Severe Hypoxia Promotes Selection of Repopulating Progenitor CELLS in Primary MDS BONE Marrow CELL Culture
Authors: Masala Erico , Buchi Francesca , Valencia Ana , Gozzini Antonella , Sanna Alessandro , Bosi Alberto , Sbarba Persio Dello , Santini Valeria 
Published: October 2019

Abstract
Abstract 4747

Introduction:
Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. In this study, we evaluated the repopulating ability of hypoxia selected cells obtained from primary MDS bone marrow cultures. We then characterized the “stemness” of MDS maintaining cells in different subtypes of this disease.

Methods:
We studied 31 samples obtained at diagnosis from patients with different WHO subtypes of MDS (6 RA, 12 RCMD, 7 RAEB, 6 AL/post MDS). Mononuclear bone marrow cells were isolated after density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 20 % FBS and a cocktail of cytokines (TPO, FLT3-L, SCF, IL-3). Cells were incubated and selected in Ruskin Concept 400 anaerobic incubator, in severe hypoxia conditions by flushing with a performed gas mixture (0,3 % O2,5%CO2,95% N2), for 10–13 days (LC1) and daily counted (by Trypan blue dye). The stem and progenitor cell potential of these cultures at different times of incubation was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, according to the Culture-Repopulating Ability assay methodology (Cipolleschi et al, Leukemia 2000). At the same time, we evaluated clonal potential of selected cells by semisolid culture in methylcellulose, in the presence of the same cocktail of cytokines. The phenotype of hypoxia selected cells was evaluated by determining the expression of CD34, CD38, CD117, CD133, as well as beta-catenin expression and localization at fluoresence microscopy. When present, we analyzed by FISH the persistence of chromosomal aberrations in MDS cells, before and after hypoxic selection.

Results:
The hypoxic culture system allowed selection of a minute cell population: in 31/31 cases viable cell number decreased of one log after 10–13 days of culture in extreme hypoxic conditions. Only 11/31 MDS cases showed a significant repopulating ability at day 17 of LC2 and in the same cases we observed parallel formation of colonies ( CFU-GM and CFU-GEMM) in semisolid cultures. Ten of these eleven cases that showed repopulating ability belonged to the Low/INT1 IPSS risk category. In the other 20, all IPSS high and INT-2 risk cases, repopulating ability was absent. In particular, MDS cases presenting > 10% blasts in the bone marrow did not show repopulating cells after selective hypoxic conditions. In 12 cases, after hypoxia selection we observed a reduction in the number of CD34+ cells. Within this shrinked CD34+ population, we could isolate highly repopulating progenitors CD34+ /CD38−, that confirmed the selection of earlier hematopoietic progenitor cells. We analyzed by interphase FISH MDS cases presenting chromosomal aberration ( +8, −Y, complex karyotype and del7q). In all cases, the marker chromosomal aberrations were maintained after hypoxic culture. In particular, in one of these cases with marker chromosomal aberration, the percentage of cells carrying –Y at interphase FISH was 66% at baseline, and 82% after hypoxic selection, while 66% of the repopulating cells, after 8 days of normoxic LC2, still had −Y.

Conclusions:
We demonstrate here that it is possible to select by severe hypoxic conditions primary MDS progenitor cells with repopulating ability. MDS cases in earlier phases of the disease still show near-normal repopulating abilityafter hypoxic selection, whereas more progressive dysplastic disease is accompanied by a loss of repopulating ability, as measured by CRA assay and colony formation. Functional characterization of the neoplastic isolated repopulating cells is warranted.

Disclosures:
Santini: Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.