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Multipotent mesenchymal stromal cells (MSC) are the key regulators of hematopoiesis. We studied changes in MSC characteristics in patients with myeloid leukemia and patients with lymphoproliferative diseases. MSC were obtained from the bone marrow of patients at the time of diagnostic puncture using a standard technique. Their proliferative potential and expression of genes associated with differentiation and regulation of hematopoiesis were studied. The total cell production of MSC in patients with leukemia at the onset of the disease did not differ from that in the group of healthy donors. The relative expression of the IL6, TGFb1 and TGFb2, PPARG genes was similar in all patients. The relative expression of the JAG1, LIF, IGF1, CSF1, IL1b, and IL1bR1 genes in MSC of patients with leukemia was enhanced and the relative expression of SDF1 was unchanged in comparison with MSC from donors. MSC from patients with leukemia were characterized by enhanced relative expression of PDGFRA and PDGFRB, and reduced expression of SOX9. Changes functions of the stromal microenvironment in patients with hemoblastoses attested to the role of stromal cells in the maintenance and spread of tumor cells.

We studied changes in the bone tissue in patients with diffuse large B-cell lymphoma at the onset of the disease (N=41; before chemotherapy) and 5-16 years after the end of treatment (N=47). Osteodensitometry, biochemical markers of osteoporosis in the blood and urine, and gene expression in multipotent mesenchymal stromal cells were analyzed. In multipotent mesenchymal stromal cells of all patients, the expression of genes associated with bone and cartilage differentiation (FGF2, FGFR1, FGFR2, BGLAP, SPP1, TGFB1, and SOX9) was changed. In primary patients, the ratio of deoxypyridinoline/creatinine in the urine and blood level of β-cross-laps were increased, while plasma concentration of vitamin D was reduced, which indicates activation of bone resorption. No differences between the groups were revealed by osteodensitometry. No direct relationship between changes in gene expression in multipotent mesenchymal stromal cells and osteoporosis markers was found. The presence of a tumor in the body affects the bone marrow stroma, but achievement of remission and compensatory mechanisms provide age-appropriate condition of the bone tissue.

Interleukin-1 beta (IL1B) is a key inducer of inflammation and an important factor in the regulation of hematopoietic stem cells and mesenchymal stromal progenitors. Irradiation of mice with ionizi...

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Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Transgenic NES-EGFP mice developed by G. Enikolopov (Tg(Nes-EGFP)33Enik), which have a green fluorescent protein (EGFP) gene under the promoter of the nestin gene, had been previously used to show that early mesenchymal progenitors are in the population of CD45–GFP+ bone marrow (BM) cells. Here we show that NES-EGFP mice can serve as a convenient model for studying MSCs capable of transferring hematopoietic microenvironment under the renal capsule of syngeneic mice. BM from one femur of NES-EGFP mouse was used to obtain Dexter-type long-time bone marrow culture (LTBMCs, n = 5). GFP+ cells had been counted weekly over the entire area of the T25 flasks for 5 weeks after culture initiation using a fluorescence microscope. Four weeks after the initiation of LTBMCs, two day long time-lapse vital photography was performed. Ectopic foci of hematopoiesis under the renal capsule (n = 6) were obtained from the BM of NES-EGFP donors in syngeneic recipients. The number of C-kit+/–GFP+ cells in the formed foci was determined using FACS analysis. GFP+ cells with stromal phenotype in the adherent cell layer of LTBMCs had been detected during 5 weeks of cultivation. The stromal layer of LTBMC contained fibroblast-like GFP-low and GFP-hi cell subpopulations. GFP-low cells were scattered across the surface and predominated. GFP-hi cells were rare and formed colonies. Ectopic foci of hematopoiesis contained 2036 ± 734 C-kit–GFP+ stromal cells. Most of these cells had GFP-low phenotype. The cellularity of the focus strongly and positively correlated with the number of C-kit–GFP+ in it. The results show that C-kit–GFP+ BM cells of NES-EGFP mice are able to survive implantation and include MSCs and early stromal mesenchymal progenitors, the number of which determines the size of the focus. NES-EGFP mice can be used for in vitro and in vivo experiments to study functional BM-derived MSCs.

