Hypoxia Increases Repopulating Ability of Myelodysplastic Syndrome Bone Marrow Cells

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. We evaluated the repopulating ability and stem cell potential of hypoxia maintained primary bone marrow (BM) progenitors derived from Myelodysplastic Syndromes (MDS) patients, and their capacity of engraftment in sublethally irradiated NOD-SCID mice.

Methods: Thirty eight BM samples were obtained at diagnosis from MDS patients (WHO: 9 RA, 12 RCMD, 7 RAEB, 8 AML/post MDS, 2 Del(5)q). Mononuclear BM cells were isolated and cultured with TPO, FLT3-L, SCF, IL-3 in severe hypoxic conditions (0,3%O₂, 5%CO₂, 95%N₂), for 10-13 days (LC1). The stem cell potential of these cultures was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, and according to the Culture-Repopulating Ability (CRA) assay methodology, cell proliferation was evaluated daily. Expression of CD34, CD38, CD117, CD133 was determined before and after hypoxia cultures, and when present, the persistence of chromosomal aberrations was analyzed by FISH at various stages of cell culture. In parallel, MDS mononuclear BM cells were intravenously injected in sublethally irradiated NOD-SCID mice before hypoxic culture and after LC1. Mice were sacrificed at day 56 and 90 after graft. The presence of CD45+ human cells in the peripheral blood of injected mice was evaluated by flow cytometry analysis every two weeks, and quantitated to evaluate the repopulating ability and the different engraftment capacity of cells before hypoxic culture and after hypoxic incubation. Mice marrow trephines as well as spleens were also evaluated morphologically to test the engraftment of human MDS cells.

Results: In all 38 MDS cases studied, cultured cell number decreased of one log or more after 10-13 days of culture in hypoxic conditions. In 12/38 MDS cases we observed a significant repopulating ability at day 17 of LC2, all classified as Low/INT-1 IPSS risk category. In IPSS high and INT-2 risk cases, repopulating ability according to CRA method was absent; CD34+ cells were always decreased after hypoxia and did not coexpress CD38, confirming the selection of earlier hematopoietic progenitor cells. We demonstrated the persistence of chromosomal aberrations in CD34pos/CD38neg cells after hypoxic culture in 5 MDS cases (del5q, +8, del20q, -Y, complex karyotype). Sublethally irradiated NOD-SCID mice transplanted with hypoxia cultured cells showed in peripheral blood an higher percentage of CD45+ human cell than mice transplanted with non hypoxia selected cells. In particular, mice transplanted with hypoxia cultured BM mononuclear cells derived from a 5q- MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with non-hypoxia selected cells at day 28 after transplant (5,5% vs 0,56 %); mice transplanted with hypoxia cultured BM mononuclear cells derived from a CRDM MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with cells before hypoxia at day 56 (13,3% vs 1,4%), and at day 71 (43,05% vs 12,7%). Mice bone marrow cytometry analysis confirmed higher percentage of CD45+ cells in bone marrow derived from mice transplanted with hypoxia cultured cells than non-hypoxia cultured (1,9 % vs 0,1%).

Conclusions: Severe hypoxic culture conditions are maintaining a sub-population of MDS cells endowed with increased repopulating capacity in vitro and in vivo. Most importantly, in our cohort of cases studied, repopulating ability was observed exclusively in IPSS lower risk MDS cases.