Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

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.

EFFECTS OF BONE AUGMENTATION MATERIALS ON PECULIARITIES OF POSTOPERATIVE PERIOD IN PATIENTS AFTER IMPACTED THIRD MOLAR EXTRACTION AND THEIR CLINICAL EFFICIENCY

The article presents a comparative analysis of the clinical effectiveness of bone augmentation materials, in particular, compositions including multipotent mesenchymal stromal cells of adipose tissue and platelet-enriched plasma, used to fill bone defects during the operation of third molars extraction. The criteria for the effectiveness of treatment at the stage of the early postoperative period in the participants included the assessment of the pain intensity, collateral oedema and hyperaemia of the oral mucosa after the surgery. The aim of the study is to determine the clinical efficiency of osteoplastic materials and to determine the feasibility of using tissue equivalents of bone tissue based on multipotent mesenchymal stromal cells of adipose tissue for healing of bone defects in patients with impacted third molars. Methods and participants. The study was conducted on the basis of the Department of Surgical Dentistry and Maxillofacial Surgery of Bukovynian State Medical University, Chernivtsi, Ukraine. Extraction of impacted third molars surgery was performed on 72 patients. After the tooth extraction, 31.94% of them underwent bone augmentation by osteoplastic material “Colapan–L” (group A); 41.67% of patients had bone augmentation with a combination of multipotent mesenchymal stromal cells of adipose tissue+”Colapan–L”+platelet-rich plasma (group B), and in the rest, 26.39% of patients, wound healing occurred under a blood clot (group C). Postoperative pain syndrome was assessed using the Numerical Rating Scale (NRS) based on patients' subjective feeling of pain. Visual assessment of the severity of collateral oedema and hyperaemia of the oral mucosa after surgery was also performed. A scoring system was used to determine the severity of collateral oedema. To assess the state of the postoperative period in patients of all study groups, a protocol was completed daily during the hospital stay period that reflected the most important data of an objective and subjective nature. During the procedure of morning dressings, we analyzed patients' complaints and evaluated general and local status: presence of appetite, quality of sleep, wound pain, postoperative oedema, hematoma, and hyperaemia of the oral mucosa, presence of secretions from the wound, body temperature, and type of wound healing. The study has demonstrated that at the final stage of postoperative follow-up the 89.31% of patients who received a combination of “Colapan–L” with multipotent mesenchymal stromal cells of adipose tissue and platelet-rich plasma reported no pain that is 1.2 times p1<0.05 and 1.3 times p2<0.05 higher than in the patients of groups A and B. Collateral oedema was absent in 98.47% of the group B patients that exceeded 1.2 times p1<0.05 the number of such individuals in group A, where the bone defect was augmented with the osteoplastic material “Colapan–L”, and in 1.4 times higher, p1<0.01, p1>0.05 that the indicators of group B, where the healing of the bone defect developed without osteoplastic materials. The absence of hyperaemia of the oral mucosa was found in 92.37% of the patients in groups B, which was 1.3 and 1.4 times higher than the values obtained in groups A and B, p1<0.05, p2<0.01.

Mesenchymal stromal cell apoptosis is required for their therapeutic function

Multipotent mesenchymal stromal cells (MSCs) ameliorate a wide range of diseases in preclinical models, but the lack of clarity around their mechanisms of action has impeded their clinical utility. The therapeutic effects of MSCs are often attributed to bioactive molecules secreted by viable MSCs. However, we found that MSCs underwent apoptosis in the lung after intravenous administration, even in the absence of host cytotoxic or alloreactive cells. Deletion of the apoptotic effectors BAK and BAX prevented MSC death and attenuated their immunosuppressive effects in disease models used to define MSC potency. Mechanistically, apoptosis of MSCs and their efferocytosis induced changes in metabolic and inflammatory pathways in alveolar macrophages to effect immunosuppression and reduce disease severity. Our data reveal a mode of action whereby the host response to dying MSCs is key to their therapeutic effects; findings that have broad implications for the effective translation of cell-based therapies.

Dynamics of Cytokines Concentration in the Blood Plasma of Patients after Multipotent Mesenchymal Stromal Cells Administration for Graft Versus Host Disease Prevention