We analyzed changes in multipotent mesenchymal stromal cells of patients with chronic myeloid leukemia before discontinuation of tyrosine kinase inhibitors. Withdrawal syndrome was significantly more common in patients who have been taking tyrosine kinase inhibitors for a longer time and in patients of older age and with lower body weight. In patients with withdrawal syndrome, the total production of mesenchymal stromal cells and expression of FGFR2 and MMP2 genes were significantly lower; loss of deep molecular response was also less frequent in this group of patients. At the same time, the expression of genes important for the maintenance of stem cells (SOX9, PDGFRa, and LIF) was significantly lower in the mesenchymal stromal cells of patients with withdrawal syndrome and loss of deep molecular response. We observed a clear-cut relationship between the development of withdrawal syndrome and the loss of deep molecular response. The decrease in the expression of FGFR2 and MMP2 genes in the mesenchymal stromal cells of patients with chronic myeloid leukemia before discontinuation of treatment can be a predictor of withdrawal syndrome, while simultaneous decrease in the expression of SOX9, PDGFRa, and LIF in these cells attests to undesirability of therapy discontinuation at the moment.

In diffuse large B-cell lymphoma, bone marrow involvement is rarely diagnosed. We compared the properties of bone marrow stromal progenitor cells and the concentration of fibroblast CFU in patients with diffuse large B-cell lymphoma without bone marrow involvement and in healthy donors. It was found that the properties of multipotent mesenchymal stromal cells in patients in the debut of the disease differed considerably from those in healthy donors. In particular, the total cell production in patients was significantly higher than in donors. In multipotent mesenchymal stromal cells of patients, some cell parameters were changes; the mean fluorescence intensity of the adhesion molecule ICAM1 on the cell surface was increased. The mean fluorescence intensity of mesenchymal stromal cell markers (HLA-ABC, CD73 and CD90) was significantly elevated. The relative expression of BMP4, MMP2, FGFR1, and ICAM1 genes in mesenchymal stromal cell was reduced, while the expression of FGFR2 gene was enhanced. Despite the absence of proven involvement of the bone marrow, the properties of mesenchymal stromal cells, the components in the stromal microenvironment niche regulating hemopoiesis are altered in patients with diffuse large B-cell lymphoma.

Analysis of changes in lymphocyte subpopulations during co-culturing with multipotent mesenchymal stromal cells (MSC) revealed two distinct MSC groups: one group (A) increased HLA-DR expression on lymphocytes during co-culturing and the other (B) did not change it in comparison with lymphocyte monoculture. In stromal cells interacting with lymphocytes, expression of HLA-DR molecules was initiated, but only in samples that induced enhanced expression on lymphocytes and irrespectively of whether allogeneic or autologous lymphocytes were used for co-culturing with MSC. In group A, the relative expression of IDO1 significantly increased in comparison with group B. The revealed individual differences in MSC can explain why not all MSC samples are effective in the treatment of autoimmune diseases, acute "graft-versus-host" disease, and other pathologies.

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate subpopulational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4+ and CD8+ expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naive cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naive state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.

We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9– 1 . 3 × 1 0 6 /kg when the blood counts indicated recovery. aGVHD of stage II–IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, ( 𝑃 = 0 . 0 0 2 ). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study.

The bone marrow stromal microenvironment that regulates normal hematopoiesis suffers during leukemia development and its treatment. In this study, we examined Multipotent Mesenchymal Stromal Cells (MMSCs) and fibroblast colony-forming units (CFU-F) derived from the Bone Marrow (BM) of 15 adult patients with acute lymphoblastic leukemia (ALL) before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The time points assessed after allo-HSCT were defined by the treatment protocol. The analogous cells obtained from the BM of 64 healthy donors were used as controls. The ability of MMSCs to proliferate, concentration of CFU-F in BM, and gene expression in both cell types were assessed. The data indicate that MMSCs of ALL patients before allo-HSCT did not differ from MMSCs of healthy donors either in cumulative cell production or in gene expression except for SDF1. The SDF1 expression was decreased 2-fold in the MMSCs of ALL patients. The MMSC cumulative cell production from ALL patients was significantly decreased during 1 year after allo-HSCT. The expression level of SDF1 was also downregulated during the observation period. We identified changes in the FGF2 and PDGF signaling pathways. The CFU-F analysis revealed that its concentration in the BM of ALL patients had been profoundly decreased for the whole year after allo-HSCT. This decrease was accompanied by the downregulation of FGFR1 and slight upregulation of differentiation marker gene expression. Thus the number of stromal precursorcells decreased and their ability to regenerate was depressed after allo-HSCT. These changes were accompanied by an increase of more mature precursor cells with reduced proliferative potential.

