P767: comparison of proliferative, differentiation potential and gene expression in multipotent mesenchymal stromal cells of patients with non-severe and severe aplastic anemia in the debut

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Aplastic anemia (AA) is a rare disease manifested in hematopoiesis disruption due to decreased number and dysfunction of hematopoietic stem cells. According to severity of pancytopenia there are 3 forms of AA: non-severe AA (NAA), severe AA (SAA) and very severe AA. AA origin is mainly of autoimmune nature, but detailed pathogenesis is not fully described. The role of bone marrow (BM) stromal cells in the pathogenesis of AA is not clear. It is known that AA patients’ BM derived multipotent mesenchymal stromal cells (MMSCs) are altered in gene expression and proliferative and differentiation potential. However, the differences of MMSCs from patients with various forms of AA were not characterized due to the AA rarity and the difficulties in MMSCs cultivation. Characterization of BM stromal precursors in various forms of AA is important for understanding its pathogenesis. Aims: To reveal differences in proliferative properties, differentiation potential and gene expression pattern of NAA and TAA patients’ MMSCs compared to donors’ ones. Methods: The study included 22 patients with NAA (11m, 11f, 21-51 y.o., median 31) and 16 patients with TAA (8m, 8f, 18-63 y.o., median 27) at the onset of the disease. The control group included 30 healthy donors (17 m, 13 f, 14-61 y.o., median 28.5). Patients’ BM was obtained during a diagnostic punctures, donors one - during planned exfusions after informed consent was signed. MMSCs were isolated according to the standard method and cultured up to passage 3. Time to passage 0 (P0), the population doubling time (PDT), and the total cell production were determined. The differentiation potential of MMSCs was analyzed by expression level of marker genes for osteogenic (ALPL, PTHR1) and adipogenic (FABP4, PPARG) lineages after corresponding induction. MMSCs incubated in the standard medium were used as controls. Gene expression was analyzed with real-time PCR in the TaqMan modification. Data are presented as mean ± standard error of the mean. For normal distribution, the significance of differences was determined with Student t-test; otherwise, Mann-Whitney test was applied. Differences were considered significant at p<0.05. Results: Differentiation potential of MMSCs from patients with NAA, TAA, and donors did not differ between the groups. We revealed no differences in the total cell production of MMSCs in patients with NAA and TAA compared to donors, therefore, the proliferative potential of patients’ MMSCs is not altered (Table 1). The PDT of NAA patients’ MMSCs was increased compared to donors ones, which indicated a decrease in the proliferation rate of MMSCs in this group. The time to P0 in both groups of AA patients was longer than in donors. Gene expression had changed similarly in NAA and TAA MMSCs when compared to donors (Table 2). The expression of growth factor receptors genes (FGFR1, FGFR2, PDGFRA, PDGFRB) and NES was upregulated; the expression of some immune regulation (IL10, HLA-DRA) and regulation of hematopoiesis (ANGPT1) genes was downregulated. Upregulation of PDGFRA in NAA patients’ MMSCs compared to TAA patients and donors ones was found, probably, associated with a decrease in their proliferation rate.Summary/Conclusion: NAA, TAA patients’ MMSCs have a similar pattern of gene expression alterations; they do not differ from donors ones in proliferative and differentiation potential. NAA patients’ MMSCs reveal a decreased proliferation rate and upregulated PDGFRA gene expression. Keywords: Gene expression, Severe aplastic anemia, Aplastic anemia, Mesenchymal stem cell