The study of molecular mechanisms of regeneration requires convenient models for in vitro and in vivo studies. In vitro cell cultures can fulfill such a function. However, their rapid aging and loss of initial specific properties in culture is a significant limitation of their use. Telomerase expression can help to overcome these limitations: it can prolong proliferative activity and stabilize the initial properties of a primary cell culture. Here, we created and studied the properties of human adipose tissue multipotent mesenchymal stromal cell (MSC) cultures that overexpress the catalytic protein subunit of human telomerase ( hTERT). We found that these MSC cultures were able to proliferate up to 38–63 PD, kept sensitivity to noradrenaline, serotonin, glutamate, γ-aminobutyric acid, parathyroid hormone, angiotensin II and histamine until at least 26 PD, retained MSC-specific immunophenotype until at least 36 PD, and preserved the ability to adipogenic, osteogenic and chondrogenic differentiation until at least 39 PD. Moreover, overexpression of hTERT in MSC cultures stabilized the qualitative and quantitative composition of their secretome at long-term passaging (at least up to 30 PD). The obtained results allow us to consider telomerase hyperexpression as a promising approach to obtaining MSC cultures with prolonged proliferative activity, which can be used as a stable and convenient object for fundamental and applied studies in the field of regenerative medicine.

INTRODUCTION:Adenylyl cyclase (AC) is an important enzyme that mediates stem cells' response to hormonal stimuli. Despite the fact that currently there are ten described isoforms of AC little is known about their individual significance in different aspects of stem cell functions control. This study focuses on elucidating the significance of AC isoforms in the control of multipotent mesenchymal stromal cells (MSCs) differentiation. We investigated changes in adenylyl cyclase expression (ADCY) during adipogenic and osteogenic differentiation of MSCs. Our results suggest that the expression only of ADCY1 elevates during MSCs' both osteogenic and adipogenic differentiations. RESULTS:We used the CRISPR/Cas9 D10A system to knock out the ADCY1 gene in MSCs. The ADCY1 knockout MSC cell line demonstrated reduced ADCY1 expression in the undifferentiated state compared to control cells. Moreover, ADCY1 expression in the ADCY1-KO cell line didn't increase during the differentiation process, which confirms the successful knockout. The ADCY1-KO cell line demonstrated an impaired differentiation potential in both adipogenic and osteogenic directions. However, the ability for adipogenic differentiation was restored by a direct PPARγ agonist addition. CONCLUSIONS:Our findings indicate that ADCY1 expression upregulation is critical for MSC differentiation into osteogenic and adipogenic lineages.

The emergence of single-cell multi-omics technologies has enabled researchers to identify rare cell populations and investigate gene regulation with unmatched resolution, advancing cancer research. However, each technique comes with its own distinct advantages and drawbacks. In this study, we compared two high-throughput droplet-based single-cell RNA sequencing (scRNA-seq) technologies—10X Chromium 3’ scRNA-seq and SeekGene scFAST-seq—using two paired samples derived from adrenocortical tumor. scFAST-seq exhibited a higher ratio of long non-coding RNAs, along with an increased number of detected genes and transcripts, while ribosomal RNA was underrepresented. Although the overlap in top cell type markers was relatively low, the relative abundances of cell populations were alike in both datasets. Also gene markers showed to be specific for scFAST-seq and 3’ scRNA-seq displayed minimal general variability in gene expression. We noted changes in RNA dynamics across the datasets, identified through RNA velocity analysis and fewer copy number variations (CNV) detected by CNV analysis. Furthermore, we found variations in regulon activity in 3’ scRNA-seq and scFAST-seq datasets. In conclusion, our research offers essential insights for selecting the most suitable scRNA-seq approach.

Traditional machine learning (TML) algorithms remain indispensable tools for the analysis of biomedical images, offering significant advantages in multimodal data integration, interpretability, computational efficiency, and robustness on smaller datasets. This review provides a comprehensive examination of TML applications across a broad spectrum of biomedical imaging modalities, highlighting its core principles, practical implementation, and unique benefits in the era of deep learning (DL). We outline the fundamental concepts of machine learning and describe key biomedical imaging tasks successfully addressed by TML. We also highlight the most popular platforms, which empower clinicians and researchers to utilize TML. DL now dominates many areas of medical image analysis due to superior performance and end-to-end feature learning. Using the most prominent examples, we analyze how TML retains unique value for applications with multimodal data processing, limited data, interpretability requirements, or rapid prototyping needs. Supported by increasingly democratized tools and validated by robust clinical studies, TML remains a vital methodology for extracting quantitative and qualitative insights from biomedical image data, ensuring its continued relevance in both research and clinical practice.

