Multipotent Mesenchymal Stromal Cells Cultures Research Articles

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.

Environmental oxygen affects ex vivo growth and proliferation of mesenchymal progenitors by modulating mitogen-activated protein kinase and mammalian target of rapamycin signaling

Background aimsStem and progenitor cells of hematopoietic and mesenchymal lineages reside in the bone marrow under low oxygen (O2) saturation. O2 levels used in ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) affect proliferation, metabolism and differentiation. MethodsUsing cell-based assays and transcriptome and proteome data, the authors compared MSC cultures simultaneously grown under a conventional 19.95% O2 atmosphere or at 5% O2. ResultsIn 5% O2, MSCs showed better proliferation and higher self-renewal ability, most probably sustained by enhanced signaling activity of mitogen-activated protein kinase and mammalian target of rapamycin pathways. Non-oxidative glycolysis-based energy metabolism supported growth and proliferation in 5% O2 cultures, whereas MSCs grown under 19.95% O2 also utilized oxidative phosphorylation. Cytoprotection mechanisms used by cells under 5% O2 differed from 19.95% O2 suggesting differences in the triggers of cell stress between these two O2 conditions. ConclusionsBased on the potential benefits for the growth and metabolism of MSCs, the authors propose the use of 5% O2 for MSC culture.

Immunomodulatory effect of cell therapy on the experimental inflammatory bowel disease model

Dysregulation of innate and adaptive immunity is a central mechanism in the pathogenesis of inflammatory bowel diseases (IBD) that include Crohn’s disease (CD) and ulcerative colitis (UC). Given the immunomodulatory properties of multipotent mesenchymal stromal cells (MMSCs), developing new pathogenetic approaches to the IBD treatment using cell therapy seems to be promising.In this article, the effect of intra-arterial and intravenous MMSCs on in vitro monocytes/macrophages cytokine-producing function and splenocytes proliferative activity in laboratory animals with experimental IBD under the conditions of nonspecific and specific stimulation (mannan – cell wall component of Saccharomyces cerevisiae yeast) was studied.MMSC cultures have been shown to possess an immunomodulatory effect against the background of improved clinical, morphometric parameters, and the pathomorphological picture of experimental IBD damage. Intra-arterial and intravenous administration of cell cultures decreased the mannan-induced TNF-α production by CD68+ cells and mitogen/mannan-stimulated splenocyte proliferation that confirms the immunosuppressive effect of MMSC cultures on autoreactive splenocyte clones in experimental IBD. The obtained results testify to the anti-inflammatory effect of cell therapy in the experimental modeling of IBD and justified a combined administration of MMSC cultures.

Towards the standardization of methods of tissue processing for the isolation of mesenchymal stromal cells for clinical use

Multipotent mesenchymal stromal cells (MSCs) are currently the most extensively studied type of adult stem cells in advanced stages of development in the field of regenerative medicine. The biological properties of MSCs have generated great hope for their therapeutic use in degenerative and autoimmune conditions that, at present, lack effective treatment options. Over the last decades, MSCs have been typically obtained from adult bone marrow, but the extraction process is highly invasive and the quality and numbers of isolated cells is drastically influenced by patient age, medication and associated comorbidities. Therefore, there is currently an open discussion on the convenience of allogeneic over autologous treatments, despite potential disadvantages such as rejection by the host. This shift to the allogeneic setting entails the need for high production of MSCs to ensure availability of sufficient cell numbers for transplantation, and therefore making the search for alternative tissue sources of highly proliferative MSC cultures with low levels of senescence occurrence, which is one of the greatest current challenges in the scale up of therapeutic cell bioprocessing. Herein we (i) present the main isolation protocols of MSCs from bone marrow, adipose tissue and Wharton’s jelly of the umbilical cord; and (ii) compare their qualities from a bioprocess standpoint, addressing both quality and regulatory aspects, in view of their anticipated clinical use.

