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INTRODUCTION: The hierarchy of stromal progenitors from the bone marrow is poorly characterized; multipotent mesenchymal stromal cells and colony-forming units of fibroblasts are isolated in culture. Mesenchymal stem cells do not have a unique combination of surface antigens, making it difficult to obtain the pure population. The expression of the nestin gene is often used as a marker of these cells. AIM: To evaluate the level of expression of the nestin gene in multipotent mesenchymal stromal cells and in colony-forming units of fibroblasts and to characterize the change in its expression during the transition from oligopotent progenitor cells to monopotent ones. MATERIALS AND METHODS: Stromal progenitors were analyzed in bone marrow samples from 19 donors by standard methods. A total of 296 individual clones of fibroblast colony-forming units were obtained from the same bone marrow samples. The cells were analyzed for the ability to differentiate toward the adipogenic and osteogenic lineages. Relative expression level of nestin gene was analyzed in all cells. RESULTS: Mean relative expression level of nestin did not differ significantly in multipotent mesenchymal stromal cells (0.410.13) and in the total population of colony-forming units of fibroblasts (0.240.05). In individual clones of colony-forming units of fibroblasts, nestin expression was not significantly higher than in the total population (0.310.04). When analyzing colony-forming units of fibroblasts differing in their differentiation potential, the highest expression of nestin was found in the group of monopotent osteogenic progenitors, while its expression was significantly lower in oligopotent progenitors. CONCLUSION: Nestin gene expression in mesenchymal stromal progenitors from the bone marrow is not specific for mesenchymal stem cells and cannot be used as a unique marker of this cell type. According to our data, a high level of nestin expression rather identifies monopotent osteogenic progenitors.

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.

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...

We analyzed multipotent mesenchymal stromal cells (MMSCs) from the bone marrow (BM) of 33 acute myeloid leukemia (AML) patients at diagnosis, after the first course of chemotherapy (day 37), and at days 100 and 180 after diagnosis. All patients were treated according to the AML 01.10 protocol. Cumulative production of MMSCs from AML patients at diagnosis was normal but increased during treatment. Most of the studied genes were upregulated at AML diagnosis, some (IL6, IL1B, LIF) remained upregulated during treatment, and others were downregulated (FGFR1, ICAM1) or normalized. A few genes were normal at diagnosis but decreased during treatment (FGF2, FGFR2, VEGF, SDF1, SOX9, TGFB1). The upregulation of proinflammatory genes both at diagnosis and during remission reflects ongoing inflammation. PDGFRB expression was upregulated in MMSCs from patients in relapse versus those in remission. The AML 01.10 protocol downregulates the expression of genes related to proliferation, differentiation and niche formation.

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

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.

BACKGROUND The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. METHODS MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. RESULTS The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. CONCLUSIONS The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored.

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.

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.

Bone marrow (BM) derived adult multipotent mesenchymal stromal cells (MMSCs) and fibroblast colony-forming units (CFU-Fs) of 20 patients with acute myeloid leukemia (AML) and 15 patients with acute lymphoblastic leukemia (ALL) before and during 1 year after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) were studied. The growth characteristics of MMSCs of all patients before allo-HSCT were not altered; however, relative expression level (REL) of some genes in MMSCs, but not in CFU-Fs, from AML and ALL patients significantly changed. After allo-HSCT, CFU-F concentration and MMSC production were significantly decreased for 1 year; REL of several genes in MMSCs and CFU-F-derived colonies were also significantly downregulated. Thus, chemotherapy that was used for induction of remission did not impair the function of stromal precursors, but gene expression levels were altered. Allo-HSCT conditioning regimens significantly damaged MMSCs and CFU-Fs, and the effect lasted for at least 1 year.

