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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.

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.

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.

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.

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.