (HMW) adiponectin on lipid droplet accumulation relative to T-cadherin. LMW adiponectin suppressed lipid droplet accumulation independently of T-cadherin, while the absence of T-cadherin enhanced susceptibility to the suppressive effects of HMW adiponectin on adipogenesis.DiscussionThese findings shed light on the role of T-cadherin in adipogenic differentiation and suggest an interplay with other receptors, such as LDLR and AdipoRs, wherein downstream signaling may be modulated through lateral interactions with T-cadherin.

Ischemic and hemorrhagic strokes, traumatic brain injury, bacterial and viral encephalitis, toxic and metabolic encephalopathies are very different pathologies. But, they have much more in common than it might seem at first glance. In this review, the authors propose to consider these brain pathologies from the point of view of the unity of their pathogenetic mechanisms and approaches to therapy. Particular attention is paid to promising therapeutic approaches, such as therapy using cells and their secretion products: an analysis of the accumulated experimental data, the advantages and limitations of these approaches in the treatment of brain damage was carried out. The review may be of interest both to specialists in the field of neurology, neurosurgery and neurorehabilitation, and to readers who want to learn more about the progress of regenerative biomedicine in the treatment of brain pathologies.

Hypertension is one of the major life-threatening complications of obesity. Recently adipose multipotent mesenchymal stromal cells (MSCs) were implicated to the pathogenesis of obesity-associated hypertension. These cells amplify noradrenaline-induced vascular cell contraction via cAMP-mediated signaling pathway. In this study we tested the ability of several cAMP-mediated hormones to affect the adrenergic sensitivity of MSCs and their associated contractility. Despite that adipose MSCs express a plethora of receptors capable of cAMP signaling activation, only 5-HT was able to elevate α1A-adrenoceptor-induced Ca2+ signaling in MSCs. Furthermore, 5-HT markedly enhanced noradrenaline-induced MSCs contractility. Using HTR isoform-specific antagonists followed by CRISPRi-mediated knockdown, we identified that the observed 5-HT effect on MSCs was mediated by the HTR6 isoform. This receptor was previously associated exclusively with 5-HT central nervous system activity. Discovered effect of HTR6 on MSCs contractility points to it as a potential therapeutic target for the prevention and treatment of obesity-associated hypertension.

Intracerebral hemorrhage is an unmet medical need that often leads to the disability and death of a patient. The lack of effective treatments for intracerebral hemorrhage makes it necessary to look for them. Previously, in our proof-of-concept study (Karagyaur M et al. Pharmaceutics, 2021), we have shown that the secretome of multipotent mesenchymal stromal cells (MSC) provides neuroprotection of the brain in a model of intracerebral hemorrhage in rats. Here, we have conducted a systematic study of the therapeutic potential of the MSC secretome in the model of hemorrhagic stroke and provided answers to the questions that need to be addressed in order to translate the secretome-based drug into clinical practice: routes and multiplicity of administration, optimal dose and door-to-treatment time. We have found that MSC secretome reveals prominent neuroprotective activity when administered intranasally or intravenously within 1–3 h after hemorrhage modeling, even in aged rats, and its multiple injections (even within 48 h) are able to reduce the delayed negative effects of hemorrhagic stroke. To our knowledge, this study provides the first systematic investigation of the therapeutic activity of a biomedical MSC-based cell-free drug in intracerebral hemorrhage and is an integral part of its preclinical studies.

To date, pulmonary fibrosis remains an unmet medical need. In this study, we evaluated the potency of mesenchymal stromal cell (MSC) secretome components to prevent pulmonary fibrosis development and facilitate fibrosis resolution. Surprisingly, the intratracheal application of extracellular vesicles (MSC-EVs) or the vesicle-depleted secretome fraction (MSC-SF) was not able to prevent lung fibrosis when applied immediately after the injury caused by bleomycin instillation in mice. However, MSC-EV administration induced the resolution of established pulmonary fibrosis, whereas the vesicle-depleted fraction did not. The application of MSC-EVs caused a decrease in the numbers of myofibroblasts and FAPa + progenitors without affecting their apoptosis. Such a decrease likely occurred due to their dedifferentiation caused by microRNA (miR) transfer by MSC-EVs. Using a murine model of bleomycin-induced pulmonary fibrosis, we confirmed the contribution of specific miRs (miR-29c and miR-129) to the antifibrotic effect of MSC-EVs. Our study provides novel insights into possible antifibrotic therapy based on the use of the vesicle-enriched fraction of the MSC secretome.

Hypertension is a major risk factor for cardiovascular diseases, such as strokes and myocardial infarctions. Nearly 70% of hypertension onsets in adults can be attributed to obesity, primarily due to sympathetic overdrive and the dysregulated renin-angiotensin system. Sympathetic overdrive increases vasoconstriction via α1-adrenoceptor activation on vascular cells. Despite the fact that a sympathetic outflow increases in individuals with obesity, as a rule, there is a cohort of patients with obesity who do not develop hypertension. In this study, we investigated how adrenoceptors’ expression and functioning in adipose tissue are affected by obesity-driven hypertension. Here, we demonstrated that α1A is a predominant isoform of α1-adrenoceptors expressed in the adipose tissue of patients with obesity, specifically by multipotent mesenchymal stromal cells (MSCs). These cells respond to prolonged exposure to noradrenaline in the model of sympathetic overdrive through the elevation of α1A-adrenoceptor expression and signaling. The extent of MSCs’ response to noradrenaline correlates with a patient’s arterial hypertension. scRNAseq analysis revealed that in the model of sympathetic overdrive, the subpopulation of MSCs with contractile phenotype expanded significantly. Elevated α1A-adrenoceptor expression is triggered specifically by beta3-adrenoceptors. These data define a novel pathophysiological mechanism of obesity-driven hypertension by which noradrenaline targets MSCs to increase microvessel constrictor responsivity.

T-cadherin is a regulator of blood vessel remodeling and angiogenesis, involved in adiponectin-mediated protective effects in the cardiovascular system and in skeletal muscles. GWAS study has previously demonstrated a SNP in the Cdh13 gene to be associated with hypertension. However, the role of T-cadherin in regulating blood pressure has not been experimentally elucidated. Herein, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene and described their phenotype. Cdh13∆Exon3 mice exhibited normal gross morphology, life expectancy, and breeding capacity. Meanwhile, their body weight was considerably lower than of WT mice. When running on a treadmill, the time spent running and the distance covered by Cdh13∆Exon3 mice was similar to that of WT. The resting blood pressure in Cdh13∆Exon3 mice was slightly higher than in WT, however, upon intensive physical training their systolic blood pressure was significantly elevated. While adiponectin content in the myocardium of Cdh13∆Exon3 and WT mice was within the same range, adiponectin plasma level was 4.37-fold higher in Cdh13∆Exon3 mice. Moreover, intensive physical training augmented the AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice as compared to WT. Our data highlight a critically important role of T-cadherin in regulation of blood pressure and stamina in mice, and may shed light on the pathogenesis of hypertension.