Reduced growth of normal myeloid progenitors on MDS stroma is unrelated to confluence

Ineffective haemopoiesis in patients with myelodysplasia (MDS) may be a consequence of defective marrow stromal cells. We have measured the growth of normal haemopoietic progenitors from cord blood mononuclear cells (MNC) in secondary long-term cultures on irradiated stromal layers grown from the marrow of normal subjects and MDS patients. Stromal layers were established in 25 cm2 flasks by inoculating 20 x 1 O6 bone marrow MNC in 10 ml of modified McCoy's 5A medium (glucose reduced to 400 mg/l and supplemented with 450 mg galactose/l) buffered with 40 mM HEPES.The proportion of the flask surface covered by stromal cells was estimated after 5 weeks primary growth (confluence %) and layers irradiated (10 Gy). Secondary cultures were established by re-inoculating with 5 x lo6 MNC from cord blood. Confluence ranged from 60-100% in normal cultures (n = 25; median = 95%) and 40-100% in MDS cultures (n = 26; median = 90%; ns). After 5 weeks secondary culture the CFU-GM from cord blood had a median value of 58.0/culture when grown on normal stroma (n = 20) and 4.3 on MDS stroma (n = 19; p = 0.0002). Median erythroid colonies numbered 15/culture with normal stroma (n = 13) and 0 with MDS stroma (n = 19; p = 0.004). In cultures with 90% confluence, or more, the median CFU-GM were 98.9hormal culture (n = 13) and 6.1IMDS culture (n = 12; p = 0.002); and the median period of CFU-GM detectability was 10 weeks on normal stroma (n = 9) and 5 weeks on MDS stroma (n = 13; p = 0.0005). There was little evidence of correlation between confluence and secondary CFU-GM numbers in weeks 4 to 7; but the period of CFU-GM detectability was correlated to confluence on normal stroma (n = 16; r, = 0.6463; p = 0.006) but not on MDS stroma (n = 20; r, = 0.2964; ns). Mean culture pH values were identical at week 5 (normal = 7.13, n = 23; MDS = 7.13, n = 19; ns) suggesting there was no difference in the underlying metabolic activity. We conclude that stromal layers from MDS marrow are defective in their ability to support normal haemopoiesis; and the variation in this defectiveness is greater than the measured variation in confluence and is independent of it