We studied the capacity of multipotent mesenchymal stromal cells isolated from human bone marrow (BM) to long-term passaging, cloning, and re-cloning. Initial multipotent mesenchymal stromal cells and cells after gene labeling were studied. Multipotent mesenchymal stromal cells were obtained from donors (13–59 years) and cultured for 7 passages. Third generation lentivector was used for delivery of green fluorescent protein marker gene. The procedure of infection revealed reduced proliferative potential of multipotent mesenchymal stromal cells from elder donors. Hierarchy of precursor cells differing by their proliferative potential was demonstrated in the culture of multipotent mesenchymal stromal cells. Three categories of multipotent mesenchymal stromal cells were identified: mature cells incapable of proliferation (75.7 ± 2.4% population) and cells with low and high proliferative potential (17.6 ± 2.1 and 6.7 ± 0.3%, respectively). The relative content of these cells insignificantly differed from passage to passage. The efficiency of cloning also remains stable, but re-cloning capacity sharply decreased after passage 3 and completely disappeared in multipotent mesenchymal stromal cells after cryopreservation. Thus, cultured multipotent mesenchymal stromal cells represent a heterogeneous and hierarchically organized population and the characteristics of this population depend of the duration of culturing and age of BM donor. This should be taken into account when using multipotent mesenchymal stromal cells in clinical practice.

Mouse mesenchymal stromal precursor cells were labeled with lentiviral vector in long-term bone marrow culture. We studied the fate of labeled cells in the stromal sublayer of the long-term bone marrow culture and in ectopic hemopoiesis foci formed from the labeled cultures. The incidence of labeled polypotent fibroblast CFU in sublayers of long-term bone marrow culture and in ectopic hemopoiesis foci formed from these sublayers under the renal capsule of syngeneic mice was also analyzed. It was shown that the marker gene was present in about 40% cells of the stromal sublayer and 30% fibroblast CFU and that effective gene transfer did not affect the total production of hemopoietic cells. The size of ectopic hemopoietic foci formed after implantation of labeled sublayers of the long-term bone marrow culture under the renal capsule did not differ from the control. Differentiated cells of the osseous shell in these foci carried the marker gene in 40% cases. Analysis of fibroblast CFU in these foci showed that despite the total concentration of fibroblast CFU was comparable to that in the bone marrow, the concentration of labeled fibroblast CFU was about 6%, which suggests that one more class of precursors probably exists in the hierarchy of stromal cells presumably between mesenchymal stem cells and fibroblast CFU. Our findings demonstrate the capacities of mesenchymal stem cells to self-maintenance and differentiation without losing the marker gene integrated into the genome.

We studied surface phenotype of tumor cells and characterized leukemia stem cells in various cell populations with phenotypes of stem and committed precursor cells in the hierarchy of hemopoietic stem cells. Transplantable murine leukemia was used as the model. Bone marrow and liver cells from mice in the terminal phase of the disease were stained with antibodies to various surface markers and analyzed on a flow cytofuorometer. The cells were sorted by various differentiation markers using a system of magnetic separation Miltenyi Biotec MACS and then transplanted to syngeneic recipients. In some cases, limiting dilutions were used for measuring the concentration of leukemia-initiating cells. All transplanted cell populations caused death of recipients: c-kit- CD45- over 23.9 days, c-kit+ over 22.2 days, c-kit- CD45+ over 15.4 days, Ter119+ over 18.2 days, and Ter119- over 17.7 days. The concentrations of leukemia cells determined by the method of limiting dilutions was 1 per 37,000 c-kit+ bone marrow cells and 1 per 45 unsorted liver cells from sick animals. Thus, leukemia stem cells retain hierarchic organization in the studied model and can differentiate at least into myeloid and erythroid cells without loosing self-maintenance capacity. This model can be used for the study of regulation of self-maintenance mechanisms in various hierarchic populations of leukemia stem cells.

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, the foremost being graft-versus-host disease (GVHD). Due to the lack of efficiency of the existing methods of GVHD prophylaxis, new methods are being actively explored, including the use of donors' multipotent mesenchymal stromal cells (MMSC). In this work, we analyzed the results of acute GVHD (aGVHD) prophylaxis by means of MMSC injections after allo-BMT in patients with hematological malignancies. The study included 77 patients. They were randomized into two groups - those receiving standard prophylaxis of aGVHD and those who were additionally infused with MMSC derived from the bone marrow of hematopoietic stem cell donors. We found that the infusion of MMSC halves the incidence of aGVHD and increases the overall survival of patients. Four of 39 MMSC samples were ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) and the MMSC properties of these donors (growth parameters, level of expression of 30 genes involved in proliferation, differentiation, and immunomodulation) revealed no significant difference between the MMSC that were effective or ineffective for preventing aGVHD. We used multiple logistic regression to establish a combination of features that characterize the most suitable MMSC samples for the prevention of aGVHD. A model predicting MMSC sample success for aGVHD prophylaxis was constructed. Significant model parameters were increased relative expression of the FGFR1 gene in combination with reduced expression levels of the PPARG and IGF1 genes. Depending on the chosen margin for probability of successful application of MMSC, this model correctly predicts the outcome of the use of MMSC in 82-94% of cases. The proposed model of prospective evaluation of the effectiveness of MMSC samples will enable prevention of the development of aGVHD in the maximal number of patients.

