Multipotent Mesenchymal Stromal Cells Research Articles

Storage conditions affect the composition of the lyophilized secretome of multipotent mesenchymal stromal cells

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton’s jelly MSCs was stored at – 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at – 80 °C, − 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at – 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at – 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at −80 °C.

Gaining momentum: stem cell therapies for HIV cure.

Durable HIV-1 remission has been reported in a person who received allogeneic stem cell transplants (SCTs) involving CCR5 Δ32/Δ32 donor cells. Much of the reduction in HIV-1 burden following allogeneic SCT with or without donor cells inherently resistant to HIV-1 infection is likely due to cytotoxic graft-versus-host effects on residual recipient immune cells. Nonetheless, there has been growing momentum to develop and implement stem cell therapies that lead to durable long-term antiretroviral therapy (ART)-free remission without the need for SCT. Most current research leverages gene editing techniques to modify hematopoietic stem cells which differentiate into immune cells capable of harboring HIV-1. Approaches include targeting genes that encode HIV-1 co-receptors using Zinc Finger Nucleases (ZFN) or CRISPR-Cas-9 to render a pool of adult or progenitor cells resistant to de-novo infection. Other strategies involve harnessing multipotent mesenchymal stromal cells to foster immune environments that can more efficiently recognize and target HIV-1 while promoting tissue homeostasis. Many of these strategies are currently in a state of infancy or adolescence; nonetheless, promising preclinical and first-in-human studies have been performed, providing further rationale to focus resources on stem cell therapies.

Impact of platelet lysate on immunoregulatory characteristics of equine mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSC) play an increasing role in the treatment of immune-mediated diseases and inflammatory processes. They regulate immune cells via cell-cell contacts and by secreting various anti-inflammatory molecules but are in turn influenced by many factors such as cytokines. For MSC culture, platelet lysate (PL), which contains a variety of cytokines, is a promising alternative to fetal bovine serum (FBS). We aimed to analyze if PL with its cytokines improves MSC immunoregulatory characteristics, with the perspective that PL could be useful for priming the MSC prior to therapeutic application. MSC, activated peripheral blood mononuclear cells (PBMC) and indirect co-cultures of both were cultivated in media supplemented with either PL, FBS, FBS+INF-γ or FBS+IL-10. After incubation, cytokine concentrations were measured in supernatants and control media. MSC were analyzed regarding their expression of immunoregulatory genes and PBMC regarding their proliferation and percentage of FoxP3+ cells. Cytokines, particularly IFN-γ and IL-10, remained at high levels in PL control medium without cells but decreased in cytokine-supplemented control FBS media without cells during incubation. PBMC released IFN-γ and IL-10 in various culture conditions. MSC alone only released IFN-γ and overall, cytokine levels in media were lowest when MSC were cultured alone. Stimulation of MSC either by PBMC or by PL resulted in an altered expression of immunoregulatory genes. In co-culture with PBMC, the MSC gene expression of COX2, TNFAIP6, IDO1, CXCR4 and MHC2 was upregulated and VCAM1 was downregulated. In the presence of PL, COX2, TNFAIP6, VCAM1, CXCR4 and HIF1A were upregulated. Functionally, while no consistent changes were found regarding the percentage of FoxP3+ cells, MSC decreased PBMC proliferation in all media, with the strongest effect in FBS media supplemented with IL-10 or IFN-γ. This study provides further evidence that PL supports MSC functionality, including their immunoregulatory mechanisms. The results justify to investigate functional effects of MSC cultured in PL-supplemented medium on different types of immune cells in more detail.

