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.

Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells.

Non-coding RNA (ncRNAs) genes have attracted increasing attention in recent years due to their widespread involvement in physiological and pathological processes and regulatory networks. The study of the function and molecular partners of ncRNAs opens up opportunities for the early diagnosis and treatment of previously incurable diseases. However, the classical "loss-of-function" approach in ncRNA function analysis is challenged due to some specific issues. Here, we have studied the potency of two CRISPR/Cas9 variants, wild-type (SpCas9wt) and nickase (SpCas9D10A) programmable nucleases, for the editing of extended DNA sequences in human mesenchymal stromal cells (MSCs). Editing the genes of fibrosis-related hsa-miR-21-5p and hsa-miR-29c-3p, we have shown that a pair of SpCas9D10A molecules can effectively disrupt miRNA genes within the genomes of MSCs. This leads not only to a decrease in the level of knockout miRNA in MSCs and MSC-produced extracellular vesicles, but also to a change in cell physiology and the antifibrotic properties of the cell secretome. These changes correlate well with previously published data for the knockdown of certain miRNAs. The proposed approach can be used to knock out ncRNA genes within the genomes of MSCs or similar cell types in order to study their function in biological processes.

The Secretome of Mesenchymal Stromal Cells in Treating Intracerebral Hemorrhage: The First Step to Bedside.

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.

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.

The Power of Gene Technologies: 1001 Ways to Create a Cell Model.

Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies.

A Novel Cre/lox71-Based System for Inducible Expression of Recombinant Proteins and Genome Editing.

In this study, we developed a novel Cre/lox71-based system for the controlled transient expression of target genes. We used the bacteriophage P1 Cre recombinase, which harbors a short, highly specific DNA-binding site and does not have endogenous binding sites within mouse or human genomes. Fusing the catalytically inactive form of Cre recombinase and the VP64 transactivation domain (VP16 tetramer), we constructed the artificial transcription factor Cre-VP64. This transcription factor binds to the lox71 sites within the promoter region of the target gene and, therefore, upregulates its expression. We tested the Cre-VP64/lox71 system for the controlled expression of several genes, including growth factors and the genome editor CRISPR/Cas9, and obtained superior efficiency in the regulation of transgene expression, achieving a high expression level upon induction together with low basal activity. This system or its modified forms can be suggested as a novel effective tool for the transitory controlled expression of target genes for functional genomic studies, as well as for gene therapy approaches.

Urokinase-Type Plasminogen Activator Enhances the Neuroprotective Activity of Brain-Derived Neurotrophic Factor in a Model of Intracerebral Hemorrhage.

Brain-derived neurotrophic factor (BDNF) is a classic neuroprotective and pro-regenerative factor in peripheral and central nervous tissue. Its ability to stimulate the restoration of damaged nerve and brain tissue after ischemic stroke and intraventricular hemorrhage has been demonstrated. However, the current concept of regeneration allows us to assert that one factor, even if essential, cannot be the sole contributor to this complex biological process. We have previously shown that urokinase-type plasminogen activator (uPA) complements BDNF activity and stimulates restoration of nervous tissue. Using a model of intracerebral hemorrhage in rats, we investigated the neurotrophic and neuroprotective effect of BDNF combined with uPA. The local simultaneous administration of BDNF and uPA provided effective neuroprotection of brain tissue after intracerebral hemorrhage, promoted survival of experimental animals and their neurological recovery, and decreased lesion volume. The study of cellular mechanisms of the observed neurotrophic effect of BDNF and uPA combination revealed both known mechanisms (neuronal survival and neurite growth) and new ones (microglial activation) that had not been shown for BDNF and uPA. Our findings support the concept of using combinations of biological factors with diverse but complementary mechanisms of action as a promising regenerative approach.

MSC Secretome as a Promising Tool for Neuroprotection and Neuroregeneration in a Model of Intracerebral Hemorrhage.

Multipotent mesenchymal stromal cells (MSCs) are considered to be critical contributors to injured tissue repair and regeneration, and MSC-based therapeutic approaches have been applied to many peripheral and central neurologic disorders. It has been demonstrated that the beneficial effects of MSC are mainly mediated by the components of their secretome. In the current study, we have explored the neuroprotective potential of the MSC secretome in a rat model of intracerebral hemorrhage and shown that a 10-fold concentrated secretome of human MSC and its combination with the brain-derived neurotrophic factor (BDNF) provided a better survival and neurological outcome of rats within 14 days of intracerebral hemorrhage compared to the negative (non-treated) and positive (BDNF) control groups. We found that it was due to the ability of MSC secretome to stimulate neuron survival under conditions of glutamate-induced neurotoxicity. However, the lesion volume did not shrink in these rats, and this also correlated with prominent microglia activation. We hypothesize that this could be caused by the species-specificity of the used MSC secretome and provide evidence to confirm this. Thus, we have found that allogenic rat MSC secretome was more effective than xenogenic human MSC secretome in the rat intracerebral hemorrhage model: it reduced the volume of the lesion and promoted excellent survival and neurological outcome of the treated rats.

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.