In single-cell biology, the main limitation has shifted from data generation to converting sparse, heterogeneous single-cell RNA sequencing (scRNA-seq) datasets into accurate cell types, interpretable multi-omic states, and reproducible conclusions. To address these challenges, we present scParadise, which transforms scRNA-seq data to a new scientific knowledge. scParadise comprises three integrated tools: scAdam, a multi-level cell type annotation tool with unknown cell type identification; scEve, a cross-tissue modality imputation tool; and scNoah, a standardized benchmarking tool. Using scParadise, we corrected annotation mistakes in the Tabula Muris Senis atlas, showing that cells labeled as granulocytes are exclusively neutrophils and that presumably annotated macrophages actually represent a range of different cell types. Moreover, we identify three previously unknown natural killer T (NKT) cell subsets by imputing protein expression across tissues, which include CD56dim CD3+, CD56dim CD3+ CD4+, and CD56dim CD3+ CD8+ cell subsets in human visceral adipose tissue, which we verify by flow cytometry. These new subsets engage in obesity-related tumor necrosis factor-centric crosstalk with myeloid and adipose progenitors, thereby illuminating a new paradigm for immune-stromal interactions that contribute to chronic inflammation and impaired adipogenesis.

Replicative or stress-induced senescence disrupts the functioning of multipotent mesenchymal stromal cells (MSCs) required for tissue renewal and regeneration. Aged MSCs demonstrate reduced proliferation, impaired differentiation, and aberrant secretory activity, defined as “senescence-associated secretory phenotype” (SASP). SASP is characterized by elevated secretion of proinflammatory cytokines and specific extracellular vesicles (SASP-EVs), which affect the cellular microenvironment and promote tissue dysfunction. However, molecular mechanisms responsible for senescent phenotype propagation remain largely obscure. Earlier, we demonstrated suppression of adipogenic differentiation and insulin sensitivity of young MSCs by SASP-EVs. In this study, we elucidated potential mechanisms underlying SASP-EVs’ effects on MSCs. Bioinformatic analysis revealed that insulin signaling components are the most probable targets of SASP-EVs microRNA cargo. We demonstrated that SASP-EVs downregulated intracellular AGO1 levels, but surprisingly, PTEN levels were upregulated. Specifically, the increase in PTEN content was provided by its nuclear fraction. We have found that the intracellular PTEN distribution in young MSCs treated by SASP-EVs was similar to senescent MSCs. Furthermore, PTEN upregulation was accompanied by increased PTENP1 expression—a molecular sponge for PTEN-targeting microRNAs. Our findings indicate that nuclear PTEN could be a hallmark of senescent MSCs, and SASP-EVs propagate the senescent phenotype in young MSCs by promoting PTEN nuclear localization.

The secretome of mesenchymal stromal cells (MSCs) can efficiently stimulate regeneration and therefore is a tempting remedy for “cell-free cellular therapy”. However, the usage of primary MSC cultures as secretome producers for translation studies has obvious obstacles, including the rapid aging of MSC cultures, the need for a large number of verified donors, and donor-to-donor variability of secretome content. MSCs immortalization makes it possible to overcome those limitations and to obtain secretome-producing cultures with a prolonged lifetime. However, the efficacy and safety of such secretomes are critical issues that limit their usage as therapeutic agents. In this study, we tested in large detail how the immortalization of MSC cultures affects the content, biological activity and safety of their secretome. MSCs immortalization via the overexpression of human TERT gene does not significantly alter the qualitative and quantitative composition of their secretome or its activity according to the results of proteomic analysis, ELISA, qPCR and functional tests in vitro. Moreover, we have demonstrated that the secretome of immortalized MSCs does not contain detectable amounts of telomerase and does not possess any transforming activity. Altogether, our data suggest that immortalized MSC cultures may become a reliable source for obtaining standardized active secretome in large-scale quantities for clinical use.

Microscopy is an essential method in modern biology, and brightfield microscopy methods (phase contrast, differential interference contrast, etc.) are being widely used and actively developed since they don’t require sample fixation and staining. But they produce low contrast images, where cells have similar intensity to the background. In this work we developed and tested a set of deep learning object detection YOLO models that detect cell nuclei in phase contrast images, which allows cell count and tracking without staining. We created a large dataset consisting of more than 100,000 640×640 pixels images with more than 3 million nuclei of 4 different cell cultures (CHO, HEK293, iPSCs, and MSCs). Using images from various microscopes and cameras, as well as full-scale augmentations, we developed a set of highly generalized models that can detect nuclei in images across different cell types and imaging conditions, including different microscopes and contrast methods. Combined with sliced inference methods, these algorithms can be applied to images of any size, allowing studies of large quantities of cells. Moreover, we developed a training-free calibration and testing algorithm based on confidence threshold optimization. It allows for fine-tuning of models for specific cell types and/or imaging options and evaluating the accuracy of the calibrated model. This provides a highly controllable and reliable method for studying cell proliferation rate, single cell tracking and other scenarios. Additionally, we developed a NuclePhaser plugin for Napari (), which allows users to calibrate, test and apply our models in code-free manner. Given that the YOLO models are fast and can run at sufficient speeds even on CPUs, this makes our work highly accessible to a wide range of researchers. ### Competing Interest Statement The authors have declared no competing interest. Non-commercial Foundation for Support of Science and Education “INTELLECT”

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.