Выбор эффективной невирусной системы доставки siPHK в культуры мультипотентных мезенхимальных стромальных клеток

Multipotent mesenchymal stromal cells (MSCs) can be used as a model for the development of gene and cell technologies and as a means of delivering nucleic acids to the body, including as part of tissue-engineering constructs. Small interfering RNA (siRNA) molecules acting by the RNA interference mechanism are a high-precision tool for genetic silencing of target mRNA transcripts. The search for low toxic and highly efficient transfection agents for delivery of siRNA or other nucleic acids to MSCs is an urgent task for the development of therapy based on these molecules. A comparative evaluation of five transfection agents showed that compounds based on cationic polymers were more efficient in delivering siRNA molecules than liposomes, while the cytotoxicity of all tested reagents was independent of their chemical structure. For two of the three transfection agents selected according to their efficiency and belonging to different classes, TurboFect and Lipofectamine® 3000, a moderate effect on cell viability was revealed. The results obtained allow us to recommend TurboFect and Lipofectamine® 3000 as highly efficient and relatively low-toxic agents for transfection of MSCs cultures. multipotent mesenchymal stromal cells, siRNA, transfection, lipofection, cationic lipids, cationic polymers, polyethyleneimine. The authors are grateful to staff of functional genomics laboratory of Research Centre for Medical Genetics Skoblova M.Yu. and Krivosheeva I.A., for technical and methodological assistance. The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation within the state assignment for Research Centre for Medical Genetics.

PB1852 PROLIFERATION CAPACITY OF STROMAL PRECURSORS FROM THE BONE MARROW OF UNTREATED APLASTIC ANEMIA PATIENTS

Background: Aplastic anemia (AA) is a rare bone marrow (BM) failure disorder, characterized by pancytopenia due to hematopoietic stem cells damage. First line treatment for AA consists of immunosuppressive drugs (anti-thymocyte globulin combined with cyclosporin A). Allogenic hematopoietic stem cell transplantation (allo-HSCT) is preferable for the patients of young age having HLA-matched BM donor. Aims: The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in the debut of AA. The changes caused by allo-HSCT would also be evaluated. Methods: The study included 12 patients in the debut of AA. Two of them later had been transplanted with allogenic hematopoietic stem cells and had also been analyzed for 1 year after the procedure. In all patients BM was aspirated after informed consent. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. The relative expression level of various genes in MMSC was determined by Taqman real-time PCR. As a control 33 MMSC samples from the BM of healthy donors of according age were used. Results: Concentration of CFU-F in the BM of AA patients in debut was higher than in donor BM, although the difference was not significant (56.8 ± 13.2 versus 29.6 ± 6.5 per 106 mononuclear BM cells in donors). The square of CFU-F colonies from AA patients was significantly larger than donor ones (27.6 ± 1.8 points versus 16.65 ± 1 in donors, p < 0.05). Analysis of proliferative potential of individual CFU-F colonies was performed for 4 newly diagnosed patients by far. CFU-F colonies from the two patients were not able to proliferate after the passage, from another two patients, on the contrary, CFU-F colonies demonstrated high proliferative potential. When compared with CFU-F colonies from 6 analyzed donors, CFU-F colonies from AA patients started to proliferate earlier, and the total clones’ number was higher. Individual CFU-F colonies both from donors and AA patients varied in their proliferation and differentiation potential. MMSC cultures were successfully obtained from the BM of 10 out of 12 newly diagnosed AA patients. The cumulative MMSC production on passages 0, 1 and 2 was significantly higher than in donor cultures. MMSC of two patients had been also analyzed for 1 year after the allo-HSCT. All values were compared with 95% CI of corresponding donor parameters. CFU-F concentration 1 month after allo-HSCT was higher than in donors. This makes a sharp contrast with the data on CFU-F concentration in patients with acute leukemia after allo-HSCT, where the dramatic decrease was observed. The cumulative MMSC production was not changed in AA patients. However, we observed a few changes in gene expression, typical for both patients and persisting for a 1 year at least. The expression of CFH, PTGES, CSF1, TGFB1, and TGFB2 was deeply downregulated; JAG1, FGF2 and IL6 were downregulated right after the allo-HSCT, but became overexpressed 1 year later. Summary/Conclusion: Stromal precursors in newly diagnosed AA patients are functionally activated and have higher proliferative capacity in comparison with donors. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the inhibited hematopoiesis. It should be scrutinized further. This work was supported by the Russian Foundation for Basic Research, projects no. 19-015-00280 and 12-04-00457.

