Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

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.

Noradrenaline Sensitivity Is Severely Impaired in Immortalized Adipose-Derived Mesenchymal Stem Cell Line.

Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called "heterologous sensitization", and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca2+-mobilization in single cells, we found that these cells did not demonstrate the sensitization seen in primary adMSCs. Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology.

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.

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.

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.