In recent years, there has been a significant expansion in the realm of processing microscopy images, thanks to the advent of machine learning techniques. These techniques offer diverse applications for image processing. Currently, numerous methods are used for processing microscopy images in the field of biology, ranging from conventional machine learning algorithms to sophisticated deep learning artificial neural networks with millions of parameters. However, a comprehensive grasp of the intricacies of these methods usually necessitates proficiency in programming and advanced mathematics. In our comprehensive review, we explore various widely used deep learning approaches tailored for the processing of microscopy images. Our emphasis is on algorithms that have gained popularity in the field of biology and have been adapted to cater to users lacking programming expertise. In essence, our target audience comprises biologists interested in exploring the potential of deep learning algorithms, even without programming skills. Throughout the review, we elucidate each algorithm's fundamental concepts and capabilities without delving into mathematical and programming complexities. Crucially, all the highlighted algorithms are accessible on open platforms without requiring code, and we provide detailed descriptions and links within our review. It's essential to recognize that addressing each specific problem demands an individualized approach. Consequently, our focus is not on comparing algorithms but on delineating the problems they are adept at solving. In practical scenarios, researchers typically select multiple algorithms suited to their tasks and experimentally determine the most effective one. It is worth noting that microscopy extends beyond the realm of biology; its applications span diverse fields such as geology and material science. Although our review predominantly centers on biomedical applications, the algorithms and principles outlined here are equally applicable to other scientific domains. Furthermore, a number of the proposed solutions can be modified for use in entirely distinct computer vision cases.

Bag3 (Bcl-2-associated athanogene 3) protein contains a number of functional domains and interacts with a wide range of different partner proteins, including small heat shock proteins (sHsps) and heat shock protein Hsp70. The ternary Bag3–sHsp–and Hsp70 complex binds denatured proteins and transports them to phagosomes, thus playing a key role in the chaperone-assisted selective autophagy (CASA). This complex also participates in the control of formation and disassembly of stress granules (granulostasis) and cytoskeleton regulation. As Bag3 and sHsps participate in multiple cellular processes, mutations in these proteins are often associated with neurodegenerative diseases and cardiomyopathy. The review discusses the role of sHsps in different processes regulated by Bag3.

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.

Stem and progenitor cells are characterized by peculiar mechanisms of hormonal regulation. Here we describe a protocol of analysis of hormonal cross-talk in adipose tissue derived multipotent mesenchymal stem cells (MSCs). Specifically, cells were treated by a “sensitizing” hormone/neuromediator followed by the measurement of cellular Ca2+ response to the “readout” hormone after various time intervals. This protocol was successfully used in studies demonstrating a permissive effect of noradrenaline and 5-HT on MSCs sensitivity to noradrenaline, which is a predictive marker of the development of obesity-associated arterial hypertension.

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.

In modern science, immortalized cells are not only a convenient tool in fundamental research, but they are also increasingly used in practical medicine. This happens due to their advantages compared to the primary cells, such as the possibility to produce larger amounts of cells and to use them for longer periods of time, the convenience of genetic modification, the absence of donor-to-donor variability when comparing the results of different experiments, etc. On the other hand, immortalization comes with drawbacks: possibilities of malignant transformation and/or major phenotype change due to genetic modification itself or upon long-term cultivation appear. At first glance, such issues are huge hurdles in the way of immortalized cells translation into medicine. However, there are certain ways to overcome such barriers that we describe in this review. We determined four major areas of usage of immortalized cells for practical medicinal purposes, and each has its own means to negate the drawbacks associated with immortalization. Moreover, here we describe specific fields of application of immortalized cells in which these problems are of much lesser concern, for example, in some cases where the possibility of malignant growth is not there at all. In general, we can conclude that immortalized cells have their niches in certain areas of practical medicine where they can successfully compete with other therapeutic approaches, and more preclinical and clinical trials with them should be expected.