Growth Factor-Mediated Tenogenic Induction of Multipotent Mesenchymal Stromal Cells Is Altered by the Microenvironment of Tendon Matrix

Age-related degenerative changes in tendon tissue represent a common cause for acute tendon pathologies. Although the regenerative potential of multipotent mesenchymal stromal cells (MSC) was reported to restore functionality in injured tendon tissue, cellular mechanisms of action remain partly unclear. Potential tenogenic differentiation of applied MSC is affected by various intrinsic and extrinsic factors. The current study presents an in vitro model to evaluate the combined extrinsic effects of decellularized equine tendon matrix, transforming growth factor beta 3 (TGFβ3) and bone morphogenetic protein 12 (BMP12) on the tenogenic fate of equine adipose tissue-derived MSC. Monolayer MSC cultures supplemented with TGFβ3 and BMP12 as well as MSC cultured on tendon matrix scaffolds preloaded with the growth factors were incubated for 3 and 5 days. Histological evaluation and real time reverse transcription polymerase chain reaction (RT-PCR) revealed that growth factor-mediated tenogenic induction of MSC was modified by the conditions of the surrounding microenvironment. While the gene expression pattern in monolayer cultures supplemented with TGFβ3 or TGFβ3 and BMP12 revealed an upregulation for collagen 1A2, collagen 3A1, tenascin c, scleraxis and mohawk (p < 0.05), the presence of tendon matrix led to an upregulation of decorin and osteopontin as well as to a downregulation of smad8 (p < 0.05). Preloading of scaffolds with either TGFβ3, or with TGFβ3 and BMP12 promoted a tenocyte-like phenotype and improved cell alignment. Furthermore, gene expression in scaffold culture was modulated by TGFβ3 and/or BMP12, with downregulation of collagen 1A2, collagen 3A1, decorin, scleraxis, smad8 and osteopontin, whereas gene expression of tenascin c was increased. This study shows that growth factor-induced tenogenic differentiation of equine MSC is markedly altered by topographical constraints of decellularized tendon tissue in vitro. While TGFβ3 represents an effective mediator for tenogenic induction, the role of BMP12 in tenogenesis may be of modulatory character and needs further evaluation.

Certain biological properties of multipotent mesenchymal stromal cells from bone marrow and adipose tissue of FVB/N mice

Multipotent mesenchymal stromal cells (MMSCs) are used for cell therapy of lesions of various genesis. The most widely used MMSCs are from two tissue sources: bone marrow and adipose tissue.The purpose of the work was to conduct a comparative assessment of the biological properties of murine bone marrow-derived and adipose tissue-derived MMSCs.Methods. The culture of MMSCs was obtained from the bone marrow and adipose tissue of 6 months-old male FVB/N mice according to standard protocols. We performed phenotyping, directed osteogenic and adipogenic differentiation, analysis of immunomodulatory properties in vitro of obtained cell cultures.Results. The cultured MMSCs from bone marrow and adipose tissue express the typical stromal markers (CD44, CD73, CD90 and Sca-1). A distinctive feature of bone marrow cells cultures of the 2nd passage was the high level of the hematopoietic markers CD45 and CD117 expression. MMSCs from both tissue sources are capable of differentiation in the osteogenic and adipogenic directions. At the same time, there were differences in the differentiation in the osteogenic direction – adipose tissue-derived MMSCs had a lower osteogenic potential. MMSCs exhibit inhibitory effect on mitogen-induced proliferation of splenocytes in vitro, expression of which does not depend on tissue origin of the MMSCs with significant inhibition of mitogen-induced proliferation of splenocytes at addition of high doses of MMSCs.Conclusions. MMSCs of bone marrow and adipose tissue express a similar level of surface markers that are characteristic of cells with multipotent properties. They are capable to differentiating in osteo- and adipogenic direction with differences in the degree of mineralization of the extracellular matrix and exhibit immunomodulatory effects in vitro, regardless of tissue origin.

Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P < 0.05), with lower percentages of CD90+ cells found in equine MSC samples cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29+ (98.60-99.90%), CD73+ (94.60-98.40%), CD90+ (99.60-99.90%), and CD105+ (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species-specific, indicating aneed for serum-free media to be optimized for MSCs from relevant animal species. © 2017 International Society for Advancement of Cytometry.

The properties of multipotent mesenchymal stromal cells samples vary depending on their ability to induce HLA-DR expression on T-cells co-cultured with them

Interactions between lymphocytes and multipotent mesenchymal stromal cells (MSCs) in vitro sometimes increase HLA-DR expression on T-cells. On lymphocytes derived from one donor the elevation of HLA-DR was observed after co-cultivation with part of MSCs samples (group A), on the others the HLA-DR expression level did not change (group B). Study of T-cell subpopulations after interactions with MSCs could explain ineffectiveness of some MSCs, as an immunomodulating agent in clinical applications. The aim of the study was to discriminate variations in T-cell subpopulations, co-cultured with MSCs from groups A and B. MSCs were isolated from bone marrow of 13 donors for allogeneic hematopoietic cells and cultured by a standard method. MSCs were seeded 100000 cells per flask, and then 1000000 allogeneic lymphocytes from single donor were added to all MSCs cultures. Lymphocytes and MSCs were immunophenotyped separately during 4 days of co-cultivation. The expression of HLA-DR, activation markers CD25, CD38, CD69, HLA-DR and PD-1 and the distribution of naïve and effector T-cells were studied by flow cytometry. Expression of HLA-DR on lymphocytes after 4 days of cultivation without MSCs did not change compared to 1st day. HLA-DR expression on lymphocytes co-cultured with MSCs from group A was 3 times greater than from group B. In lymphocytes co-cultured with MSCs there were higher number of naïve cells compared to control, p < 0.001. Group B showed lower number of EM and TM cells. Differences between groups were more pronounced when lymphocytes were activated. In group B proportion of HLA-DR CD4+ and CD8+ cells was significantly lower, compared to group A and control samples. At the same time the number of CM and PD-1+ CD4+ cells was lower in group A, but number of TE was increased. These data indicate that MSCs from group A became more immunogenic after interaction with lymphocytes and could not show immunomodulating properties in the same way as MSCs from group B. Data obtained can explain why administration of MSCs is not always successful. Preliminary study of MSCs prior to their administration may be used to predict their efficiency in the future.

Effects of autologous gingiva-derived cells with myogenic potential on regeneration of skeletal muscle

In our recent studies we found for the first time the ability of human multipotent mesenchymal stromal cells (MSCs) derived from alveolar gingiva (alveolar mucosa) to differentiate into myogenic direction. The aim of the present study was to evaluate the effects of autologous gingiva-derived MSCs with myogenic potential on the regeneration of muscular tissue after mechanical damage. The study was conducted on 11 male rabbits. Biopsy of alveolar gingiva was performed at each animal before experiment for autologous MSCs obtainment. Cultures of MSCs were induced in vitro into myogenic direction. To model the damage, the medial heads of the gastrocnemius muscles were intersected on both pelvic limbs of the rabbit. Injection of autologous MSCs was performed on the seventh day after injury into the damaged muscle of one of the extremities, while equal volume of saline (control) was injected into the muscle of the contralateral limb. The animals were sacrificed on 0, 21, and 35 days after the administration of cells. MSCs transplantation led to significant reduction of the area of muscle damage. Immunohistochemical analysis revealed earlier increase in the proportion of MyoD- and myogenin-positive cells, as well as decrease in the expression of Ki-67 in damaged tissue, in experimental group compared to the control. Autologous cells did not significantly affect the composition of muscle fibers. Significant decrease in the proportion of fibrous tissue was also observed in the experimental group. The results indicate the effectiveness of autologous alveolar gingiva-derived MSCs for treatment of mechanical damage of muscle tissue. Local administration of cells accelerated reparative regeneration and prevented fibrosis.