Multipotent mesenchymal stromal cells (MMSCs) have been demonstrated to produce mature stromal cells and maintain hematopoietic progenitor cells (HPC). It was previously demonstrated that interleukin-1 beta (IL-1 beta) stimulates the growth of the stromal microenvironment in vivo. The aim of this study was to investigate the effect of IL-1 beta treatment of human MMSCs on their proliferative potential, gene expression, immunomodulating properties, and their ability to support HPCs in vitro. Human bone marrow-derived MMSCs were cultivated in standard conditions or with IL-1 beta. The cumulative cell production was assessed for five passages. After withdrawal of IL-1 beta, MMSC clonal efficiency was investigated, and the maintenance of HPCs on top of MMSCs layers was estimated using cobblestone area forming cell (CAFC) and long-term culture initiating cell (LTC-IC) assays. The effect of untreated MMSCs or MMSCs pretreated with IL-1 beta on lymphocyte proliferation was studied by CFSE staining. The relative expression level of various genes by MMSCs was analyzed using RT-qPCR. The administration of IL-1 beta elevated MMSCs clonal efficiency and total cell production but did not affect lymphocyte proliferation. MMSCs pretreatment with IL-1 beta enhanced their ability to maintain HPCs, as detected by CAFC assay, and it altered the expression levels of genes participating in HPC regulation by stromal cells, e.g., adhesion molecules (ICAM1) and growth factors (SDF1). This study revealed the ability of IL-1 beta to stimulate MMSCs proliferation and enhance their potential to maintain HPCs. MMSCs are considered a stromal niche component in vitro. The combined in vitro and previous in vivo data suggest that IL-1 beta is a systemic regulator of the stromal microenvironment.

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.

Background Bone 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. Aims To 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. Methods 74 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. Results At 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. Conclusion The 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. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

