ALBERT

Beschreibung: 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.

Beschreibung: 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

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: Background. Aplastic anemia (AA) is a disorder characterized by pancytopenia, hypoplastic bone marrow (BM), and the absence of underlying malignancy. It is believed to be of autoimmune nature. However, some patients fail to respond to the immunosuppressive therapy. The impaired hematopoietic microenvironment could be another reason for BM failure. The severity of AA varies widely from mild, chronic pancytopenia to total hematopoietic failure. The diagnosis of severe (SAA) and non-severe AA (NAA) is based on an absolute neutrophil count as an essential criterion. The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in BM of untreated SAA and NAA patients. Methods. The study included 22 AA patients (8 with SAA and 14 with NAA) in the debut of the disease. In all patients BM was aspirated after informed consent at diagnostic punctures. The proportion of non-hematopoietic CD45-CD34-CD71-CD235-CD90+CD73+CD105+ cells was estimated by FACS. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. Individual CFU-F-derived colonies were analyzed for their proliferative and differentiation potential. Adipogenic and osteogenic differentiation potential was analyzed with standard techniques. Relative expression level (REL) of several genes had been estimated with RT-PCR in Taqman modification. As a control 19 BM samples of healthy donors of according age were used. Results. The data are presented in the table. The proportion of non-hematopoietic cells was higher in the BM of AA patients than in healthy donors. We recalculated the proportion of CFU-F among non- hematopoietic cells; it was similar in the BM of AA patients and healthy donors. However, the concentration of -CFU-F was much higher in the BM of patients with SAA then in the BM of patients with NAA. Among NAA patients, 2 had PNG clone and unlike other NAA patients increased CFU-F concentration, comparable to patients with SAA. It seems that the character of stromal cell damage depends on the severity of AA. Individual CFU-F- derived clones from the BM of NAA patients had very limited proliferative potential, while those of SAA patients did not differ from colonies of healthy donors. The analysis of CFU-F-derived colonies differentiation ability revealed that the proportion of the precursors that did not respond to the differentiation induction was higher in the BM of AA patients than in donors. It reflects the involvement of a certain subpopulation of stromal precursors that are either pre-differentiated into fibroblasts, or, conversely, earlier precursors of the hematopoietic microenvironment, which were not able to differentiate into osteoblasts and/or adipocytes within standard time. The analysis of the MMSC growth characteristics revealed that the time required for MMSC from SAA and NAA patients to form a confluent monolayer after the initial seeding and the population doubling time, were significantly higher than in MMSC of healthy donors. Thus, the proliferation rate of MMSC of AA patients is reduced. Nevertheless, the total cell production for 3 passages did not differ in cultures of AA patients and healthy donors. Therefore, the proliferative potential of MMSC of AA patients is not altered. Probably MMSC being analyzed ex vivo can restore their function. However, the analysis of REL of genes regulating the proliferation (FGF2, FGFR1, FGFR2) in MSCs had revealed the differences in comparison with donors and between SAA and NAA. Moreover, the analysis of the polymorphism in CFH gene, participating in immunomodulation, showed that the distribution differs between NAA and SAA patients. Conclusions. Stromal precursors in BM of untreated NAA and SAA patients are impaired and differ between the two subtypes of AA. It seems that the differences between NAA and SAA may lay not only in the absolute neutrophil count but also in the BM stroma itself. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the hematopoiesis failure. This work was supported by the Russian Foundation for Basic Research, project no. 19-015-00280. Table Disclosures No relevant conflicts of interest to declare.