MEASUREMENT OF THE GROWTH-PARAMETERS OF PRECURSOR B-ACUTE LYMPHOBLASTIC LEUKEMIC-CELLS IN COCULTURE WITH BONE-MARROW STROMAL CELLS - DETECTION OF 2 CD10 POSITIVE POPULATIONS WITH DIFFERENT PROLIFERATIVE CAPACITIES AND SURVIVAL

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent pro be which becomes incorporated into the plasma membrane upon the staini ng of cells. As the amount of probe per cell reduces at each cell divi sion, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were staine d with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cel l layers. Viable leukaemic cells from these cultures were identified o n the basis of forward light scatter, 90 degrees light scatter, propid ium iodide exclusion, PKH 26 GL staining and CD10 expression by flow c ytometry at the beginning of the culture period and on days 2, 6, 10 a nd 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with s tromal cells compared with control cultures, without stromal cells. in the presence of stromal cells, survival and proliferation continued t hroughout the culture period; in contrast in five of seven control cul tures no viable cells could be detected after 6-10 days. Interestingly , two populations of leukaemic cells were distinguished on the basis o f their different rates of proliferation, when co-cultured with stroma l cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stro ma-dependent system.