Multifunctional platform for future applications in cell and tissue engineering based on silicate phosphate hydroxyapatite co-doped with Li+, Eu3+ and Gd3+ ions

Although hydroxyapatite has established itself as a promising biomaterial for the simultaneous diagnosis and treatment of bone defects, several physicochemical properties can be further refined to achieve a specialized theranostic platform for cell and tissue engineering. In this context, we synthesized silicate-substituted hydroxyapatites co-doped with Li+, Eu3+, and Gd3+ ions (abbr. as Si-HAp-LEG) using a microwave-assisted hydrothermal method. The aim was to create a combination of dopants to stimulate bone regeneration (Li+ ion) while enabling bioimaging (Eu3+ and Gd3+ ions). Structural and morphological assessments included, e.g., XRPD (X-ray Powder Diffraction), HR-TEM (High-resolution Transmission Electron Microscopy) imaging, and BET (Brauner-Emmet-Teller) analysis. Unit-cell parameters were estimated via Rietveld refinement. The luminescence properties (emission, excitation spectra, and emission kinetics) were recorded depending on the varying Li+-ion concentration. The cytocompatibility of biomaterials was tested using hBMSCs (multipotent human bone-marrow mesenchymal stromal cells). In vitro assays evaluated biomaterials’ effects on morphology, metabolic activity (Alamar blue assay), and transcriptomic activity (RT-qPCR). The results indicate that co-doping with Li+, Eu3+, and Gd3+ ions introduces distortions while preserving the crystal structure of hydroxyapatite. Additionally, the cells maintained proper morphology and ultrastructure, internalizing biomaterials without significant cytotoxicity. The biomaterials also triggered the expression of key transcriptional factors. Si-HAp-LEG biomaterials, particularly LEG-222 and LEG-221, exhibit desired physicochemical and biological features, rendering them highly bioactive and promising for cell and tissue engineering applications.

(106) Stem Cell Therapy Leads to an Increase in Testosterone Levels in Infertile Men

Abstract Introduction Sexual dysfunction is frequently encountered in infertile males. This may be related to psychosexual aspects of infertility and to some common factors of pathogenesis, such as hypogonadism. For example, many patients with non-obstructive azoospermia (NOA) have low testosterone levels, which may lead to low sex drive, erectile dysfunction and metabolic abnormalities later in life. Patients with NOA typically undergo surgical sperm retrieval procedures, which may lead to further decrease in androgen production, which may be transient or even permanent in some cases. Stem cell therapy is an investigative mode of treatment for patients with NOA, but its influence on Leydig cell function is currently unknown. Objective To assess the impact of stem cell therapy on testosterone levels in men. Methods Sixty patients with NOA were included in this study. All of them underwent unsuccessful MicroTESE more than 12 months prior to the inclusion in order to justify the use of stem cell therapy. Multipotent mesenchymal stromal cells (MMSC) were derived from healthy donor placenta following physiologic childbirth. MMSC product was used for systemic and local treatment via intravenous and intratesticular injection, respectively. Endocrine profiling was performed at baseline and 3 months after the procedure. Results Median testosterone level prior to MMSC therapy was 8.7 nmol/l (interquartile range [IQR]: 4.2–12.1), and 3 months after it was 12.3 nmol/l (IQR: 8.8–16.3) (p < 0.00001). This may be related to an increase in luteinizing hormone levels from 6.4 mIU/ml at baseline (IQR: 3.6–11.3) to 9.6 mIU/ml (IQR: 5.2–14.1) (p = 0.00026). Paradoxically, follicle-stimulating hormone levels declined from 12.5 mIU/ml (IQR: 4.7–24.7) to 7.4 mIU/ml (IQR: 4.2–12.3) (p < 0.00001). Prior to MMSC therapy 43 patients (71.7%) had testosterone levels lower than 12 nmol/l, after MMSC therapy 24 patients (40%) had testosterone levels lower than this threshold. Thirty patients (50%) and 14 patients (23.3%) had testosterone level below 8 nmol/l before and after MMSC therapy respectively. Conclusions MMSC therapy may increase testosterone levels in patients with NOA, though the exact mechanisms and its possible impact on quality of life should be elucidated. Disclosure No.

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.

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors.

Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47×109 cells in a real-scale 2.4-L batch.

