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.

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.

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.

Correlations between biomarkers of senescent cell accumulation at the systemic, tissue and cellular levels in elderly patients.

The aim of the study was to investigate the relationship between established clinical systemic biomarkers of ageing and the development of age-associated diseases and senescent cell biomarkers at tissue and cellular levels. Thirty-eight patients (mean age 70 ± 4.9 years) who were assessed for traditional risk factors for cardiovascular diseases were included. From all patients we obtained biomaterials (peripheral blood, skin, subcutaneous fatty tissue) and isolated different cell types (peripheral blood mononuclear cells (PBMC), fibroblasts (FB) and mesenchymal stem/stromal cells (MSC)). Isolated cells were analyzed using several senescent cells biomarkers such as telomere length and telomerase activity, proliferation rate, cell cycle inhibitor expression (p16 and p21), b-galactosidase activity, gH2AX expression. CD34+ cell content in peripheral blood was determined by flow cytometry. Systemic senescent cell-associated factors (insulin-like growth factor 1 (IGF-1), fibroblast growth factor 21 (FGF-21), osteoprogerin, ferritin, soluble vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule 1 (ICAM-1)) in peripheral blood as well as senescence-associated secretory phenotype (SASP) components in MSC and FB secretome were evaluated by ELISA. Skin and adipose tissue biopsy samples were analyzed histologically to assess senescent cell markers. A strong significant association of tissue p16 expression with age (r = 0.600, p < 0.001), pulse wave velocity (PWV) (r = 0.394, p = 0.015), vascular cell adhesion molecule (VCAM-1) content (r = 0.312, p = 0.006) in the systemic blood stream and p16 mRNA level in the blood mononuclear cells (MNCs) (r = 0.380, p = 0.046) were confirmed by correlation analysis. Statistically significant correlations were found with indicators of FBs and MSCs proliferation in culture and acquisition of SASP by the cells. Thus, p16 expression in tissues correlated significantly with interleukin-6 (IL-6) (r = 0.485, p < 0.05) and monocyte chemoattractant protein type 1 (MCP-1) (r = 0.372, p < 0.05) secretion by isolated cells. The results of regression analysis confirmed that, regardless of age, the expression of p16 was associated with the proliferation of isolated cells and IL-6 within SASP. Based on these findings, two models have been proposed to predict the level of p16 expression in tissues from the levels of other markers of senescent cell accumulation determined by non-invasive methods and available in clinical practice.

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.].

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.

Therapeutic Angiogenesis by a "Dynamic Duo": Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle.

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels' formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need-peripheral artery disease and associated limb ischemia.

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3-7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

Secretome of Mesenchymal Stromal Cells Prevents Myofibroblasts Differentiation by Transferring Fibrosis-Associated microRNAs within Extracellular Vesicles.

Fibroblasts differentiation into myofibroblasts is a central event of tissue fibrosis. Multipotent mesenchymal stromal cells (MSCs) secretome can interfere with fibrosis development; despite precise underlying mechanisms remain unclear. In this study, we tested the hypothesis that MSC secretome can affect fibroblast' differentiation into myofibroblasts by delivering regulatory RNAs, including microRNAs to these cells. Using the model of transforming growth factor-beta (TGFbeta)-induced fibroblast differentiation into myofibroblasts, we tested the activity of human MSC secretome components, specifically extracellular vesicles (MSC-EV). We showed that MSC-EV down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts. High-throughput sequencing of RNAs extracted from MSC-EV has revealed many fibrosis-associated microRNAs. Fibroblast treatment with MSC-EV led to direct transfer of microRNAs, which resulted in the elevation of most prominent fibrosis-associated microRNAs, including microRNA-21 and microRNA-29c. Using MSC-EV transfection by antagomirs to these microRNAs we demonstrated their involvement in the suppression of fibroblast differentiation in our model. Taken together, MSC secretome can suppress fibrosis by prevention of fibroblast differentiation into myofibroblasts as well as induce de-differentiation of the latter by direct transfer of specific microRNAs.

Conditioned Medium from Human Mesenchymal Stromal Cells: Towards the Clinical Translation.