Background In patients with acute leukemia the stromal microenvironment is deeply modified. Disturbances in signaling pathways, genetic abnormalities and functional changes in mesenchymal cells of these patients have been previously described. Chemotherapy also affect stromal progenitor cells. A damaged microenvironment might impair hematopoiesis in acute leukemia patients. Aims To investigate the relative expression level in MMSCs and CFU-Fs, derived from the bone marrow (BM) of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients before and over the course of chemotherapy. Methods 54 newly diagnosed cases (33 AML, 21 ALL) 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. MMSCs were cultured in aMEM with 10% fetal calf serum, CFU-Fs, in aMEM with 20% fetal calf serum. The relative expression level (REL) of different genes was measured by TaqMan RQ-PCR. As a control MMSCs and CFU-Fs from 88 healthy donors were used. Results At the time of the disease manifestation the analysis of gene expression in MMSCs from acute leukemia patients revealed a significant increase in the REL of genes which regulate immune system responses and thereby can influence on the leukemic cell proliferation and migration (IL-6, IL-8, IL-1b and IL-1R1) (Pic.1). Also at the time of the diagnosis an increase in REL of genes, that are responsible for hematopoiesis regulation, was observed. For example, the REL of CSF1 that can influence on leukemic cells proliferation was increased at the disease manifestation and became normal during the treatment. The same dynamics was observed in the REL of JAG1 that has an antiapoptotic effect on leukemic cells. The REL of LIF had been also significantly increased at the disease manifestation, reflecting the efforts of MMSCs to inhibit leukemic proliferation. Chemotherapy affected REL of the studied genes differently. The treatment lead to the downregulation of IGF, TGFB1 and TGFB2 (Pic.2). As far asTGFB1 and 2 inhibit the differentiation of mesenchymal stem cells, and IGF is associated with myelodysplastic changes in elderly bone marrow, so their downregulation may refer to the effectiveness of therapy. The REL of genes regulating MMSC proliferation (PDGFRa and PDGFRb, FGF2, FGFR1 and 2) increased during chemotherapy. Exploring cell adhesion molecules, the decrease in the REL of their encoding genes was observed. As far as VCAM facilitate the leukemic cell extravasation and ICAM was shown to depress the Th17 cell differentiation, the down-regulation of their genes may reflect the microenvironment restoration. The influence of chemotherapy lead to decrease in REL of genes, associated with MMSCs differentiation (BGLAP and SOX9 (Pic.3)), reflecting the mechanism of the blocking of MMSCs migration and differentiation under the stress conditions. The alterations of bone marrow stroma were more pronounced in patients who didn't achieve remission. The REL of 9 genes was studied in CFU-F colonies. There were no differences in gene expression in CFU-Fs before the treatment, except for an increase in the REL of PPARg in acute leukemia CFU-Fs. During the treatment, a decrease in the REL of SPP1 and an increase in the REL of FGFR1 and 2 were observed. Conclusion Therefore, chemotherapy used does not impair the functional ability of MMSCs and CFU-Fs, but influence on their gene expression profile. The two types of precursors are affected differently, indicating their different differentiation level and functions. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Multipotent mesenchymal stromal cells (MMSCs) are a heterogeneous population consisting of cells with a distinct proliferative potential. The aim of this study was to define clonal composition in MMSCs and trace the dynamics of individual clones in MMSC subpopulations with different proliferative potentials during the process of cultivation. The investigation was performed at single-cell level using genetically marked cells. Specifically, human bone marrow MMSCs were infected with a lentiviral vector–bearing marker gene. Integration site analysis was performed for clones at each passage by ligation-mediated polymerase chain reaction and Southern blot hybridization. Sibling connections between clones and clonal composition of MMSC culture at each passage were revealed. The MMSC population contained multiple, different, mainly small, clones. It was found that large long-living clones with a high, but limited proliferative potential could be detected rarely in MMSCs population. These data suggest that the human MMSC population does not fit the "stem cell" criteria, however, MMSCs may contain a subpopulation of large clones with a high proliferative potential.

Objective. Massive liver infiltration by leukemic cells is an indicator of poor prognosis in some hemoblastoses. The aim of this study was to determine the mechanism of liver invasion by leukemic cells using the mouse model of transplantable myeloproliferative disease-like myeloid leukemia characterized by liver invasion. Materials and Methods. CD45 + cells from the liver of mice transplanted with leukemic cells were sorted by magnetic separation. Gene expression alterations in CD45 + cells invading the liver were examined by polymerase chain reaction arrays and quantitative real-time polymerase chain reaction (including polymerase chain reaction arrays) analysis of selected genes. Results. Liver chemokine receptors (Ccr1, Ccr2, Ccr5, and others) were expressed in cells invading the liver. The expression level of Ccr1 was increased 149-fold in comparison with CD45 + cellsderived from the liversof healthy mice. Expression levels of several genesresponsible for proliferation and self-renewal were elevated dramatically, which is in accordance with a high concentration of leukemia stem cells in the livers of moribund animals. The nuclear factorLkB signaling pathway and several oncogenes are also activated in these leukemia cells. Conclusions. Overexpression of liver-specific cytokine receptors allowed the leukemic cells to invade the liver. The high concentration of leukemia stem cells in the liver suggests the cells of this leukemia are able to adapt to new extramedullar niches. The model for the investigation and development of preventative strategies against massive liver invasion are described here. 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Mechanisms of hematopoietic failure in patients with aplastic anemia (AA) are obscure. We investigate alterations in the hematopoietic microenvironment in AA patients. We present the results of studying mesenchymal stromal cells (MSC), fibroblastic colony-forming units (CFU-F), and adherent cell layers (ACL) of long-term bone marrow cultures (LTBMC) from bone marrow (BM) samples of AA patients. MSC of AA patients proliferated longer than those of donors. In half of the patients' MSC cultures, adipogenesis was impaired. Osteogenic differentiation was not achieved in 36% of AA MSC. CFU-F formed enlarged colonies, and their concentration in the BM of AA patients was significantly increased. Our data suggest that the physiological activation of the stromal microenvironment is characteristic of AA. We detected a decrease in the expression of the angiopoetin-1 (ANG-1) and vascular cell adhesion molecule-1 (VCAM-1) genes, together with an increase in the expression of vascular endothelial growth factor (VEGF) in ACL of AA patients. This indicates abnormal regulatory patterns in both osteoblastic and vascular contexts. Addition of AA patients' serum to donors' LTBMC for 3 weeks induced similar gene expression alterations. The addition of parathyroid hormone (PTH) resulted in the expression levels of analyzed genes returning to normal, in both AA LTBMC and donor cultures treated with AA serum. The physiologic status of the BM stromal microenvironment (MSC, CFU-F, and ACL of LTBMC) of AA patients was altered.