Alterations of the Bone Marrow Stromal Microenvironment in Adult Patients with Leukemia before and after the Treatment

BackgroundBone marrow (BM) microenvironment is involved in the initiation and propagation of normal hematopoiesis as well as hematological diseases. Leukemia and high dose chemotherapy affect both hematopoietic and stromal precursor cells. Multipotent mesenchymal stromal cells (MMSCs) are the essential element of both healthy and leukemic hematopoietic microenvironment.AimsTo investigate two types of stromal precursor cells, MMSCs and their more differentiated progeny CFU-Fs, derived from the BM of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) patients before and after therapy.Methods74 newly diagnosed cases (33 AML, 21 ALL, 20 CML) were involved in the study after informed consent. BM was aspirated prior to any treatment (time-point 0) and at days 37, 100 and 180 since the beginning of treatment of acute leukemia (AL) and +3, +6 and +12 months for CML (time-points 1-3). MMSCs were cultured in aMEM with 10% fetal calf serum. Time to P0 and cumulative MMSC production after 3 passages were evaluated. CFU-F concentration was analyzed in standard conditions.ResultsAt the time of the diagnosis the percentage of blast cells in the bone marrow of patients with AML was 22-86,6% (median 64%). After the induction therapy complete remission (CR) was achieved in 58% of patients. Patients who didn't achieve CR were switched to the alternative chemotherapy regimens. The percentage of blast cells in the bone marrow of patients with ALL at the diagnosis was 68-97% (median 88%). After the induction therapy complete remission (CR) was achieved in 90% of patients.Time needed to reach P0 reflects the quantity of MMSC in the BM sample. The time to P0 in control group was 13.7 ± 0.3 days. The elongation of time to P0 in AL MMSC cultures at the time of the diagnosis (Table 1) suggested the reduction of MMSC number or their decreased proliferative potential due to leukemic expansion. After the induction therapy time to P0 reached the normal level, but subsequently lengthened during the consolidation therapy and before the maintenance therapy, that can be explained by the chemotherapy influence. In CML MMSC cultures time to P0 was also significantly longer during the whole observation period due to the continuous therapy and maintaining disease.Cumulative MMSC production in control group was 7.1 ± 1 x 106 cells. In patients with AML it was 1/3 of the donor’s at the time of the diagnosis with no difference at time-points 1, 2 and 3, indicating the impaired proliferative abilities of MMSC at the AML manifestation due to the disease aggressiveness or the patient’s elder age. Cumulative MMSC production in patients with ALL and CML didn’t differ from donor’s. BM blast count did not correlate with MMSC production. Among patients with AL, who didn’t achieve CR, the time to P0 and total MMSC production did not differ significantly from that of the patients in remission.CFU-F concentration in the BM of AL patients was significantly lower (almost halved) than in donors (25.4 ± 3.1 per 106 BM cells) at the time-point 0 with no difference at time-points 1, 2 and 3. CFU-F concentration in the BM of CML patients was also nearly 40% lower than in control group at the time-point 0 with its following restoration at time-point 1 and subsequent drop at next time-points (up to 5 fold lower) at time-point 3) (Table 1), reflecting the long-lasting lesion of these group of precursors during the course of the disease. There were no correlations between BM blast count and CFU-F concentration in all nosologies studied.ConclusionThe study supports the major influence of leukemic cells and chemotherapy on the BM microenvironment. The two types of studied precursors are affected differently. Future studies are needed to evaluate the role of MMSCs in leukemia pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immune Privileged Features of Multipotent Mesenchymal Stromal Cells Are Lost after Co-Cultivation with Allogeneic Lymphocytes in Vitro

Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed.The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics.Materials and methodsMSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2.After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA).Data are presented as mean ± standard error. Statistical analysis was performed using Student’s t-test (considered reliable p <0.05).ResultsThe number of cells in cultures of MSCs and IFN-g-MSCs did not differ significantly during the 4 days of observations. The presence of non-activated lymphocytes had no effect on the parameters of growth and viability of MSCs. Co-cultivation of activated lymphocytes with MSCs resulted in a reduction of viable MSCs, to 51.6 ± 5.5% compared with IFN-g-MSCs (68,1 ± 6,2%, p = 0.02). The viability of MSCs cultures without lymphocytes was 74.5% ± 4.8 on day 4 from the beginning of the experiment.HLA-DR expression level gradually increased in IFN-g-MSCs (see table). Co-culturing MSCs with lymphocytes also leads to a gradual increase in HLA-DR expression on MSCs apparently due to IFN-g, produced by lymphocytes. However, in IFN-g-MSCs co-cultured with lymphocytes HLA-DR MFI was significantly lower (p = 0.05) than without lymphocytes. HLA-DR MFI increased about 10 times in MSCs co-cultured with activated lymphocytes regardless of their IFN-g pretreatmentThe data obtained indicate that IFN-g-MSCs did not change in their growth characteristics and had increased immunomodulating properties. IFN-g-MSCs have a greater resistance to activated lymphocytes, which makes them more effective for cell therapy. The results suggest that injected to patient MSCs could have increased level of HLA-DR expression regardless of their initial treatment with IFN-g. The increase of HLA-DR expression on MSCs co-cultured with lymphocytes indicates a possibility of loss of immune privilege by these cells when injected to patient. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Morphological and functional characteristics of cryopreserved multipotent mesenchymal stromal cells from bone marrow, adipose tissue and tendons

The aim of study was to comparatively evaluate the morphological and functional properties of cryopreserved multipotent mesenchymal stromal cells (MMSCs) from bone marrow, fat and tendon.Materials and methods. MMSC cultures obtained from rat bone marrow, fat and tendon. The cells was cryopreserved under protection of 10 % DMSO and 20% FBS with cooling rate of 1 deg/min down to -80°C followed by plunging into liquid nitrogen. In the studied cultures the membrane integrity, immunophenotype, ability to colony formation, proliferative characteristics (MTT-test), directed differentiation and type I collagen synthesis were evaluated.Results. Investigated cryopreserved cell culture derived from bone marrow, tendon and adipose tissue had high membrane integrity indicators, colony formation and proliferation as well as the ability to directional adipogenic and chondrogenic differentiation. The analysis of immunophenotype showed that the tested cryopreserved MMSCs culture characterized by high levels of expression (≥90 %) of CD44, CD90, CD105, CD73 and low expression (≤1 %) of hematopoietic marker CD45. Cryopreserved bone marrow MMSCs were characterized by a high content of cells that synthesized type I collagen as compared to cultures which were derived from fat and tendon.Conclusions. Cell cultures derived from all studied sources have immunophenotype of precursor cells of mesenchymal origin. The MMSC of tendon tissue characterized by a greater capacity for colony formation and proliferation, and lower capacity for directed adipogenic differentiation, than MSCs from bone marrow and adipose tissue.

404. Development of Human Artificial Chromosomes for Gene Cell Therapy of Muscular Dystrophies

Muscular dystrophies are clinically heterogeneous group of muscle diseases related to different genes defects. The results of defects impair myogenic differentiation and histophysiology of skeletal muscles. One of major forms of disease – limb-girdle muscular dystrophy 2B (LGMD2B), caused by dysferlin gene mutations. Both big size of gene and wide variety of mutations complicate development of genetically engineered drugs. Human artificial chromosomes (HAC) could be used as alternative approach. This vector can contain unlimitedly large DNA region for transfer, is retained during cell division, and does not have carcinogenic and immunogenic properties.HAC matrix was developed earlier by Larionov V. et al. (http://www.ncbi.nlm.nih.gov/pubmed/22123967). Optimized DYSF gene sequence (optDYSF) from pAd/CMV/V5-DEST vector (kindly provided by A.A. Rizvanov) was transferred into backbone vector also containing loxP sites for further transfer into HAC. Transfer correctness confirmed by sequencing. After that Chinese hamster ovary cells carrying HAC were transfected by backbone vector containing optDYSF. Selection performed in HAT medium. 7 clones were selected and analyzed at day 10. Expression of optDYSF observed in all obtained clones. Episomal HAC localization determined using fluorescent hybridization in sity by colocalization of label at optDYSF and a-satellite DNA. Episomal localization of HAC determined only in 2 clones from 7. Dysferlin production observed in all clones.On other hand described vector requires somatic myogenic vector cell. Human myoblasts could be successfully used for this purpose. The major source of myoblasts – skeletal muscle tissue is not optimal due to limited proliferative potential, especially in patients with muscular dystrophies. Our team revealed presence of myoblasts in gingival mucosa specimens and developed new method of myoblasts obtainment from it. Primary cultures of multipotent mesenchymal stromal cells (MSC) were derived from gingiva biopsy samples. The cells expressed typical mesenchymal stem cells markers and had ability for 3-way differentiation. Myogenic induction of gingiva derived MSC in culture was demonstrated. Gingiva derived MSC are capable to express markers of myogenic differentiation (skeletal actin, sceletal myosin, MyoD1) and form multicellular elongated fibers – myotubes. Ability of gingiva derived MSC cultures to differentiate in myogenic direction was preserved during up to 10th passage. Such features make this cell source attractive for using in treatment of inherited muscular degenerative diseases.In our future research we are planning to transfer HAC containing optDYSF gene into clonogenic gingiva derived MSC using microcell-mediated chromosome transfer. This method could be used for further development of gene cell therapy of LGMD2B.

