INFLUENCE OF CYTOKINES ON THE GROWTH-KINETICS AND IMMUNOPHENOTYPE OF DAUGHTER CELLS RESULTING FROM THE FIRST DIVISION OF SINGLE CD34(-1(+)LIN(-) CELLS()THY)

There is a need to determine whether culture conditions may exist for ex vivo expansion of hematopoeitic stem cells (HSC), which favor solel y proliferative self-renewal of HSC as opposed to proliferation with d ifferentiation. Using single cells, we studied the effects of individu al and combinations of cytokines in serum-free medium on the kinetics of the first cell doubling and the resulting phenotype of each of indi vidual daughter cell. CD34(+)Thy-1(+)lin(-) cells were plated 1 cell p er well in Terasaki plates in serum-free medium containing cytokines. Each well containing a single cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individ ual well, the phenotype of the daughter cells was determined by staini ng with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)conjugated lineage specific antibodies. The cumulative percent of wells with an undivided single cell, wells in which the cell had divi ded, and wells in which the cell had died were scored. The number of d oublets with conserved phenotype (CD34(+)lin(-)) was compared to those wells with one or more differentiated daughter cells (CD34(+)lin(+)). Over 7 days, cells cultured in single factors showed that between 13% (interleukin-6 [IL-6]) and 29% (thrombopoietin [TPO]) of the cells we re undivided, between 13% (IL-l) and 35% (TPO) of the cells doubled, a nd between 35% (TPO) and greater than 60% (IL-11, IL-1, or hepatocyte growth factor [HGF]) died. When combinations of cytokines were used ov er 7 days, between 5% (FLT-3 ligand [FLT-3L], stem cell factor [SCF], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], beta nerve growth factor [beta NGF]) and 22% (FLT-3L + HGF) of the cells remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68% (SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64.6%) of cells with conserved phenotype (perce nt conserved doublets + percent with 1 cell conserved), followed by SC F + TPO, (50%) and the combination of FLT-3L, SCF, IL-3, IL-6, G-CSF, beta NGF (53%). These combinations also produced the highest yield of cells with conserved phenotype after one division (FLT-3L + TPO - 81 c ells/100 initial cells, SCF + TPO - 68 cells/100 initial cells) (P = . 01). Observation of the time of the initial cell division and phenotyp e of the daughter cells allowed us to identify candidate combinations of cytokines that promote maintenance of lin(-) cells (TPO), or recrui t the primitive cells to divide and undergo phenotypic self-renewal (F LT-3L + TPO, SCF + TPO). (C) 1998 by The American Society of Hematolog y.