The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

Four sides to the story: A proteomic comparison of liquid-phase and matrix-bound extracellular vesicles in 2D and 3D cell cultures.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

NANOVESICULAR THERAPEUTICS AND DRUG DELIVERY SYSTEMS IN ORTHOPAEDICS

Nanovesicle-based therapy is increasingly being pursued as a safe, cell-free strategy to combat various immunological, musculoskeletal and neurodegenerative diseases. Small secreted extracellular vesicles (sEVs) obtained from multipotent mesenchymal stromal cells (MSCs) are of particular interest for therapeutic use since they convey anti-inflammatory, anti-scarring and neuroprotective activities to the recipient cells. Cell-derived vesicles (CDVs) produced by a proprietary extrusion process are surrounded by a lipid bilayer membrane with correct membrane topology, display biological activities similar to MSC-derived EVs and may find specific application for organ-targeted drug delivery systems. Translation of nanovesicle-based therapeutics into clinical application requires quantitative and reproducible analysis of bioactivity and stability, and the potential for GMP-compliant manufacturing. Manufacturing and regulatory considerations as well as preclinical models to support clinical translation will be discussed.

Применение секретома мультипотентных мезенхимальных стромальных клеток для стимуляции регенерации костной ткани в эксперименте

The article is devoted to a pressing issue in regenerative medicine about the use of cellular products to stimulate reparative regeneration of bone defects. The article presents the results of a study of the structural characteristics of the regenerated rat femoral diaphysis with an induced defect using the secretome of multipotent mesenchymal stromal cells. White outbred rats (males, 120 g) were taken as the object of the study. The study material included femur samples taken on the 7th, 14th, 30th, 60th, 90th and 120th days. The work used methods of computed tomography, anatomical dissection and light microscopy. It has been shown that the use of the secretome of multipotent mesenchymal stromal cells in the early stages activates angiogenesis in the healing area. Against this background, stimulation of reparative osteogenesis occurs with better consolidation of the regenerate with the edges of the defect. In this case, the structure of the regenerate corresponds typical structure of the diaphysis: external and internal systems of bone plates and an osteon layer are observed. All this may indicate earlier remodeling of the regenerate, as well as restoration of the structural and functional characteristics of the bone. The data obtained can be used for further fundamental research in the field of regenerative medicine, as well as in clinical practice.

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-β, 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.

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.

M2-Macrophage-Induced Chronic Inflammation Promotes Reversible Mesenchymal Stromal Cell Senescence and Reduces Their Anti-Fibrotic Properties

Fibrosis and the associated decline in organ functionality lead to an almost 50% mortality rate in developed countries. Multipotent mesenchymal stromal cells (MSC) were shown to suppress the development and progression of fibrosis through secreted factors including specific non-coding RNAs transferred within extracellular vesicles (EV). However, age-associated chronic inflammation can provoke MSC senescence and change secretome composition, thereby affecting their antifibrotic properties. Alternatively activated macrophages (M2-type) are key players in chronic inflammation that may interact with MSC through paracrine mechanisms and decrease their antifibrotic functions. To confirm this hypothesis, we evaluated the M2-macrophage conditioned medium (CM-M2) effect on human adipose-tissue-derived MSC senescence in vitro. We found that CM-M2, as well as a pro-senescence agent, hydrogen peroxide (H2O2), increased p21+–MSC number and secretion of IL-6 and MCP-1, which are considered main senescence-associated secretory phenotype (SASP) components. Thus, both exposures led to the senescent phenotype acquisition of MSC. EV from both CM-M2 and H2O2-exposed MSC, which showed a decreased effect on the suppression of TGFβ-induced fibroblast-to-myofibroblast differentiation compared to EV from control MSC according to αSMA level and the αSMA+–stress fiber reduction. After two weeks of subsequent cultivation under standard conditions, MSC demonstrated a decrease in senescence hallmarks and fibroblast differentiation suppression via EV. These results suggest that M2-macrophage-induced chronic inflammation can reversibly induce MSC senescence, which reduces the MSC’s ability to inhibit fibroblast-to-myofibroblast differentiation.

Biogenesis of Mitochondria in Multipotent Mesenchymal Stromal Cells in Patients with Acute Leukemia.

