Code-Free Machine Learning Solutions for Microscopy Image Processing: Deep Learning.

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.

Noradrenaline and serotonin-dependent sensitization of MSCs to noradrenaline.

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.

Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling.

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.

Peripheral 5-HT/HTR6 axis is responsible for obesity-associated 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.

Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives.

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.

Novel Potential Markers of Myofibroblast Differentiation Revealed by Single-Cell RNA Sequencing Analysis of Mesenchymal Stromal Cells in Profibrotic and Adipogenic Conditions.

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.

Alpha1A- and Beta3-Adrenoceptors Interplay in Adipose Multipotent Mesenchymal Stromal Cells: A Novel Mechanism of Obesity-Driven Hypertension.

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.

Corrigendum: Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.

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

Balance between Pro- and Antifibrotic Proteins in Mesenchymal Stromal Cell Secretome Fractions Revealed by Proteome and Cell Subpopulation Analysis.

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-ß, 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.

Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.

Multipotent mesenchymal stromal cells (MSCs) maintain cellular homeostasis and regulate tissue renewal and repair both by differentiating into mesodermal lineage, e.g., adipocytes, or managing the functions of differentiated cells. Insulin is a key physiological inducer of MSC differentiation into adipocytes, and disturbances in MSC insulin sensitivity could negatively affect adipose tissue renewal. During aging, regulation and renewal of adipose tissue cells may be disrupted due to the altered insulin signaling and differentiation potential of senescent MSCs, promoting the development of serious metabolic diseases, including metabolic syndrome and obesity. However, the potential mechanisms mediating the dysfunction of adipose-derived senescent MSC remains unclear. We explored whether aging could affect the adipogenic potential of human adipose tissue-derived MSCs regulated by insulin. Age-associated senescent MSCs (isolated from donors older than 65 years) and MSCs in replicative senescence (long-term culture) were treated by insulin to induce adipogenic differentiation, and the efficiency of the process was compared to MSCs from young donors. Insulin-dependent signaling pathways were explored in these cells. We also analyzed the involvement of extracellular vesicles secreted by MSCs (MSC-EVs) into the regulation of adipogenic differentiation and insulin signaling of control and senescent cells. Also the microRNA profiles of MSC-EVs from aged and young donors were compared using targeted PCR arrays. Both replicatively and chronologically senescent MSCs showed a noticeably decreased adipogenic potential. This was associated with insulin resistance of MSCs from aged donors caused by the increase in the basal level of activation of crucial insulin-dependent intracellular effectors ERK1/2 and Akt. To assess the impact of the paracrine cross-talk of MSCs, we analyzed microRNAs profile differences in MSC-EVs and revealed that senescent MSCs produced EVs with increased content of miRNAs targeting components of insulin-dependent signaling cascade PTEN, MAPK1, GAREM1 and some other targets. We also confirmed these data by differentiation of control MSCs in the presence of EVs from senescent cells and vice versa. Thus, aging attenuated the adipogenic potential of MSCs due to autocrine or paracrine-dependent induction of insulin resistance associated with the specific changes in MSC-EV cargo.

Dynamic Balance between PTH1R-Dependent Signal Cascades Determines Its Pro- or Anti-Osteogenic Effects on MSC.

Parathyroid hormone (PTH) is one of the key regulators of calcium and phosphate metabolism in the body, controlling bone metabolism and ion excretion by the kidneys. At present, attempts to use PTH as a therapeutic agent have been associated with side-effects, the nature of which is not always clear and predictable. In addition, it is known that in vivo impairment of PTH post-receptor signaling is associated with atypical differentiation behavior not only of bone cells, but also of connective tissues, including adipose tissue. In this work, we studied the functional responses of multipotent mesenchymal stromal cells (MSCs) to the action of PTH at the level of single cells. We used MSCs isolated from the periosteum and subcutaneous adipose tissue to compare characteristics of cell responses to PTH. We found that the hormone can activate three key responses via its receptor located on the surface of MSCs: single transients of calcium, calcium oscillations, and hormone-activated smooth increase in intracellular calcium. These types of calcium responses led to principally different cellular responses of MSCs. The cAMP-dependent smooth increase of intracellular calcium was associated with pro-osteogenic action of PTH, whereas phospholipase C dependent calcium oscillations led to a decrease in osteogenic differentiation intensity. Different variants of calcium responses are in dynamic equilibrium. Suppression of one type of response leads to increased activation of another type and, accordingly, to a change in the effect of PTH on cell differentiation.

