ALBERT

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: 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.

Description: 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

Description: The stromal microenvironment regulating hematopoiesis in patients with hematologic malignancies undergoes significant alterations. The changes in the concentration of colony-forming fibroblast units (CFU-F) in the bone marrow (BM) and disruption in the functioning of multipotent mesenchymal stromal cells (MSCs) are shown in many studies for patients with acute leukemia. Often it is not possible to distinguish the cause of changes in stromal progenitor cells after treatment: interaction with tumor cells or the effects of therapy. Most of the patients with diffuse large B-cell lymphoma (DLBCL) do not have BM involvement. It was assumed that the properties of MSCs in these patients were not changed, and so this could be an attractive model for investigation the effect of antitumor drugs on human BM stromal microenvironment. The aim of the study was to compare the properties of MSCs in patients with DLBCL in the onset of the disease and a month after the end of therapy. Methods The study included 20 patients with DLBCL (11 male, 9 female) aged 42-60 years in the onset of the disease and a month after the end of treatment. 3-5 ml of BM were collected during diagnostic punctures after informed consent. MSCs and CFU-F were cultured by standard methods. The total MSCs production, the doubling-population level per day, the concentration of CFU-F, the relative gene expression level (REL) in MSC by real-time PCR and the mean fluorescence intensity (MFI) by flow cytometry were analyzed. The control group included 31 donors of the corresponding age. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Results The total cell production for 4 passages in primary patients' MSCs was significantly higher than in donors (11.4 ± 2 x 106 per flask versus 6.9 ± 1.1, p = 0.04). It remained elevated after therapy (10.2 ± 1.5). At the same time, the MSCs population-doubling level per day was significantly decreased in patients in comparison with donors (0.6 ± 0.03 vs. 0.4 ± 0.04, p

Description: Background: Recent evidence suggests that MSCs might improve survival during sepsis in animal models. However, no study has investigated the effects of MSC therapy on the survival of pts with sepsis and SS, especially severe-neutropenic pts. Aim: The aim of this study was to investigate the efficacy of the administration of MSCs for the treatment of SS in neutropenic pts. Patients and Methods: This prospective, single-center, randomized Russian clinical trial of MSCs in severe neutropenic pts with SS (RUMCESS) (NCT 01849237) was approved by the local ethics committee and was begun in December 2012. Neutropenic pts (WBC 〈 0.5x109/l) with SS were enrolled on to the study. The pts were randomly assigned (1:1) to receive either conventional therapy (CT) of SS (CT group), or CT plus donor MSCs at a dose of 106/kg intravenously within the first 10 hours after SS onset (CT+MSCs group). Written informed consent was obtained for all pts. All pts were admitted and treated in the ICU of the National Research Center for Hematology (Moscow). The Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sepsis-related Organ Failure Assessment (SOFA) score were employed to determine the severity of illness. Pts were followed up for 28 days after enrolment in the study, and 28-day all-cause mortality was assessed. Pts characteristics and complication rates were compared using Fisher's exact test. The Kaplan-Meier method with the log-rank test, and Cox proportional hazard regression model were used to determine the statistical significance of the relationship between overall survival (OS) and the treatment regimen. Statistical analyses were performed using SAS 9.1. Results: Of the 27 neutropenic pts with SS, 13 received CT and 14 received CT+MSCs. There were no statistically significant differences in the demographic variables between groups . The CT group included 7 males, 6 females, aged 33-81 yrs, median 55 yrs. The CT+MSCs group included 6 males, 8 females, aged 30-75 yrs, median 48 yrs. Hematological disorders were also similar in the two groups: AML (4), NHL (4), HL (1), MM (3), MDS (1) in the CT group, and AML (5), NHL (7), MM (1) in the CT+MSCs group. In all pts, except for one with MDS, neutropenia developed after chemotherapy. In 8/13 pts in the CT group and 9/14 pts in the CT+MSCs group blood cultures were found positive, mostly gram-negative. Baseline APACHE II scores (34.2 [95% CI 28.3-43.1] and 32.2 [95% CI 26.2-37.5] in the CT- and CT+MSC-groups, respectively), and SOFA scores (17.9 [95% CI 13.5-22.2] and 15.1 [95% CI 11.0-19.2] respectively), were similar in the two groups. 