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.

Abstract 4554 Background Severe graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic-stem cell transplantation (allo-HSCT). Steroids are the first-line treatment for established GvHD with a response rate of 30–50%. The efficacy of multipotent mesenchymal stromal cells (MMSCs) in the treatment of steroid-resistant GvHD was reported to be 71–94%. Although it has been shown that the immunomodulatory effect of MMSCs mainly occurs through the secretion of soluble mediators, the exact mechanism of their action remains controversial. Aim The aim of the study was to investigate the influence of immunomodulating factors expressed by donor's MMSCs in vitro on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. IL-6, IL-10, colony stimulating factor 1 (CSF1), indoleamine 2, 3 dioxygenase (IDO1), prostaglandin E synthase (PTEGS) and complement factor H (CFH) were measured as factors playing substantial role in GvHD development. Methods Five patients with steroid resistant acute GvHD were treated with bone marrow derived MMSCs from hematopoietic stem cells donors. MMSCs were infused intravenously at the dose 1×106 per kg of body weight. The efficiency of GvHD therapy by means of MMSCs was scored as: complete response – 3, partial response - 2, clinical improvement – 1, no response – 0. MMSCs were cultured in aMEM with 4% human platelet lysate for 2–5 passages. Total RNA was extracted from MMSCs at 2–3 passages by standard protocol. Relative level of gene expression was estimated by the real-time PCR with previous reverse transcription in MMSCs from 31 donors and determined by normalizing the expression of each target gene to b-actin and GAPDH, calculated using ΔΔCt method for each MMSCs sample. Results Characteristics of patients, donors and graft MMSCs are shown in the table 1. Correlation between relative level of gene expression and efficacy of MMSCs infusion was investigated. The existence of inverse negative relationship between IL-6, CSF1 expression level and treatment score was revealed. The increased level of IL-6 in donor's MMSCs in patients with low and no response (treatment score 1 – 0) could be related to main functions of this factor in inflammation. Increased level of CSF1 in donors' MMSCs could further enhance macrophage activation resulting in GvHD progression instead of inhibition. The augmentation of PTGES expression could be associated with improvement of response to MMSCs therapy, but the correlation was not found. There were no relationship between the expression levels of IL-10, IDO1 and CFH and efficiency of MMSCs infusion. In a patient with no response the relative expression level of all studied factors was altered in comparison with average level of their expression in MMSCs from studied donors: IL-6 and CSF1 increased about 2 fold, while the expression level of IL-10, CHF and PTGES decreased 1.7, 12 and 11 fold correspondingly. So not all MMSCs fit to GvHD therapy that could be associated with their characteristics. Conclusions The data demonstrate the influence of IL-6, CSF1 and PTGES expression on the efficacy of MMSCs in the treatment of steroid-resistant GvHD. As most clinical trials involve non-relative allogeneic MMSCs, analysis of expression level of IL6, CSF1 and PTGES in available MMSCs before infusion could predict the efficiency of acute GvHD therapy and permit the choice of the most proper samples. For successful clinical use of MMSCs further investigation of their properties on cellular and molecular levels are needed. Disclosures: No relevant conflicts of interest to declare. Disclosures: No relevant conflicts of interest to declare.

