Novel missense variants in brain morphogenic genes associated with depression and schizophrenia.

Introduction: Impaired 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. Methods: We 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. Results: Whole-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. Discussion: However, 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.

Morphogenetic theory of mental and cognitive disorders: the role of neurotrophic and guidance molecules.

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.

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.

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.

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.

Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies.

Gene editing tools have become an indispensable part of research into the fundamental aspects of cell biology. With a vast body of literature having been generated based on next generation sequencing technologies, keeping track of this ever-growing body of information remains challenging. This necessitates the translation of genomic data into tangible applications. In order to address this objective, the generated Next Generation Sequencing (NGS) data forms the basis for targeted genome editing strategies, employing known enzymes of various cellular machinery, in generating organisms with specifically selected phenotypes. This review focuses primarily on CRISPR/Cas9 technology in the context of its advantages over Zinc finger proteins (ZNF) and Transcription activator-like effector nucleases (TALEN) and meganucleases mutagenesis strategies, for use in agricultural and veterinary applications. This review will describe the application of CRISPR/Cas9 in creating modified organisms with custom-made properties, without the undesired non-targeted effects associated with virus vector vaccines and bioactive molecules produced in bacterial systems. Examples of the successful and unsuccessful applications of this technology to plants, animals and microorganisms are provided, as well as an in-depth look into possible future trends and applications in vaccine development, disease resistance and enhanced phenotypic traits will be discussed.

Pharmacological and Genetic Suppression of VDAC1 Alleviates the Development of Mitochondrial Dysfunction in Endothelial and Fibroblast Cell Cultures upon Hyperglycemic Conditions.

Prolonged hyperglycemia related to diabetes and its complications leads to multiple cellular disorders, the central one being the dysfunction of mitochondria. Voltage-dependent anion channels (VDAC) of the outer mitochondrial membrane control the metabolic, ionic, and energy cross-talk between mitochondria and the rest of the cell and serve as the master regulators of mitochondrial functions. Here, we have investigated the effect of pharmacological suppression of VDAC1 by the newly developed inhibitor of its oligomerization, VBIT-4, in the primary culture of mouse lung endotheliocytes and downregulated expression of VDAC1 in human skin fibroblasts on the progression of mitochondrial dysfunction upon hyperglycemic stress. The cells were grown in high-glucose media (30 mM) for 36 h. In response to hyperglycemia, the mRNA level of VDAC1 increased in endotheliocytes and decreased in human skin fibroblasts. Hyperglycemia induced overproduction of mitochondrial ROS, an increase in the susceptibility of the organelles to mitochondrial permeability transition (MPT) pore opening and a drop in mitochondrial membrane potential, which was accompanied by a decrease in cell viability in both cultures. Treatment of endotheliocytes with 5 µM VBIT-4 abolished the hyperglycemia-induced increase in susceptibility to spontaneous opening of the MPT pore and ROS generation in mitochondria. Silencing of VDAC1 expression in human skin fibroblasts exposed to high glucose led to a less pronounced manifestation of all the signs of damage to mitochondria. Our data identify a mitochondria-related response to pharmacological and genetic suppression of VDAC activity in vascular cells in hyperglycemia and suggest the potential therapeutic value of targeting these channels for the treatment of diabetic vasculopathies.

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.

Comparative Study of Cytotoxic and Membranotropic Properties of Betulinic Acid-F16 Conjugate on Breast Adenocarcinoma Cells (MCF-7) and Primary Human Fibroblasts.

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G0/G1 phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA-F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

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.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

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.

Impact of p53 knockdown on protein dataset of HaCaT cells.

The HaCaT line of immortalized non-tumor cells is a popular model of keratinocytes used for dermatological studies, in the practice of toxicological tests, and in the study of skin allergic reactions. These cells maintain a stable keratinocyte phenotype, do not require specific growth factors during cultivation, and respond to keratinocyte differentiation stimuli. HaCaT cells bear two mutant p53 alleles - R282Q and H179Y. At least two mechanisms of GOF (gain-of-function) of mutant p53 are known: it affects functions of p63/p73 by inhibiting their binding to DNA; or it binds to new DNA sites by interacting with other transcription factors (NF-Y, E2F1, NF-KB, VDR, p63). Proteins of the P53 family play an important role in the regulation of proliferation and differentiation processes of human keratinocytes. Proteomic study of HaCaT cells with TP53 gene knockdown provides new data for understanding the limitations of HaCaT cells when using them as an experimental model of normal human keratinocytes. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of human immortalized HaCaT keratinocytes and p53 knockdown HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS/MS measurements followed by their processing with MaxQuant software (version 1.6.3.4). The "RAW" files were deposited to the ProteomeXchange with identifier PXD033538.

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.

Urokinase Receptor uPAR Downregulation in Neuroblastoma Leads to Dormancy, Chemoresistance and Metastasis.

uPAR is a membrane receptor that binds extracellular protease urokinase, contributes to matrix remodeling and plays a crucial role in cellular adhesion, proliferation, survival, and migration. uPAR overexpression in tumor cells promotes mitogenesis, opening a prospective avenue for targeted therapy. However, uPAR targeting in cancer has potential risks. We have recently shown that uPAR downregulation in neuroblastoma promotes epithelial-mesenchymal transition (EMT), potentially associated with metastasis and chemoresistance. We used data mining to evaluate the role of uPAR expression in primary and relapsed human neuroblastomas. To model the decreased uPAR expression, we targeted uPAR using CRISPR/Cas9 and shRNA in neuroblastoma Neuro2a cells and evaluated their chemosensitivity in vitro as well as tumor growth and metastasis in vivo. We demonstrate that the initially high PLAUR expression predicts poor survival in human neuroblastoma. However, relapsed neuroblastomas have a significantly decreased PLAUR expression. uPAR targeting in neuroblastoma Neuro2a cells leads to p38 activation and an increased p21 expression (suggesting a dormant phenotype). The dormancy in neuroblastoma cells can be triggered by the disruption of uPAR-integrin interaction. uPAR-deficient cells are less sensitive to cisplatin and doxorubicin treatment and exhibit lower p53 activation. Finally, low uPAR-expressing Neuro2a cells formed smaller primary tumors, but more frequent metastasis in mice. To the best of our knowledge, this is the first study revealing the pathological role of dormant uPAR-deficient cancer cells having a chemoresistant and motile phenotype.

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.

Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism.

Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.

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.