GPCRs in the regulation of the functional activity of multipotent mesenchymal stromal cells.

Adipose tissue is one of the tissues in the human body that is renewed during the whole life. Dysregulation of this process leads to conditions such as obesity, metabolic syndrome, and type 2 diabetes. The key role in maintaining the healthy state of adipose tissue is played by a specific group of postnatal stem cells called multipotent mesenchymal stromal cells (MSCs). They are both precursors for new adipocytes and key paracrine regulators of adipose tissue homeostasis. The activity of MSCs is tightly adjusted to the needs of the organism. To ensure such coordination, MSCs are put under strict regulation which is realized through a wide variety of signaling mechanisms. They control aspects of MSC activity such as proliferation, differentiation, and production of signal molecules via alteration of MSC sensitivity to hormonal stimuli. In this regard, MSCs use all the main mechanisms of hormonal sensitivity regulation observed in differentiated cells, but at the same time, several unique regulatory mechanisms have been found in MSCs. In the presented review, we will cover these unique mechanisms as well as specifics of common mechanisms of regulation of hormonal sensitivity in stem cells.

COVID-19 and metabolic disease: mechanisms and clinical management.

Up to 50% of the people who have died from COVID-19 had metabolic and vascular disorders. Notably, there are many direct links between COVID-19 and the metabolic and endocrine systems. Thus, not only are patients with metabolic dysfunction (eg, obesity, hypertension, non-alcoholic fatty liver disease, and diabetes) at an increased risk of developing severe COVID-19 but also infection with SARS-CoV-2 might lead to new-onset diabetes or aggravation of pre-existing metabolic disorders. In this Review, we provide an update on the mechanisms of how metabolic and endocrine disorders might predispose patients to develop severe COVID-19. Additionally, we update the practical recommendations and management of patients with COVID-19 and post-pandemic. Furthermore, we summarise new treatment options for patients with both COVID-19 and diabetes, and highlight current challenges in clinical management.

Decreased Insulin Sensitivity in Telomerase-Immortalized Mesenchymal Stem Cells Affects Efficacy and Outcome of Adipogenic Differentiation in vitro.

Modern biomedical science still experiences a significant need for easy and reliable sources of human cells. They are used to investigate pathological processes underlying disease, conduct pharmacological studies, and eventually applied as a therapeutic product in regenerative medicine. For decades, the pool of adult mesenchymal stem/stromal cells (MSCs) remains a promising source of stem and progenitor cells. Their isolation is more feasible than most other stem cells from human donors, yet they have a fair share of drawbacks. They include significant variability between donors, loss of potency, and transformation during long-term culture, which may impact the efficacy and reproducibility of research. One possible solution is a derivation of immortalized MSCs lines which receive a broader use in many medical and biological studies. In the present work, we demonstrated that in the most widely spread commercially available hTERT-immortalized MSCs cell line ASC52telo, sensitivity to hormonal stimuli was reduced, affecting their differentiation efficacy. Furthermore, we found that immortalized MSCs have impaired insulin-dependent and cAMP-dependent signaling, which impairs their adipogenic, but not osteogenic or chondrogenic, potential under experimental conditions. Our findings indicate that hTERT-immortalized MSCs may present a suboptimal choice for studies involving modeling or investigation of hormonal sensitivity.

Hematopoietically-expressed homeobox protein HHEX regulates adipogenesis in preadipocytes.

Obesity is a key health problem and is associated with a high risk of type 2 diabetes and other metabolic diseases. Increased weight as well as dysregulation of adipocyte homeostasis are the main drivers of obesity. Pathological adipogenesis plays a central role in obesity-related complications such as type 2 diabetes, hypertension and others. Thus, an understanding of the molecular mechanisms involved in physiological and pathogenic adipogenesis can help to develop new strategies to prevent or cure obesity and related diseases. Previously, genetic polymorphisms in the HHEX gene that encodes the homeobox transcription factor HEX (PRH) were found to be associated with type 2 diabetes and high body mass index at birth by GWAS in distinct human populations. To understand whether HHEX has a regulatory function in adipogenesis, we performed RNAi-mediated knockdown of Hhex in preadipocyte cell line 3T3-L1 in vitro, and studied changes in the efficacy of adipogenesis. We found that Hhex knockdown blocks adipogenesis in preadipocytes in a dose-dependent manner and leads to a significant decrease of PPAR-gamma protein - the main regulator of adipogenesis. We also propose that Hhex can play an important role in adipocyte differentiation by affecting the level of the PPAR-gamma protein. Our study supports the claim that Hhex plays an important role in adipocyte differentiation program and can contribute to fat tissue homeostasis.