Abstract 4569 Background It is considered that leukemias posses a rare population of leukemia stem cells (LSCs) capable of the limitless self-renewal necessary for cancer initiation and maintenance. These cells also are believed to be responsible for the relapses of leukemias in patients and thus it is important to find the differences between normal HSCs and LSCs to create curative treatment of leukemias. We previously reported a model of transplantable myeloid leukemia in mice. In this model bone marrow (BM) and liver cells of affected mice were fully transplantable; recipients became moribund within 17-32 days since cells injection. Limiting dilutions analysis revealed that the concentration of LSCs in the BM of moribund mice was one LSC per 37000 c-kit+ cells and one per 45 cells from affected liver. Concentration of LSCs in other cell populations was calculated on the base of mice lifespan after transplantation and was shown to be one LSC per 2500000 c-kit-CD45-, 200 c-kit-CD45+, 4500 Ter119+ and 2600 Ter119- cells. Thus LSCs of this disease retain hierarchical organization and are able to differentiate at least among myeloid and erythroid lineages without the loss of self-renewing ability. Extremely high concentration of LSCs in the liver suggests the population of these cells being scaled up during invasion. The aim of this work was to investigate dynamics of liver repopulation by LSCs and to study additional molecular characteristics of leukemia initiating cells. Methods To trace the development of the disease female mice (C57Bl/6 x CBA) F1 were injected i.v. with 106 BM cells from syngeneic moribund donors. Each day after the injection the liver cells of mice were sorted into CD45+ and CD45- populations. RNA was isolated from both populations and expression of some genes was evaluated using real time PCR and conventional PCR. To assess the role of chemokines secreted by liver in the migration of LSCs the expression of chemokine receptors was analysed by means of PCR in the liver tissue of moribund mice. Results Taking into consideration that CD45+ cells comprise 45 percent of total cells in the liver by the terminal stage of the disease the concentration of LSCs among CD45+ cells in the liver may reach 1 per 23 and even higher. It allows isolating relatively homogenious population of LSCs for studying genes that play role in neoplastic transformation. The investigation of the dynamics of the disease showed that the weight of the liver and spleen began to grow on day 10 after cells injection. At the same time the dramatic rise of CD45+ cells occured in the liver and continued till death. Overexpression of genes responsible for self-renewal and proliferation (Bmi-1 and Myc) was revealed since the day 7. The expression of Bmi-1 and Myc in CD45+ cells in leukemic liver from moribund mice was 20-40 and 25-50 times higher than in CD45+ cells of control liver. The expression of Csf1r (M-CSFr) was elevated 100-fold, so this surface antigen may be served as a marker of LSCs in given disease. The analysis of house-keeping genes Rpl13a, Ubc, Hprt1 and Actb expression have shown that none of these genes fits for the normalization of cDNA amount, because the expression of them (by the terminal stage) in CD45+ cells of leukemic liver was elevated 45-, 25-, 98- and 59-fold as compared to CD45+ cells of control liver. It means that LSCs are essentially different from normal hematopoietic precursor cells. The expression analysis of genes coding the receptors of chemokines in the liver of moribund mice has shown that Ccr1 expression appeared and the expression of Ccr2 and Ccr5 increased in comparison with normal liver cells. It allowed to state that the tumor cells in liver express Ccr1 on their surface. This data suggests the role of chemokines secreted by liver and primarily the role of Ccl3 (Mip-1a) and Ccl5 (Rantes) in the migration of tumor cells into the liver. On the other hand this data allowed speculating on the nature of LSCs, because though Ccl3 and Ccl5 are chemoattractants for different subsets of leukocytes, they mainly attract granulocytes and monocytes. Conclusion Thus the model used is unique for the study of LSCs properties, the mechanisms of leukemogenesis, the migration and retention of LSCs extranodally in the dependence of specific 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 In acute and chronic leukemia, the changes in the basic properties of multipotent mesenchymal stromal cells (MSCs), including