Post-thaw non-cultured and post-thaw cultured equine cord blood mesenchymal stromal cells equally suppress lymphocyte proliferation in vitro.

Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001). Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13). These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies.

Evaluation of ultrasmall superparamagnetic iron oxide contrast agent labeling of equine cord blood and bone marrow mesenchymal stromal cells.

To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for 5 passages and assessed for viability and proliferative capacity, labeling efficacy, and labeled cell proportion. For MRI detection, 5 × 10(6) labeled BM MSCs from passage 1 or 2 were injected into a collagenase-induced superficial digital flexor tendon defect of an equine cadaveric forelimb from 2 horses. For passages 1, 2, and 3, labeling efficacy and cell proportion for UCB MSCs (99.6% [range, 98.8% to 99.9%], 16.6% [range, 6.5% to 36.1%], and 1.0% [range, 0.4% to 2.8%], respectively) were significantly higher than for BM MSCs (99.2% [range, 97.8% to 99.7%], 4.5% [range, 1.6% to 11.8%], and 0.2% [range, 0.1% to 0.6%], respectively). Labeling was not detectable after passage 3. Viability of MSCs was not affected, but cell doubling time increased in labeled MSCs, compared with that of unlabeled MSCs. On MRI 3-D T2*-weighted fast gradient echo sequences, decreased signal intensity was observed for BM passage 1 MSCs. Equine UCB and BM MSCs were labeled with SPIO at high efficiencies.

Toll like receptor 3 & 4 responses of human turbinate derived mesenchymal stem cells: stimulation by double stranded RNA and lipopolysaccharide.

Background and objectivesMultipotent mesenchymal stromal cells (MSCs) represent a promising cell-based therapy for a number of inflammatory or autoimmune diseases. Herein, Toll like receptor (TLR) expression by MSCs and their immune regulatory roles are investigated. In this study, we investigated the influence of TLR on the immune response, proliferation, and differentiation potential of human turbinated MSC (hTMSC) cultures in vitro.Subjects and MethodsAfter isolating hTMSCs from discarded inferior turbinate tissue, FACS analysis was used to assess the expression of TLRs such as TLR2, TLR3, TLR4, and TLR5 in hTMSCs and cell proliferation was assessed using a cell counting kit (CCK)-8. Cytokine and chemokine secretions were analyzed with multiplex immunoassays for IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-a, GM-CSF, and IFN-γ. The differentiation potential of hTMSCs was evaluated in the osteogenic, chondogenic, and adipogeinc media and analyzed by histology and gene expression related to differentiation.ResultsFACS analysis revealed that TLR3 and TLR4 expression consisted of a relatively high percentage of the surface proteins expressed by hTMSCs. The proliferation of hTMSCs was influenced and significantly increased by the presence of TLR4 agonists. In particular, hTMSCs produced a set of cytokines and chemokines and the expression of IL-6, IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, and GM-CSF were up-regulated in response to the TLR4 agonist LPS. The osteogenic and adipogeinc differentiation potential of hTMSCs was not affected by TLR agonists.ConclusionsWe conclude that TLR4 stimulation affects TLR expression, proliferation, and the immunomodulation potential of hTMSCs. Understanding the mechanism behind TLR's influence on hTMSCs and their immunomodulating properties would be useful for providing a novel target to exploit in the improvement of stem cell-based therapeutic strategies.

