PB1852 PROLIFERATION CAPACITY OF STROMAL PRECURSORS FROM THE BONE MARROW OF UNTREATED APLASTIC ANEMIA PATIENTS
Authors: I. Shipounova, N. Petinati, A. Dorofeeva, N. Drize, A. Luchkin, Z. Fidarova, E. Mikhailova, V. Savchenko
Journal: HemaSphere (2019)

Background: Aplastic anemia (AA) is a rare bone marrow (BM) failure disorder, characterized by pancytopenia due to hematopoietic stem cells damage. First line treatment for AA consists of immunosuppressive drugs (anti-thymocyte globulin combined with cyclosporin A). Allogenic hematopoietic stem cell transplantation (allo-HSCT) is preferable for the patients of young age having HLA-matched BM donor. Aims: The aim of the study was to analyze the multipotent mesenchymal stromal cells (MMSC) and fibroblasts colony forming units (CFU-F) in the debut of AA. The changes caused by allo-HSCT would also be evaluated. Methods: The study included 12 patients in the debut of AA. Two of them later had been transplanted with allogenic hematopoietic stem cells and had also been analyzed for 1 year after the procedure. In all patients BM was aspirated after informed consent. From the BM, MMSC were isolated by the standard method and the concentration of CFU-F was determined. The relative expression level of various genes in MMSC was determined by Taqman real-time PCR. As a control 33 MMSC samples from the BM of healthy donors of according age were used. Results: Concentration of CFU-F in the BM of AA patients in debut was higher than in donor BM, although the difference was not significant (56.8 ± 13.2 versus 29.6 ± 6.5 per 106 mononuclear BM cells in donors). The square of CFU-F colonies from AA patients was significantly larger than donor ones (27.6 ± 1.8 points versus 16.65 ± 1 in donors, p < 0.05). Analysis of proliferative potential of individual CFU-F colonies was performed for 4 newly diagnosed patients by far. CFU-F colonies from the two patients were not able to proliferate after the passage, from another two patients, on the contrary, CFU-F colonies demonstrated high proliferative potential. When compared with CFU-F colonies from 6 analyzed donors, CFU-F colonies from AA patients started to proliferate earlier, and the total clones’ number was higher. Individual CFU-F colonies both from donors and AA patients varied in their proliferation and differentiation potential. MMSC cultures were successfully obtained from the BM of 10 out of 12 newly diagnosed AA patients. The cumulative MMSC production on passages 0, 1 and 2 was significantly higher than in donor cultures. MMSC of two patients had been also analyzed for 1 year after the allo-HSCT. All values were compared with 95% CI of corresponding donor parameters. CFU-F concentration 1 month after allo-HSCT was higher than in donors. This makes a sharp contrast with the data on CFU-F concentration in patients with acute leukemia after allo-HSCT, where the dramatic decrease was observed. The cumulative MMSC production was not changed in AA patients. However, we observed a few changes in gene expression, typical for both patients and persisting for a 1 year at least. The expression of CFH, PTGES, CSF1, TGFB1, and TGFB2 was deeply downregulated; JAG1, FGF2 and IL6 were downregulated right after the allo-HSCT, but became overexpressed 1 year later. Summary/Conclusion: Stromal precursors in newly diagnosed AA patients are functionally activated and have higher proliferative capacity in comparison with donors. This effect could participate in the pathogenesis of AA or be the consequence of compensatory reaction of stromal microenvironment to the inhibited hematopoiesis. It should be scrutinized further. This work was supported by the Russian Foundation for Basic Research, projects no. 19-015-00280 and 12-04-00457.