Adipocyte Biology from the Perspective of In Vivo Research: Review of Key Transcription Factors.

Obesity and type 2 diabetes are both significant contributors to the contemporary pandemic of non-communicable diseases. Both disorders are interconnected and associated with the disruption of normal homeostasis in adipose tissue. Consequently, exploring adipose tissue differentiation and homeostasis is important for the treatment and prevention of metabolic disorders. The aim of this work is to review the consecutive steps in the postnatal development of adipocytes, with a special emphasis on in vivo studies. We gave particular attention to well-known transcription factors that had been thoroughly described in vitro, and showed that the in vivo research of adipogenic differentiation can lead to surprising findings.

Mesenchymal Stromal Cell-Produced Components of Extracellular Matrix Potentiate Multipotent Stem Cell Response to Differentiation Stimuli.

Extracellular matrix (ECM) provides both structural support and dynamic microenvironment for cells regulating their behavior and fate. As a critical component of stem cell niche ECM maintains stem cells and activates their proliferation and differentiation under specific stimuli. Mesenchymal stem/stromal cells (MSCs) regulate tissue-specific stem cell functions locating in their immediate microenvironment and producing various bioactive factors, including ECM components. We evaluated the ability of MSC-produced ECM to restore stem and progenitor cell microenvironment in vitro and analyzed the possible mechanisms of its effects. Human MSC cell sheets were decellularized by different agents (detergents, enzymes, and apoptosis inductors) to select the optimized combination (CHAPS and DNAse I) based on the conservation of decellularized ECM (dECM) structure and effectiveness of DNA removal. Prepared dECM was non-immunogenic, supported MSC proliferation and formation of larger colonies in colony-forming unit-assay. Decellularized ECM effectively promoted MSC trilineage differentiation (adipogenic, osteogenic, and chondrogenic) compared to plastic or plastic covered by selected ECM components (collagen, fibronectin, laminin). Interestingly, dECM produced by human fibroblasts could not enhance MSC differentiation like MSC-produced dECM, indicating cell-specific functionality of dECM. We demonstrated the significant integrin contribution in dECM-cell interaction by blocking the stimulatory effects of dECM with RGD peptide and suggested the involvement of key intracellular signaling pathways activation (pERK/ERK and pFAK/FAK axes, pYAP/YAP and beta-catenin) in the observed processes based on the results of inhibitory analysis. Taken together, we suppose that MSC-produced dECM may mimic stem cell niche components in vitro and maintain multipotent progenitor cells to insure their effective response to external differentiating stimuli upon activation. The obtained data provide more insights into the possible role of MSC-produced ECM in stem and progenitor cell regulation within their niches. Our results are also useful for the developing of dECM-based cell-free products for regenerative medicine.

Growth Factors in Regeneration and Regenerative Medicine: "the Cure and the Cause".

The potential rapid advance of regenerative medicine was obstructed by findings that stimulation of human body regeneration is a much tougher mission than expected after the first cultures of stem and progenitor cells were established. In this mini review, we focus on the ambiguous role of growth factors in regeneration, discuss their evolutionary importance, and highlight them as the "cure and the cause" for successful or failed attempts to drive human body regeneration. We draw the reader's attention to evolutionary changes that occurred in growth factors and their receptor tyrosine kinases (RTKs) and how they established and shaped response to injury in metazoans. Discussing the well-known pleiotropy of growth factors, we propose an evolutionary rationale for their functioning in this specific way and focus on growth factors and RTKs as an amazing system that defines the multicellular nature of animals and highlight their participation in regeneration. We pinpoint potential bottlenecks in their application for human tissue regeneration and show their role in fibrosis/regeneration balance. This communication invites the reader to re-evaluate the functions of growth factors as keepers of natively existing communications between elements of tissue, which makes them a fundamental component of a successful regenerative strategy. Finally, we draw attention to the epigenetic landscape that may facilitate or block regeneration and give a brief insight into how it may define the outcome of injury.

Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells.

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.