Introduction Despite the large number of clinical studies on the use of multipotent mesenchymal stromal cells (MSCs) for the treatment and prevention of graft-versus-host disease (GVHD), the mechanism of their action in the organism is not well understood. The known data refer either to clinical effects or obtained in vitro. Due to the immunomodulatory effect of MSCs in the body, subpopulations of T cells and the concentration of cytokines involved in the immune response can change. The role of T cells and certain cytokines (TNF alpha, IL6, IL8, etc.) in the pathophysiology of GVHD is described. The aim of this investigation was to study the composition of T cells subpopulations and the concentration of cytokines in the peripheral blood of patients who received MSCs for GVHD prophylaxis. Methods The study included 21 patients who received hematopoietic stem cells donor's derived MSCs for the prevention of GVHD as part of the ClinicalTrials.gov Identifier NCT01941394 trial. The control group included 16 patients who did not receive MSCs. After signing informed consent, blood samples were taken from all patients during routine examinations on the day of restoration of the number of leukocytes to 1000 in μl (day 0), after 3 and after 30 days. MSCs were injected on day 0. None of the patients developed GVHD during this time. To analyze plasma cytokines and chemokines, the Bio-Plex Pro Human Cytokine Panel kit, 27-Plex (BioRad) was used, to determine the concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, bFGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-bb, RANTES, TNF-α, VEGF according to manufacturer's recommendations. Forward and side scattering parameters determined the peripheral blood lymphocytes population and then CD4+ or CD8+ lymphocytes were gated. For each of this population the composition of memory cells subset were determined by flow cytometry. Results Significant differences were found between the 2 groups only on day 30 in the concentration of IL8 (17.0±3.2 pg/ml in the control group versus 32.8±4.1 pg/ml in the MSC group, p&lt;0.0001). It has been shown that 30 days after MSCs, the number of CD4+ T cells in the peripheral blood of patients significantly increases compared to the group without MSCs (CD4 CM 38.9±9.0 vs 22.4±7.8, CD4 TM 97.0±32.5 vs 91.0±43.2, CD4 TE 5.0±1.9 vs 1.4±1.0, CD4 EM 34.6±20.1 vs 19.9±7.9, CD4CD25+ 27.6±6.6 vs 12.9±5.0). These cells produce IL8, which play an important role in immune cell homeostasis by activating antimicrobial neutrophils. Without the introduction of MSCs, the concentration of this protective against GVHD cytokine practically did not change within a month, whereas after the introduction of MSCs, it gradually increased almost 2 times. However, the dynamics of changes in the levels of the studied cytokines differed greatly between the 2 groups (Table). The concentration of IP10, which is involved in the development of GVHD, increased significantly faster and stronger in the group without MSCs. An increase in the concentration of other investigated cytokines associated with the activation of macrophages (MCP-1, MIP-1a, MIP-1b) did not depend on the MSCs administration. At the same time, in the MSC group, the concentration of the growth factor PDGF-bb necessary for the HSC proliferation increased significantly more actively. The IL9 concentration on day 0 was comparable to the level in healthy donors, and then gradually increased in both groups and after 30 days it was significantly higher than on day 0 in the MSC administration group. The concentration of G-CSF changed in a similar way. Conclusion Changes in the dynamics of T cells subpopulations and the concentration of cytokines in the blood after MSCs administration contribute to a faster recovery of patients after allogeneic bone marrow transplantation. The work were supported by the Russian Foundation for Basic Research, Project No. 19-29-04023. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions. We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes. The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p&lt;0.05 when Student's unpaired t-test or Mann-Whitney test was applied. MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA. Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun. Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression. The work is supported by the RFBR, project 19-015-00280. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Activation of reparative liver regeneration following the combined transplantation of multipotent mesenchymal stromal cells and hepatic stellate cells

Aim. To study the effect of combined transplantation of multipotent mesenchymal stromal and hepatic stellate cells on the reparative liver regeneration. Methods. Laboratory mice were given intravenous administration of multipotent mesenchymal stromal and hepatic stellate cells after partial hepatectomy. The mice were divided into four groups: control, experimental 1 (injection of multipotent mesenchymal stromal cells), experimental 2 (co-transplantation of multipotent mesenchymal stromal cells and hepatic stellate cells), the comparison group. Comparison of the experimental groups with the control group and the comparison group was carried out. Each group consisted of 14 animals. The control and experimental groups underwent partial hepatectomy. The experimental mice were injected with the cells into the lateral tail vein 1 hour after the operation. Multipotent mesenchymal stromal cells were administered at a dose of 4 million cells/kg (120 thousand cells/mouse), hepatic stellate cells in the amount of 9 million cells/kg (270 thousand cells/mouse), suspended in 0.2 ml 0.9% NaCl solution. The control group animals were injected with 0.2 ml 0.9% NaCl solution into the lateral tail vein. The comparison group consisted of mice without partial hepatectomy, injected with 0.2 ml 0.9% NaCl solution. To assess reparative regeneration of the liver, morphometric parameters of the liver, blood biochemical parameters on the 3rd and 7th days after partial hepatectomy were studied. The severity of apoptosis was assessed by the immunohistochemical method, the activity of deoxyribonucleic acid (DNA) repair enzymes of the poly (ADP-ribose) polymerases was determined by flow cytometry. The number of micronucleated hepatocytes was also determined. The hepatocyte growth factor (HGF) content was measured by using an enzyme-linked immunosorbent assay in serum. The significance of differences in the compared samples was determined by using the Student's t-test. Statistical data processing was performed by using the SPSS Statistics software version 17.0. Results. It was found that the combined transplantation of multipotent mesenchymal stromal and stellate liver cells causes restoration of the activity of alanine aminotransferase (a decrease of 30.3%, p=0.016), aspartate aminotransferase (a decrease of 27.7%, p=0.021), alkaline phosphatase (a decrease of 21.1%, p=0.036), an increase in the protein synthetic function of the liver (increase in albumin level by 36.6%, p=0.009), an increase in hepatocyte growth factor level by 74.3%. These changes were accompanied by the restoration of liver morphometric parameters: there was an increase in the mitotic activity of hepatocytes by 28.7% (p=0.008), the nuclear area of hepatocytes by 26.7% (p=0.006), the number of binucleated hepatocytes by 26.1% (p=0.004), which led to the restoration of liver mass. There was a decrease in the level of apoptosis by 28.8% (p=0.006) and a decrease in the number of micronucleated hepatocytes by 22.7% (p=0.001) compared with the control group, which may be related to an increase in the activity of Poly (ADP-ribose) polymerase repair enzymes detected in the study. The deviations were presented as a difference relative to the indicators of the control group (operated animals that were injected with 0.9% NaCl solution). Conclusion. Combined transplantation of multipotent mesenchymal stromal and hepatic stellate cells activates reparative liver regeneration after partial hepatectomy.Keywords: multipotent mesenchymal stromal cells, MSC, hepatic stellate cells, HSC, liver regeneration, partial hepatectomy.