Introduction Hematopoiesis is maintained in close contact with bone marrow (BM) stroma. Malignant hematopoietic cells probably affect the hematopoietic microenvironment, as well as high dose chemotherapy. The goal of this study was to analyze the alterations in the characteristics of human multipotent mesenchymal stromal cells (MSC) and their more differentiated progeny – fibroblastic colony forming units (CFU-F), derived from the BM of acute myeloid and lymphoid leukemia (AML and ALL) patients. Methods 26 newly diagnosed cases (18 AML, 8 ALL) and 35 patients (20 AML, 15 ALL) before and during 1 year after allogeneic hematopoietic stem cell transplantation (alloHSCT) were involved in the study after informed consent. BM was aspirated prior to any treatment in the newly diagnosed group and before the conditioning and at 6 time points during 1st year after the alloHSCT. MSC were cultured in aMEM with 10% FCS. Cumulative MSC production was counted after 3 passages. CFU-F concentration was analyzed in BM samples by standard protocol. The relative expression level (REL) of genes was measured by RT2 Profiler PCR Array (Qiagen) and TaqMan RQ-PCR. MSC and CFU-F from 50 healthy donors of BM for alloHSCT were used as a control after informed consent. Results The CFU-F concentration in the BM of patients at the moment of diagnostics was 1/3 of the donor’s for AML and ≈1/10 of ALL (9±5.2 and 3.9±2.3 versus 31±3.5 per 106 nucleated cells in donor BM, p Conclusions During the leukemia development malignant cells alter the stromal precursor cells leading to the decrease in the REL of many regulatory genes and in the number of more differentiated stromal precursor cells in the BM. Chemotherapy used for induction of the remission in these patients restore functional ability of MSC and CFU-F, while gene expression levels remained altered. Conditioning regiments used for the alloHSCT significantly damage both types of studied stromal precursors, and the effect lasted at least for 1 year. So, both leukemic cells and chemotherapy affect BM hematopoietic microenvironment. Disclosures No relevant conflicts of interest to declare.

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow (BM). Lymphocytes connect tightly with stromal cells in BM. Leukemia and high dose chemotherapy affected both hematopoietic and stromal precursor cells. Changes in the hematopoiesis that occur during acute lymphoblastic leukemia (ALL) probably correlate with variation in the composition of stromal microenvironment. The aim of the study was to analyze the alterations occurring in stromal precursor cells - colony forming unit fibroblasts (CFU-F) and multipotent mesenchymal stromal cells (MSC) in patients with ALL before and after allogeneic hematopoietic stem cell transplantation (HSCT). HSCT following myeloblative conditioning was performed in 7 ALL patients (6 male, 1 female). After informed consent BM was aspirated before conditioning, and during the year after transplantation. MSC were cultured in aMEM with 10% fetal calf serum. Cumulative MSC production was counted after 5 passages. CFU-F was analyzed in standard conditions. The relative expression level (REL) of different genes was measured by RT-PCR. All data obtained at different time points were analyzed summarily. As a control MSC and CFU-F from 20 healthy donors of BM for HSCT were used after informed consent. The concentration of CFU-F in BM of ALL patients before HSCT was reduced by 12% in comparison with donors. After HSCT the concentration of CFU-F decreased further in 5.2 fold compared to the level before HSCT (p=0.02). Colonies formed by CFU-F depend on autocrine secretion of FGF2. It signals through 2 types of receptors, FGFR1 is considered to be the most important one. REL of FGFR1 was reduced by 27% in colonies from BM of patients before HSCT compared to donors. After HSCT it further decreased 1.2 fold. REL of FGFR2 in colonies of patients decreased 16 fold before and 50 fold after HSCT compared to donors (p=0.01 in both cases). REL of FGF2 in colonies of patients before HSCT doubled compared to donors and decreased 16 fold after HSCT. These data offer the molecular basis of decrease in CFU-F concentration in BM of patients after HSCT. In CFU-F colonies from patients REL of FGFR2 was decreased dramatically, however before HSCT it did not lead to decrease in CFU-F concentration due to increase in REL of FGF2. After HSCT REL of FGF2 and both receptors considerably decreased that led to significant reduction of CFU-F concentration in patients BM. REL of genes-markers of adipogenic and osteogenic differentiations were significantly decreased in CFU-F colonies of patients before HSCT (SPP1 in 8.6 fold, p=0.02, PPARG in 4.7 fold, p=0.01) pointing to less differentiated status of CFU-F progeny. After HSCT REL of these genes increased up to levels higher (PPARG in 3.5 fold, p=0.02) or comparable with donor ones, thus CFU-F seemed to regain “normal” differentiation status. REL of chondrogenic marker SOX9 did not change in CFU-F of ALL patients before and after HSCT, probably due to block of this differentiation lineage in CFU-F. REL of BMP4 in patients’ CFU-F colonies was reduced 30 fold compared to donors (p=0.002), apparently due to damage made by leukemic сells. It increased 100 fold compared to donors after HSCT, that might reflect the intensive recovery of stromal microenvironment by interaction with non-malignant hematopoietic cells. Thus multiple alterations in CFU-F concentration and gene expression were revealed in CFU-F from BM of patients. MSC were also affected in ALL patients. Cumulative cell production in cultures from patients before HSCT was reduced by 35%, while after HSCT the decrease was more pronounced (by 61%, p=0.01). REL of genes regulating the proliferation (FGFR1, FGFR2 and FGF2) in patients MSC was halved in comparison to donors. After HSCT REL of both types of receptors did not change, while REL of FGF2 increased 1.5 fold not reaching donors’ level. The decrease in cumulative cell production could depend on both FGF2 pathway alteration and on the increase of differentiation status in MSC population after HSCT. REL of differentiation markers in MSC before HSCT did not differ from donors’ ones, while after HSCT REL of SPP1 increased 3.8 fold, PPARG – 2.3 fold (p=0.05) and SOX9 in 1.6 fold compared to donors. In patients MSC were less affected by leukemia and its treatment than CFU-F. This data suggest significant damage of stromal precursor cells in ALL patients that lasted at least for one year since HSCT. Disclosures: No relevant conflicts of interest to declare.