In patients with acute leukemia, not only normal hematopoiesis, but also bone marrow stromal microenvironment is damaged. Multipotent mesenchymal stromal cells (MSC) are essential for the formation and function of the stromal microenvironment. Analysis of changes in MSC is important for the development of new approaches to leukemia therapy. The metabolism of mitochondria in MSC, relative content of mitochondrial DNA, and expression levels of genes encoding PGC-1α and Nrf2 proteins, important regulators of biogenesis, were studied using real-time PCR. Relative content of mitochondrial DNA does not change in MSC of acute leukemia patients at the onset of disease or in remission. Relative expression level of the gene encoding PGC-1α protein in MSC does not change significantly. However, relative expression level of the gene encoding Nrf2, an important antioxidant activity regulator, insignificantly decreases in patients at the onset of acute leukemia, and this decrease becomes significant upon reaching remission.

Multipotent mesenchymal stromal cells of various origins reduce reactive gliosis in the hippocampal CA1 area during acute ischemia-reperfusion of the rat brain

Neuroprotective therapy in acute cerebrovascular disorders is directly aimed at preserving neurons in the penumbra zone, but it also emphasizes the importance of gliogenesis in the affected area. Glial cells exhibit rapid reactivity and are highly sensitive to cerebral ischemic damage. Recent experimental studies have demonstrated the successful application of multipotent mesenchymal stromal cells (MMSCs) in stroke to modulate microglial activation. The aim of the study was to investigate the impact of MMSCs of different origins, MMSC lysate, and citicoline on glial components in a model of cerebral ischemia-reperfusion in rats. Materials and methods. The experiments were conducted on 190 male Wistar rats aged 4 months, weighing 160-190 g. After modeling cerebral ischemia-reperfusion through bilateral 20-minute occlusion of the internal carotid arteries, rats were intravenously administered MMSCs derived from human Wharton’s jelly, human adipose tissue, and rat Wharton’s jelly at a dose of 106 cells per animal. Other groups received fetal rat fibroblasts (106 cells/animal in 0.2 ml of physiological solution) and lysate from human Wharton’s jelly at a dose of 0.2 ml per animal. Control animals received 0.2 ml of physiological solution intravenously. The last group of rats received a single dose of the reference drug citicoline at a dose of 250 mg/kg. On the 7th and 14th days, the area and fluorescence intensity of cells expressing markers of astrocytes (GFAP), microglia (Iba1), and oligodendrocytes (Rip) were quantitatively assessed in CA1 hippocampal region slices using immunohistochemical examination and confocal microscopy. Results. On the 7th and 14th days after cerebral ischemia-reperfusion in rats, the intensity of fluorescence of GFAP-positive astrocytes and Iba1-positive microglial cells increased, indicating pronounced reactive astrogliosis and microglial activation in the CA1 region of the hippocampus. Meanwhile, ischemia-reperfusion had a significant impact on the content of Rip-positive oligodendrocytes in brain slices. The application of all treatments (transplantation of MMSCs of different origins, their lysate, or the reference drug citicoline) had a cytoprotective effect, reducing reactive astrogliosis and microgliosis both on the 7th and 14th days after injury. The best result was demonstrated with the treatment using MSCs from human Wharton’s jelly.s. Conclusion. Cerebral ischemia-reperfusion induces reactive gliosis through the activation of GFAP- and Iba1-positive glial cells in all layers of the hippocampus. The application of MSCs from human Wharton’s jelly and fetal rat fibroblasts significantly reduces its intensity on both the 7th and 14th days after injury modeling.

The effects of human umbilical cord-derived multipotent mesenchymal stromal cells transplantation in mice of different strains with an experimental model of parkinsonism