Co-culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes

Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy. This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.

Mesenchymal Stromal Cells Suppress T-and B-Cells via Galectin-9 in a Donor Dependent Manner

AbstractAbstract 1248Therapeutic approaches using multipotent mesenchymal stromal cells (MSCs) are advancing in regenerative medicine, transplantation and autoimmune diseases. Until now the way of action for MSC-mediated immune suppression is still controversial and relies most probably on a multifactorial mechanism. MSCs have been demonstrated to produce the suppressive molecules hepatocyte growth factor (HGF), tumor growth factor-β (TGF-β), prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO). Furthermore, it has been described that immunosuppression by MSCs is enhanced via stimulation with interferon-γ (IFN-γ). Recently, galectin-1, a β-galactoside binding lectin with immune modulatory properties, has been added to the group of immune modulatory molecules that are responsible for MSC-mediated immune suppression. Here, we identified galectin-9 (Gal-9) as a new molecule involved in MSC-mediated immune modulation.First, we isolated MSCs from bone marrow of randomly selected donors and performed several in vitro experiments regarding their immune modulatory potential (e.g proliferation and IgG production). Interestingly, Gal-9 was the only investigated protein, which was strongly upregulated in MSCs upon activation with IFN-γ. We moreover demonstrate that Gal-9 is a major mediator of the anti-proliferative effect of MSCs on T-cells. Although a B-cell suppressive function of Gal-9 has previously not been reported, we were surprised to detect the same inhibitory effect on isolated B-cells. Proliferation of immune cells was triggered upon either stimulation with either PHA and LPS, or CD40L and PHA. Activation of MSCs with IFN-γ resulted in a major decrease of proliferation of both T-cells and B-cells. In addition, Gal-9 and activated MSCs contribute to the suppression of VZV triggered immunoglobulin release as well. Again activation of MSCs with IFN-γ decreased the IgG release, whereas blocking Gal-9 with lactose, a well characterized inhibitor of Gal-9 function, reversed the effect almost completely. Further, we determined that Gal-9 expression levels (mRNA and protein) distinguish between MSC cultures from different donors after activation. Among donors, we could differentiate between individuals with high Gal-9 levels and higher immune modulatory potential and such with low Gal-9 expression and lower immune modulatory potential. Compared to untreated MSCs we demonstrated a three- to fifty-fold rise in Gal-9 levels after prior activation with IFN-γ. In addition, we demonstrated the upregulation of Gal-9 in MSCs by cell-cell contacts with either T-or B-cells. The upregulation was additionally at least two fold increased by previeously activating MSCs with IFN-γ.Because our group is interested in the therapy of hemophilia A and because of the unxpected suppressive effect of Gal-9 on B-cells and B-cell function, we next tested the effect of MSCs and Gal-9 on the induction of inhibitory antibodies to coagulation factor VIII (FVIII). Mice were immunized with human coagulation factor VIII (FVIII) in the presence or absence of either human MSCs, anti-murine Gal-9 or human Gal-9. As predicted, MSCs suppressed and anti-Gal-9 antibodies anhanced antibody formation. However in contrary to the expected, human Gal-9 co-treatment enhanced the anti-FVIII antibody response. A set of additional experiments revealed, that human Gal-9 suppresses murine regulatory T-cells in vivo. Further, in contrast to human immune cells, murine-derived T- and B-cells did not respond to human recombinant Gal-9 in vitro, but human IFN-γ activated MSCs were able to suppress proliferation of murine immune cells. Because of only 60% homology of murine and human Gal-9 we assume that the murine model cannot predict the function of human Gal-9 and that MSC-mediated immune modulatory functions are exerted via alternative pathways in this setting. Experiments with murine Gal-9 to demonstrate the in vivo function of Gal-9 are ongoing.In conclusion, Gal-9 is novel mediator of MSC immunomodulatory functions and affectsmultiple immune cell types including B-cells. Gal-9 is differentially expressed in MSCs from different donors and may therefore serve as a predictive indicator for clinical MSC functionality. Disclosures:No relevant conflicts of interest to declare.