28-day survival rates were 15% (2 out of 13 pts) in the CT group and 57% (8 out of 14 pts) in the CT+MSCs group (P=0.04) (Figure 1). The significant increase in 28 days OS of the pts in CT+MCSs group was associated with SOFA score decrease, which was started in three days after onset of SS. Despite higher 28-day survival rates only 3 pts treated with CT+MSCs remained alive after 3 months, and 5 of 8 pts from the CT+MSCs-group who survived 28 days died later because of sepsis-related organ dysfunction. Conclusions: Administration of MSCs in the first hours of SS might improve short-term survival in neutropenic pts, but does not prevent death from sepsis-related organ dysfunction in the long term. Perhaps repeated administration of MSC is required. Figure 1. Comparison of OS rates between the two groups of pts in the ICU. There was a statistically significant increase of the 28-day OS rates (42% vs. 15%; P=0.04) and a statistically significant decrease of the risk of death (HR 0.35; 95% CI 0.13 - 0.91; P=0.04) in the CT+MCSs group vs. the CT group. Figure 1. Comparison of OS rates between the two groups of pts in the ICU. There was a statistically significant increase of the 28-day OS rates (42% vs. 15%; P=0.04) and a statistically significant decrease of the risk of death (HR 0.35; 95% CI 0.13 - 0.91; P=0.04) in the CT+MCSs group vs. the CT group. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 4899 Background: Abnormal karyotype in bone marrow (BM) cells is detected in 30–70% of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias with myelodysplasia-related changes (AML). The same cytogenetic abnormalities are found in isolated CD34+ hematopoetic progenitor cells (HPCs) of these patients. According to recent studies cytogenetic abnormalities are described in 10–70% MDS and AML patients in BM derived mesenchymal stromal cells (MSCs) [Flores-Figueroa et al. 2008, Blau et al. 2011]. These abnormalities can be clonal and nonclonal (spontaneous). The goal of the study was to characterize and compare the cytogenetic changes in BM derived MSCs and in CD34+ HPCs isolated from BM and peripheral blood (PB) in MDS and AML patients. Patients and methods: The data from 35 patients is presented: 6 pts with AML (1 pt with therapy-related AML), 29 pts with MDS (refractory anemia − 4 pts, refractory cytopenia with multilineage dysplasia − 13 pts, refractory anemia with ringed sideroblasts − 2 pts, refractory anemia with excess blasts − 7 pts, 5q-syndrome – 3 pts). Median age was 60 years (range 19 to 77). Patients' assignment to different groups was made according to the 2008 World Health Organization (WHO) classification. Cytogenetic analysis was performed using G-banding and FISH. MSCs were derived from bone marrow mononuclear cells then plated in 75 cm3flacks and harvested after 2–5 passages. CD34+ HPCs were collected from BM and PB by magnetic separation with anti-CD34 MicroBead Kit human (Miltenyi Biotec). Results: BM karyotype was normal in 17 pts, cytogenetic abnormalities were found in 18 pts − 4 AML and 14 MDS: in 9 pts (2 AML/7 MDS) – isolated del(5q), monosomy 7, i(14), inv(3) or trisomy 8; in 8 pts (2 AMl/6 MDS) – two or more abnormalities; and 1 pt displayed only constitutional abnormality (inv(9)(p13q21)). It is worth to note that routine cytogenetic analysis didn't present any mitosis in 3 MDS pts however FISH analysis revealed del(5) and del(7) simultaneously in one of these pts, trisomy 8 in one pt and normal karyotype in another one pt. Cytogenetic analysis of MSCs was carried out in 23 pts – 4 AML and 19 MDS pts. BM karyotype was normal in 10 pts and abnormal in 13 pts. Karyotype in MSCs was normal in all AML pts and in 16 MDS pts. There were cytogenetic changes in 3 MDS pts: in 1 pt - constitutional inversion (inv9(p13q21)), in 1 pt - nonclonal translocation and constitutional inversion (46XY,t(2;22)(p10;q11),inv(9)(p13q21)[1]/46XY,inv(9)(p13q21)[19]) and in 1 pt - clonal abnormalities (46XY,add(2q)[7]/46XY[13]). Patient with clonal cytogenetic abnormalities in MSCs had complex karyotype in BM (45–46XY,−5,−13,der(19),add(q13?or p13?),−20,+marx2,+mar del(13q21)?[15]/45–46XY idem, +dmin[2]/46XY[3]), but these changes were different. Constitutional inversion was confirmed in both cases by cytogenetic analysis in PB lymphocytes. FISH analysis was performed in CD 34+ HPCs magnetically isolated from BM and PB in 24 pts. Fourteen of these pts had normal BM karyotype and we confirmed that using FISH with LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8 (Vysis). We used the same DNA probes for evaluation of CD34+ HPCs from BM and PB in pts with normal BM karyotype. We used FISH in 10 pts with ascertained cytogenetic abnormalities in BM to confirm these findings with DNA probes LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8, LSI AML1/ETO, CEP X/CEP Y, LSI (20q12) (Vysis). FISH analysis didn't reveal any aberration in BM and PB CD34+ HPCs in patients with normal BM karyotype. In pts with distinguished abnormalities in BM the same pattern was demonstrated by FISH in CD34+ HPCs. We counted the percent of abnormal nuclei per 200. The means of percentages by FISH of BM, BM CD 34+ HPCs and PB CD34+ HPCs didn't differ and constituted 65.8%, 73.1% and 74.8% respectively. Conclusion: Cytogenetic analysis in MSCs revealed aberrations only in MDS patients (3 from 16). No cytogenetic abnormalities in MSCs in AML pts was found. Karyotype in MSCs didn't coincide with BM karyotype in the same patients. Hematopoetic progenitor cells from BM and PB of MDS and AML patients displayed the same cytogenetic abnormalities as the full population of bone marrow cells. The data shows that hematopoetic and mesenchymal precursors in MDS and AML are cytogenetically distinct. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1256 Acute and chronic graft-versus-host disease (GvHD) develops in more than 50% patients after hemopoietic stem cell transplantation (HSCT) and remains one of main causes of mortality. That's why it is still very important to find new methods of GvHD prophylaxis and treatment. There is a wellknown algorithm of GvHD treatment with glucocorticoids as first line and there are many other options in the treatment of steroid-resistant GvHD, including multipotent mesenchymal stromal cells (MMSC). The efficacy of MMSC in the treatment of steroid-resistant GvHD was reported to be 71–94% (Le Blanc et al., Lancet, 2008; Kebriaei et al., Biol Blood Marrow Transplant, 2009). There are few data on using MMSC as GvHD prophylaxis. So we designed a trial based on a random patient allocation in two groups: receiving standard GvHD prophylaxis (cyclosporine A + methotrexate, cyclosporine A + methotrexate + prednisolon, or cyclosporine A +methotrexate+ mycophenolate mofetil) and receiving MMSC in addition to the standard GvHD prophylaxis. The goal of the study was to investigate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. MMSC were derived from the bone marrow of hemopoietic stem cells donors and cultured in alpha-MEM with 4% human platelet lysate derived from peripheral blood of the same donors. The bone marrow sample was aspirated at the day of HSCT. The trial was approved by local ethic committee and started in October, 2008. Currently 22 patients with stem cell transplantation from related donors are randomised. 10 patients received standard GvHD prophylaxis and 12 received MMSC in addition. 5 patients received fresh cultivated MMSCs while others – MMSCs after freezing and thawing procedures, carried out in standard conditions. Patients characteristics are presented in the Table 1. MMSCs were administered at the time of blood counts recovery. The dose of MMSC was 0.9–1.25×106/kg. The median day of administration was + 30 day after HSCT (25-54 dd). Most of the patients had fever and chills after MMSC administration. There were no other complications. The clear difference in acute GvHD development was observed in two groups. In standard prophylaxis group acute GvHD II-IV developed in 50% patients, in MMSC group no acute GvHD II-IV occurred (p=0,009). Two patients in the second group had acute GvHD I developed before MMSC administration. The regression of symptoms was observed after MMSC injection. Chronic GvHD was diagnosed in 40% patients in the first group and in 16.7% patients in the second group. There was no difference in disease recurrence rate and graft rejection rate. More infectious complications occurred in patients from MMSC prophylaxis group, mostly of viral origin. There were no lethal infections in this group. The overall mortality was similar in both groups (20% compared to 8.4% in MMSC group). The results of our small study clearly demonstrate the efficacy and safety of MMSC administration as graft-versus-host disease prophylaxis. We intend to continue the study and we incorporated the MMSC as treatment option in steroid-resistant GvHD. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice. At 112, 3, 6, 9, and 12 months after reconstitution, the bone marrow cells were repeatedly collected from each mouse under light anesthesia and the number of colony-forming unit-spleen (CFU-S), spleen repopulating ability (SRA), and the integration of human ADA gene were studied in CFU-S–derived colonies by polymerase chain reaction (PCR) and Southern blot hybridization analyses. After 9 months, the proportion of donor CFU-S detected by PCR with a Y-chromosome–specific probe in mice reconstituted with mobilized PBSC was 75.3% ± 6.0%, which is similar to the concentration of donor CFU-S seen after bone marrow transplantation. Similarly, there was no difference in the concentration of CFU-S in mice reconstituted with transduced mobilized PBSC or bone marrow cells. However, in both cases the CFU-S content in the bone marrow was reduced fivefold to 10-fold compared with the concentration of CFU-S in mice transplanted with nontransduced bone marrow. The SRA of CFU-S in mice reconstituted with peripheral blood and bone marrow cells was the same 1.5 months posttransplantation, but after an additional 4 months, SRA of mice reconstituted with bone marrow cells was fivefold higher as compared with those engrafted by PBSC. The integration of the human ADA gene was observed during 9 months in about 60% of studied CFU-S. The proportion of marked colonies sharply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridization in the same reconstituted mice during the whole period of observation. The time of clone existence was about 3 months. We conclude that long-term marrow repopulating cells mobilized into circulation by treatment with granulocyte colony-stimulating factor (G-CSF ) and stem cell factor (SCF ) are capable of maintaining lifelong polyclonal hematopoiesis in reconstituted mice.