Mesenchymal stem/stromal cells (MSC) remain a promising tool for regenerative medicine as the efficacy of MSC-based cell therapy has been demonstrated for a broad spectrum of indications. Their therapeutic potency is mainly associated with their ability to secrete multiple factors critical for tissue regeneration. Due to comparable effects along with superior safety MSC conditioned medium (MSC-CM) containing a complex of MSC-secreted products is considered a reasonable alternative to cell therapy. However, the lack of standards regulating bioprocessing, use of proper auxiliary materials, and quality control complicates the development of MSC secretome-based therapeutics. In this study, we suggested several approaches addressing these issues. We manufactured 36 MSC-CM samples based on different xeno-free serum-free chemically defined media (DMEM-LG or MSC NutriStem® XF) using original protocols and considered total concentrations of regeneration-associated paracrine factors secreted by human adipose-derived MSC at each time-point of conditioning. Using regression analysis, we retrospectively predicted associations between concentrations of several components of MSC-CM and its biological activity to stimulate human dermal fibroblast and endothelial cell migration in vitro as routine examples of potency assays for cell-based products. We also demonstrated that the cell culture medium might affect MSC-CM biological activity to varying degrees depending on the potency assay type. Furthermore, we showed that regression analysis might help to overcome donor variability. The suggested approaches might be successfully applied for other cell types if their secretome was shown to be promising for application in regenerative medicine.

The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1.

Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic ß-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to search new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability.

Data supporting that miR-92a suppresses angiogenic activity of adipose-derived mesenchymal stromal cells by down-regulating hepatocyte growth factor.

This article contains the full list of miRNAs expressed in cultured mesenchymal stromal cells, which were isolated from human adipose tissue. We provide here data regarding the effect of miR-92a overexpression on MSCs viability and cellular content of HGF and angiopoietin-1. These are followed by the data regarding the effect of conditioned medium of MSC transfected with pre-miR-92a, anti-miR-92a or scramble oligos on HUVEC viability as well as their tube formation efficiency. We also demonstrate here data regarding the effect of extracellular vesicle depletion from MSCs conditioned medium on its ability to stimulate the tube formation by HUVEC. Data interpretation and discussion can be found in Kalinina et al. (2015) [1].

miR-92a regulates angiogenic activity of adipose-derived mesenchymal stromal cells.

Mesenchymal stromal cells including those from adipose tissue (MSCs) regulate angiogenesis in adult tissues. MicroRNAs (miRs), small noncoding RNAs that control gene expression by binding to target mRNAs, reducing their stability and/or inhibiting translation, appear to be important regulators of blood vessel growth. In this study, we examined the impact of angio-miRs on paracrine activities of MSCs. Using Illumina microarrays we found that miR-92a is one of the most abundant angio-miRs in human MSCs. We transfected MSC with pre-miR-92a or anti-miR-92a which led to the coordinated changes of known miR-92a target mRNA levels. Then we tested the ability of conditioned medium from transfected cells to stimulate tube formation by HUVECs. MSC overexpressing miR-92a completely lost the ability to stimulate tubes formation by endothelial cells. However, knocking-out miR-92a by transfection with anti-miR-92a did not increase the ability of MSC to stimulate tube formation. Secretion of hepatocyte growth factor (HGF) and angiopoetin-1 was significantly lower in the medium of miR-92a overexpressing MSC, whereas VEGF secretion did not change significantly. The replenishment of HGF but not angiopoietin-1 has restored the ability of conditioned medium from miR-92a overexpressing MSC to stimulate the tube formation. We conclude that overexpression of miR-92a in MSC suppresses angiogenic properties of these cells by down-regulation of HGF secretion.

Autologous Stem Cell Therapy: How Aging and Chronic Diseases Affect Stem and Progenitor Cells.

During recent years different types of adult stem/progenitor cells have been successfully applied for the treatment of many pathologies, including cardiovascular diseases. The regenerative potential of these cells is considered to be due to their high proliferation and differentiation capacities, paracrine activity, and immunologic privilege. However, therapeutic efficacy of the autologous stem/progenitor cells for most clinical applications remains modest, possibly because of the attenuation of their regenerative potential in aged patients with chronic diseases such as cardiovascular diseases and metabolic disorders. In this review we will discuss the risk factors affecting the therapeutic potential of adult stem/progenitor cells as well as the main approaches to mitigating them using the methods of regenerative medicine.