Introduction Allogeneic bone marrow transplantation (allo-BMT) is currently the only way to cure many hematoproliferative disorders. However, allo-BMT use is limited by severe complications, among which the most challenging is graft-versus-host disease (GVHD). As the conventional methods of GVHD prophylaxis are often inefficient new method involving the use of donors’ multipotent mesenchymal stromal cells (MSC) was developed. In some cases prophylaxis of acute GVHD (aGVHD) failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or characteristics of MSC samples. The results of the aGVHD prophylaxis with donors’ MSC injections after allo-BMT in patients with hematological malignancies included in the randomized clinical trial (Clinicaltrials.gov NCT01941394) were analyzed. In order to discriminate between effective and ineffective for aGVHD prophylaxis MSC samples were thoroughly analyzed. The growth and differentiation characteristics, relative expression levels of different genes were investigated in all MSC samples. Methods The study included 77 patients who received allo-BMT from related donors after informed consent. The patients were randomized into 2 groups: the first received standard prophylaxis of aGVHD and the second were additionally infused with MSC from the bone marrow of corresponding hematopoietic stem cells donor at day of WBC reconstitution >1*109/l. MSC were cultivated in aMEM with 4% donors’ platelet lysate. MSC were administered intravenously when the blood counts indicated recovery (peripheral blood leukocytes reached 1x109/l). MSCs and colony-forming unit-fibroblasts (CFU-Fs) from the bone marrow of those donors were analyzed. For this purpose MSC were cultivated in standard conditions (aMEM, 10% fetal calf serum) for 5 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR in all MSC samples. Results The infusion of MSC reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (p=0.047). Four of 39 MSC samples have been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index), the MSC properties of these donors (growth parameters, REL of 30 genes involved in proliferation, differentiation and immunomodulation) found no significant differences between the MSC, effective and ineffective for preventing aGVHD. However the analysis revealed that cumulative MSC production and CFU-F concentrations in bone marrow decreased with donor age. MSC populations revealed the hierarchy that changed during cultivation, resulting in an increase in the impact of mature cells and a decrease in the subpopulation of cells with high proliferation potential. The combination of predictors that characterize the most suitable for the prevention of aGVHD MSC samples was revealed by multiple logistic regression analysis. A model calculating the probability of the success of MSC samples application was proposed: logit(P)=0,75+10,897*FGFR1-4,272*PPARG-2,014*IGF1, where logit(P) = ln[P/(1-P)], P – probability of successful prophylaxis, FGFR1, PPARG and IGF1 – REL of corresponding genes in tested MSC sample. Chi-square goodness of fit test p= 0.0053. The calculated efficiency of this model was 94%. The following parameters of MSC were essential for the success of aGVHD prophylaxis: increased REL of FGFR1 combined with reduced REL of PPARG and IGF1 genes. Depending on the chosen value for probability of successful application of MSC, this model can correctly predict the outcome of the use of MSC in 82-94% of cases. Conclusions These data confirm the presence of hierarchy as well as heterogeneity in MSC population. The high variability of all analyzed characteristics among MSC from different donors was shown. The mathematical model revealed the combination of parameters enabling to distinguish effective and ineffective MSC samples. By means of the proposed model ineffective MSC samples could be discharged and replaced by effective MSC sample from the third part donor. Such strategy hopefully will prevent the development of aGVHD in the maximum number of patients. Disclosures No relevant conflicts of interest to declare.