Multipotent mesenchymal stromal cells (MSCs) regulate tissue repair through paracrine activity, with secreted proteins being significant contributors. Human tissue repair commonly results in fibrosis, where fibroblast differentiation into myofibroblasts is a major cellular mechanism. MSCs’ paracrine activity can inhibit fibrosis development. We previously demonstrated that the separation of MSC secretome, represented by conditioned medium (CM), into subfractions enriched with extracellular vesicles (EV) or soluble factors (SF) boosts EV and SF antifibrotic effect. This effect is realized through the inhibition of fibroblast-to-myofibroblast differentiation in vitro. To unravel the mechanisms of MSC paracrine effects on fibroblast differentiation, we performed a comparative proteomic analysis of MSC secretome fractions. We found that CM was enriched in NF-κB activators and confirmed via qPCR that CM, but not EV or SF, upregulated NF-κB target genes (COX2, IL6, etc.) in human dermal fibroblasts. Furthermore, we revealed that EV and SF were enriched in TGF-β, Notch, IGF, and Wnt pathway regulators. According to scRNAseq, 11 out of 13 corresponding genes were upregulated in a minor MSC subpopulation disappearing in profibrotic conditions. Thus, protein enrichment of MSC secretome fractions and cellular subpopulation patterns shift the balance in fibroblast-to-myofibroblast differentiation, which should be considered in studies of MSC paracrine effects and the therapeutic use of MSC secretome.

Mesenchymal stromal cells (MSCs) are the key regulators of tissue homeostasis and repair after damage. Accumulating evidence indicates the dual contribution of MSCs into the development of fibrosis induced by chronic injury: these cells can suppress the fibrotic process due to paracrine activity, but their promoting role in fibrosis by differentiating into myofibroblasts has also been demonstrated. Many model systems reproducing fibrosis have shown the ability of peroxisome proliferator-activated receptor (PPAR) agonists to reverse myofibroblast differentiation. Thus, the differentiation of multipotent cells into myofibroblasts and adipocytes can be considered as processes that require the activation of opposite patterns of gene expression. To test this hypothesis, we analyzed single cell RNA-Seq transcriptome of human adipose tissue MSCs after stimulation of the myofibroblast or adipogenic differentiation and revealed several genes that changed their expression in a reciprocal manner upon these conditions. We validated the expression of selected genes by RT-PCR, and evaluated the upregulation of several relevant proteins using immunocytochemistry, refining the results obtained by RNA-Seq analysis. We have shown, for the first time, the expression of neurotrimin (NTM), previously studied mainly in the nervous tissue, in human adipose tissue MSCs, and demonstrated its increased gene expression and clustering of membrane receptors upon the stimulation of myofibroblast differentiation. We also showed an increased level of CHD3 (Chromodomain-Helicase-DNA-binding protein 3) in MSCs under profibrotic conditions, while retinol dehydrogenase-10 (RDH10) was detected only in MSCs after adipogenic induction, which contradicted the data of transcriptomic analysis and again highlights the need to validate the data obtained by omics methods. Our findings suggest the further analysis of the potential contribution of neurotrimin and CHD3 in the regulation of myofibroblast differentiation and the development of fibrosis.

Regulation of the functional activity of stem cells is extremely important for their coordinated action in the processes of tissue repair and regeneration, as well as maintenance of its homeostasis. We now understand that stem cells are not just progenitors but are capable of regulating their host tissue’s function. This includes regulation of proliferation, migration, and differentiation of other cells, as well as secretion of paracrine factors, extracellular vesicles, and regulatory microRNAs. A wide variety of mechanisms and forms of regulation of stem cells hormonal sensitivity ensures harmonious interaction between the body, tissue, and stem cells, finely adjusting their functioning to dynamically changing conditions. This diversity is based on the functional heterogeneity of stem cell populations in the human body. The functional heterogeneity of stem cells manifests itself at several levels. First, stem cells differ in their properties when they are isolated from different individuals, even from the same line of animals. This is especially noticeable and important in practical terms when it comes to the use of primary cultures of human stem cells. Secondly, stem cells of even the same type differ when they are isolated from different organs. For example, multipotent mesenchymal stromal cells (MSCs) of adipose tissue differ in their proliferative, and differentiation potential, as well as in their immunomodulatory ability when isolated from various adipose tissue depots (Kulebyakin et al., 2022). Further, stem cells are heterogeneous in their sensitivity to regulatory hormones. Thus, it was shown that individual MSCs are sensitive to various hormones that activate GPCR (Kotova et al., 2014). At the same time, cells show plasticity, signal heterogeneity reappears in clones of a single cell (Tyurin-Kuzmin et al., 2020). As a result, at the level of the whole organism, functional heterogeneity allows us to achieve two goals: to ensure that stem cells are in different states (for example, part of the stem cells is in an undifferentiated state as a tissue regenerative potential), and on the other hand, to provide sensitivity to a wide range of regulatory influences at the population level. This Research Topic has invited original research articles and reviews focused on all aspects of functional heterogeneity of stem cells including but not limited to the links between the phenotype of stem cells and their functions, functional analysis of subpopulations of stem cells, regulation of stem cell heterogeneity by signaling molecules and microRNAs, and influence of cellular senescence and metabolic disorders on the subOPEN ACCESS

[This corrects the article DOI: 10.3389/fcell.2022.1050489.].

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.