One of the promising directions in cell therapy for Parkinson's disease or parkinsonism is the transplantation of multipotent mesenchymal stromal cells from various sources, including human umbilical cord (hUC-MMSCs), the effectiveness of which may depend on the recipient's genotype. Objective. To compare the impact of transplanted MMSC-P on behavior, T-lymphocytes, and macrophages in the brain and lymphoid organs of mice from different lines with a toxic model of parkinsonism. Materials and methods. Adult (6-7 months old) male mice of FVB/N (genotype H-2q) and 129/Sv (genotype H-2b) strains were administered the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg (control group), and after 7 days, hUC-MMSCs (500,000 cells) were transplanted into the tail vein. Behavioral reactions were assessed in open field, rigidity, and rotarod tests. The relative content of T-lymphocytes and activated macrophages in the brain was measured by flow cytometry, and the mass of lymphoid organs was determined. Results. Under the influence of MPTP, the number of rearings, "sniffs into the nest," body length, and step length decreased, the number of boluses increased in FVB/N and 129/Sv mice, and the number of squares crossed in the open field test decreased in 129/Sv mice. In the brain of mice from both lines, the content of activated macrophages increased, and in FVB/N mice, the number of T-lymphocytes also increased. The thymus mass decreased in mice from both lines, while the spleen mass decreased only in 129/Sv mice. The transplantation of hUC-MMSCs improved predominantly motor activity in FVB/N mice, while in 129/Sv mice, emotional activity improved, and manifestations of rigidity decreased in mice from both lines. The content of T-lymphocytes and activated macrophages in the brain of mice from both lines, as well as the thymus mass, corresponded to the values of intact animals. MMSC transplantation promoted the survival of FVB/N and 129/Sv mice with the MPTP-induced parkinsonism model. Conclusions. The manifestations of behavioral disorders, changes in the content of T-lymphocytes and activated macrophages in the brain, and the mass of lymphoid organs in mice with the MPTP-induced parkinsonism model, as well as the positive effects of transplanted hUC-MMSCs in these animals, largely depend on their genotype according to the H-2 system (analogous to the HLA system in humans). The results may provide a basis for developing personalized cell therapy for this pathology using multipotent mesenchymal stromal cells.

Comparison of the therapeutic effect of native and preconditioned human umbilical cord-derived multipotent mesenchymal stromal cells on a rat model of acute pancreatitis

Pancreatitis is a disease characterized by an acute or chronic inflammatory process of the pancreas, induced by the activation of trypsin inside acinocytes. These processes result in necrosis of the organ's parenchyma, abscess formation, and systemic complications, contributing to patient mortality rates ranging from 30-47%. As of now, there is no globally recognized effective treatment for acute pancreatitis. Multipotent mesenchymal stem cells (MMSCs) are potential candidates for treating this disease due to their immunomodulatory properties. The aim of this study was to compare the therapeutic effects of transplanting native human umbilical cord-derived MMSCs (hUC-MMSCs) with hUC-MMSCs preconditioned with H2O2 in a rat model of acute pancreatitis. Materials and methods. Acute pancreatitis in rats was induced by intraperitoneal injection of L-arginine at a dose of 3.5 g/kg body weight at an interval of 1 hour. The rats were then categorized into four groups. Group 1 – negative control, where animals received a physiological solution; group 2 – positive control (pancreatitis). Three days after the onset of signs of developed acute pancreatitis, group 3 rats were intraperitoneally transplanted with native hUC-MMSCs, meeting the minimum criteria of the International Society for Cell and Gene Therapy, at a dose of 1.5×106 cells/kg. Group 4 received the same dose of hUC-MMSCs, but these cells were preconditioned with H2O2 at a concentration of 30 µM for 18 hours. The effects of both native and preconditioned hUC-MMSCs were evaluated at 3, 7, and 14 days after their introduction. Assessment criteria included the α-amylase index, identification of insulin in the islets of Langerhans, and histological analysis. Results. It was demonstrated that, three days post L-arginine administration, the α-amylase level surged by five times compared to the negative control, and blood glucose levels increased by 2.5 times, indicating damage to both the exocrine and endocrine parts of the pancreas. Microscopic examination revealed a 3.5-fold increase in fibrosis of the pancreatic parenchyma compared to the negative control. Three days after the transplantation of native hUC-MMSCs, the α-amylase level in the blood decreased by 1.7 times, and in the variant with preconditioned hUC-MMSCs, it decreased by 2 times compared to Group 2. By day 7, the α-amylase index in the native hUC-MMSCs group decreased by 2.4 times compared to the positive control, and in the preconditioned hUC-MMSCs group, it approached normal levels. A gradual recovery of the pancreatic architecture was observed by day 7, with a faster recovery in the preconditioned MMSCs group. Histological sections revealed a replacement of nuclei-free areas with acinocytes, indicated by a 3-fold decrease in the number of nuclei-free cells in the native hUC-MMSCs transplantation, and almost normal levels in the preconditioned hUC-MMSCs group. Both variants exhibited a positive histochemical PAS-reaction for the identification of insulin in the islets of Langerhans, and blood glucose levels corresponded to the norm. After 14 days, complete recovery of the pancreas was observed in both variants. Conclusion. The study results indicate that hUC-MMSCs transplantation in rats with acute pancreatitis contributes to the restoration of pancreatic parenchyma architecture and function. Moreover, the administration of MMSCs preconditioned with H2O2 significantly enhances the therapeutic effect, expediting the recovery process of the pancreas in animals.