morphology, immunomodulatory abilities, and the expression of various genes, were described. The aim of the investigation was to study the ability of MSCs derived from the bone marrow (BM) of patients with acute lymphoblastic (ALL), myeloid (AML) leukemia and chronic myeloid leukemia (CML) to maintain normal hematopoietic progenitor cells. Methods The study included 14 patients with ALL, 25 with AML and 15 with CML. All work was conducted in accordance with the Declaration of Helsinki (1964). BM was aspirated from the patient during diagnostic punctures before the treatment and for patients with acute leukemia 37 and 100 days; for patients with CML on average at the 125th day and 220th day after the start of the treatment. The BM samples of 22 healthy donors were used as controls. MSCs were derived from 5-10 ml of BM and cultivated at the density 3 х 106 cells in T25 culture flasks in aMEM with 10% FCS. The ability of MSCs from patients to maintain normal hematopoietic precursor cells - coble stone area forming cells (CAFC) was performed by the limiting dilution method. One day prior to assay, MSCs from donors and patients were explanted with 1000 cells per well of 96-well plates. As a control, the cell line MS5 supporting growth of hematopoietic precursor cells was used. On the following day, with complete medium change, BM cells from healthy donors were implanted in four serial dilutions. The frequency of CAFC in the normal BM was performed using Poisson's equation and presented as the percent of control. The total RNA was extracted from MSCs at passage 1 using the standard method. The relative gene expression levels (REL) were determined by normalizing the expression of each target gene to the levels of beta-actin and glyceraldehyde 3-phosphate dehydrogenase and calculated using the ΔΔCt method. Results Total cell production of MSCs from ALL (5.6±1.5) x 106 and AML (5.4±1.0) x 106 patients decreased at the moment of diagnosis, whereas the production of MSCs from CML (7.9±1.9) x 106 patients did not differ from the donors (7.1±1.04) x 106. Reduced cell production is likely associated with a significant decrease in the expression level of FGF2, VEGF, BGLAP and SOX9 genes (Table). The ability of MSCs derived from the BM of AML patients at the onset of the disease to maintain normal CAFC (59.3±6.8) was significantly decreased (p = 0.02) when compared to donors (74.9±9.5). At the end of the first course of chemotherapy, the ability to maintain CAFC in patients' MSCs reached that of the donors(80.7±5.8); 2 months later, the CAFC frequency on MSCs from AML patients doubled (107.9±18.4) in comparison with the start of the disease (p = 0.04). The ability of MSCs derived from ALL patients to support CAFC was lower than that of MSCs of AML patients both before treatment (57.8±12) and 37 days after the start (57.2±7.4). Three months after the initiation of treatment of these patients, the ability of MSCs to maintain CAFC recovered (73.7±13.3) and reached that of the donors' MSCs. MSCs of CML patients (100.8±9.9) at the disease onset maintained CAFC better than donors' MSCs. This ability increased with treatment at 125th day (109.6±14.4) and at 220th day (169.6) after the start of the treatment. The expression levels of LIF, IGF1, IL6, CSF1 increased significantly in CML-derived as well as AML-derived MSCs, but changes were more pronounced in the case of CML. Table. REL of different genes in MSCs derived from patients at the onset of the disease. Gene AML ALL CML Donors FGF2 2.8±0.2* 2.0 ±0.4* 4.2±0.5 6.0±0.9 VEGF 1.5±0.1* 1.5±0.2* 1.7±0.2 2.5±0.9 LIF 9.1±1.3* 7.1±2.3* 21.8±7.5 2.2±0.4 IL6 12.7±1.8* 11.3±4.4 22.5±4.8* 6.2±1.8 CSF1 3.6±0.9* 2.1±1.2 0.96±0.1* 0.7±0.1 IGF1 1.3±0.3* 1.49±0.7 2.6±0.5* 0.6±0.1 BGLAP 0.6±0.2* 0.8±0.4* 1.7±0.4 2.7±0.1 SOX9 1.2±0.1* 0.9±0.3* 1.2±0.2 1.7±0.2 (*significantly different from donors) Conclusions Functional changes in MSCs, which are the part of BM stromal microenvironment and, in particular, a niche for the HSCs was revealed. The ability of MSCs to support CAFC is dramatically changed in patients with hematological malignancies; the nature of the functional alterations of MSCs depends on the diagnosis. In cases of acute leukemia, MSCs' ability to maintain CAFC normalized with the treatment, whereas the strengthening of this capacity was revealed in cases of CML. Disclosures Turkina: Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.

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.