European Society for Paediatric Research Abstract

CLONOGENIC RESPONSES OF SELECTED CD 34+LI- PRECURSORS IN STROMA DEPENDENT SYSTEMS FROM PATIENTS WITH MYELODYSPLASTIC SYNDROMES

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Abstract 16

The clonogenic properties of bone marrow (BM) CD34+ progenitors in 12 adults with MDS (FAB: RA: 4, RARS: 1, RAEB: 2; RAEB-T: 2, CMML:3; median age: 60 range 19-74) and 4 children (CMML/JMML; age 3.5, range 1,6-4) was studied. All had features of dysplasia in at least 2 lines and were transfusion dependent. BM mononuclear cells (MNC) from normal individuals or MDS patients were lymphocyte and monocyte depleted and CD34+ population selected with paramagnetic beads (Dynal, Oslo). From another aliquot 5 × 105/mL MNC were used to prepared stromal layers in aMEM in the presence of hydrocortisone, foetal calf and horse sera. At 4-6 weeks confluent stroma was seeded in cross-culture assays with 5 × 103 control or MDS CD 34+cells and cultured for 5 days without any growth factors. Aggregates (>20 cells) were scored as CFU-bl. Stroma non-adherent cells were eluted and cultured in the CFU-GM assay. CD 34+ Lin-cells (median 92%) from MDS patients produced significantly fewer CFU-bl (median 28[SD 52.82]) than control cells (median 147.5 [SD 54.49]; p=0.0002). The stroma non adhesive progenitors were also significantly reduced (median 24 [SD 82.19]) compared to control samples (230 [SD5.05]; p=7.3-5). The median % surface area covered by stroma was 80 (SD 30.42) compared to normal values (98 [SD 4.54]; p=0.001) and they supported poorly the proliferation of control CFU-bl (86[SD 58.96]; p=0.05). Stroma also survived poorly and lifted in6 patients at 6 week. When cells from patients with CMML were compared to those from JMML, the latter produced significantly higher number of CFU-bl (128.5 [SD 21.85] vs 21[SD 13.5]; p=0.0003) while their stroma non-adhesive precursors were significantly reduced (35.5 [SD 10.87] vs 179 [SD 144.85]; p=0.05). It is concluded that CD 34+Li- precursors from MDS patients have a poor proliferative capacity and their stroma supports heaematopoiesis suboptimally. However, in JMML patients, stroma adherent precursors gave appropriate clonogenic responses and the stroma behaved normally.

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