Alteration of PBMC transcriptome profile after interaction with multipotent mesenchymal stromal cells under “physiological” hypoxia

Multipotent mesenchymal stromal cells (MSCs) have demonstrated a pronounced immunosuppressive activity, the manifestation of which depends on the microenvironmental factors, including O2 level.Here we examined the effects of MSCs on transcriptomic profile of allogeneic phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) after interaction at ambient (20%) or “physiological” hypoxia (5%) O2. As revealed with microarray analysis, PBMC transcriptome at 20% O2 was more affected, which was manifested as differential expression of more than 300 genes, whereas under 5% O2 220 genes were changed. Most of genes at 20% O2 were downregulated, while at hypoxia most of genes were upregulated. Altered gene patterns were only partly overlapped at different O2 levels. A set of altered genes at hypoxia only was of particular interest. According to Gene Ontology a part of above genes was responsible for adhesion, cell communication, and immune response. At both oxygen concentrations, MSCs demonstrated effective immunosuppression manifested as attenuation of T cell activation and proliferation as well as anti-inflammatory shift of cytokine profile.Thus, MSC-mediated immunosuppression is executed with greater efficacy at a “physiological” hypoxia, since the same result has been achieved through a change in the expression of a fewer genes in target PBMCs.

The sensitivity of multipotent mesenchymal stromal cells to short-term hypoxic stress in vitro depends on the efficiency of homotypic communication through gap junctions

Gap junctions (GJ) provide metabolic cooperation between cells through the direct exchange of cytoplasmic components. We analyzed the effect of short-term hypoxic stress on the efficiency of communication through the GJs in cultured multipotent mesenchymal stromal cells (MSCs) and characterized the sensitivity of MSCs to short-term hypoxic stress depending on the GJ function. Mitotically inactive MSCs were used in the experiments, in which the GJs were blocked with a specific inhibitor – carbenoxolone. The MSCs were continuously cultured at 20% O2. Further, MSCs with blocked and working GJs were subjected to hypoxic stress (0.1%, 24 hours). The efficiency of GJ communication was attenuated under hypoxic stress. The combined action of GJ inhibition and hypoxic stress was accompanied by an increase in ROS level as compared to the MSCs after hypoxic stress only. MSCs with blocked GJs were less sensitive to short-term hypoxic stress in comparison with MSCs integrated into the common network through working GJs. It was manifested in attenuation of hypoxia-induced angiogenic activity of MSCs. The angiogenic effects of conditioned medium from the MSCs with blocked GJs were almost twice less, which seems to be related to differences in the angiogenic mediators’ profiles: VEGF level decreased and FGF-2 level increased, while the monocyte chemoattractant protein 3 (MCP-3) level was unchanged. Thus, a decrease in the efficiency of direct MSCs- MSCs communication had a negative effect on mostly requested MSCs activity – the ability to induce angiogenesis. We conclude that blocking of GJ communication in MSCs is a negative event that impairs the coordination of MSCs’ response to the microenvironmental factors, in particular hypoxic stress, and reduces their functional plasticity.