Introduction. Combined deficiency of factors V and VIII is a rare hereditary bleeding disorder with a prevalence of 1:1,000,000 in the general population, but the disease is more common in regions where consanguineous marriages are acceptable. Data on this hereditary coagulopathy in the Russian Federation are limited. Aim: to analyze clinical and laboratory characteristics of the course of the disease in patients with hereditary combined deficiency of factors V and VIII in the Russian population. Materials and methods. The retrospective and prospective study involved 6 patients with hereditary combined deficiency of factors V and VIII in the Russian population. Results. The average age of patients was 50 years (32-72 years). The average age at the time of diagnosis was 40 years. Bleeding scores on the ISTH-BAT scale ranged from 17-29, with an average value of 23.5. The average value of activated partial thromboplastin time was 85 seconds, the prothrombin by Quick was 35 %, and the activity of FV and FVIII was 5.7 % and 9.0 %, respectively. The course of the disease was characterized more or less by cutaneous-mucous hemorrhagic syndrome, postoperative, obstetric-gynecological, and life-threatening bleeding. Conclusion. Clinical and laboratory characteristics of patients expand the understanding of hereditary combined deficiency of factors V and VIII and make it possible to accelerate diagnosis verification.

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

Factor FXI is an essential participant of the blood coagulation cascade and is coded by the F11 gene, mutations in which lead to an extremely rare (1 : 1 000 000) autosomal disease-FXI deficiency, also known as hemophilia C. The most frequently, FXI deficiency is diagnosed in Ashkenazi Jews with three major mutations. The aim of this study was a primary description of the F11 gene mutational spectrum in the Russian population. During the study, we sequenced all functionally important regions of the F11 gene for 11 unrelated patients with hemophilia C. In total, ten different gene defects were revealed: five missense mutations, one nonsense mutation, three frameshift deletions, and one inframe deletion. All of them were uniformly distributed across the gene. Among the most frequent genetic defects in the world population, we found only type II mutation p.Glu135Ter common in Ashkenazi Jews in our sample. Two previously undescribed variants (c.1768del and p.His53Tyr) were evaluated as probably pathogenic. There was a typical picture of incomplete dominance by laboratory parameters-a significant decline of FXI activity level and an increase in APTT when both copies of the gene were damaged and slight deviations from the norm if only one of the copies was impaired. To sum up, we described F11 defects in Russian patients with FXI deficiency. Our findings indicate a high level of heterogeneity of the mutational spectrum leading to hemophilia C in Russia.

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.

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.

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.

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.

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.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

The present study involved three patients with graft failure following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We obtained multipotent mesenchymal stromal cells (MSCs) from the original hematopoietic cell donors and implanted these cells in the periosteum to treat long-term bone marrow aplasia. The results showed that in all patients endogenous blood formation was recovered 2 weeks after MSC administration. Donor MSCs were found in recipient bone marrow three and 5 months following MSC implantation. Thus, our findings indicate that functional donor MSCs can persist in patient bone marrow.

Multipotent mesenchymal stromal cells (MSCs) are applied for prophylaxis of acute graft versus host disease (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Not all samples of MSC used in National Research Center for Hematology were efficient for aGvHD prevention. The suitabilityof MSCs for aGvHD prophylaxis was studied. MSCs derived from the bone marrow of HCT donor were injected intravenously precisely at the moment of blood cell reconstitution. MSCs were cultivated for 3 passages. The characteristics of donor bone marrow samples including colony forming unit fibroblast (CFU-F) concentration, growth parameters of MSCs and the relative expression levels (REL) of different genes in them were analyzed. MSCs infusion induced a decrease in aGvHD development in patients with related and unrelated donors compared with the standard prophylaxis group. aGvHD prophylaxis with MSCs was ineffective in 13.5% of cases. In these MSC samples, a significant decrease in total cell production and the REL of CFH, FGFR1, PDGFRa and ICAM1 were observed. This study showed that MSCs injection resulted in a significant 2-fold decrease in aGvHD development compared with patients in the standard prophylaxis group. The effective MSC samples are characterized by higher total cell production and REL of CFH, FGFR1, PDGFRa and ICAM1 than ineffective.

Murine mesenchymal stem cells in long-term bone marrow culture were genetically labeled using lentiviral vector carrying enhanced green fluorescent protein (eGFP) reporter gene under SFFV promoter or without it. We studied the developmental fate of labeled mesenchymal stem cells in stromal cell layers of long-term bone marrow culture and in ectopic hemopoietic foci formed by these stromal layers under the renal capsule of syngeneic mice. The frequency of labeled polypotent stromal precursors (fibroblast CFU) was analyzed in adherent cell layers of long-term culture and ectopic foci formed from them. The proportion of labeled fibroblast CFU in ectopic foci increased by 10 times in case of implantation of adherent cell layers infected with lentivirus containing eGFP reporter gene without promoter. eGFP expression leads to rejection of labeled stromal cells. Labeling with eGFP-carrying vector without promoter makes possible long-term tracking of mesenchymal stromal cells.

Green fluorescent protein (eGFP) gene was transferred into mouse mesenchymal stem cells in vivo using a lentiviral vector. In 2 months after injection of the lentivirus into the cavity of the femoral bone, up to 30% fibroblast CFU in the bone marrow of infected mice contained the alien gene. The transferred gene was found in more than 50% of adherent layers of longterm bone marrow cultures formed by mesenchymal stem cells from the infected mice bone marrow; 4% fibroblast CFU obtained from these layers were labeled. Ectopic hemopoiesis foci developed after transplantation of the bone marrow from infected mice under the renal capsule of syngeneic recipients contained bone tissue labeled with the alien gene in 57% cases and labeled fibroblast CFU in 11%. The data confirm the possibility of gene transfer with the lentiviral vectors into the mesenchymal stem cells in vivo .

Biological characteristics of bone substituting tissue engineering construction (TEC) that contained porous calcium phosphate ceramic granulate (CPC) of phase structure ((tricalcium phosphate (TCP)), fibrin hydrogel and autologous multipotent mesenchymal stromal cells (auto-MMSC) induced and non-induced to osteogenic differentiation were studied in vivo. The following characteristics of TEC were determined: ability to transfer within its structure the viable auto-MMSC with preservation of their regeneration potential; ability to osteogenesis only under conditions of orthotopic implantation; ability of induced to osteogenic differentiation auto-MMSC to participate in the reparative processes for not more than within 6 weeks after implantation; negative affect of fibrin hydrogel on the osteoinductive properties of CPC within TCP structure. It was shown that to provide osteogenesis in the implanted TEC not only the viable auto-MMSC but simultaneous presence of osteoinductive and osteoconductive factors was required. No bone formation in a critical bone defect and in ectopic implantation takes place without observance of these conditions.