Influence of Cytokines on the Growth Kinetics and Immunophenotype of Daughter Cells Resulting From the First Division of Single CD34+Thy-1+lin- Cells

There is a need to determine whether culture conditions may existfor ex vivo expansion of hematopoeitic stem cells (HSC), which favorsolely proliferative self-renewal of HSC as opposed to proliferationwith differentiation. Using single cells, we studied the effects ofindividual and combinations of cytokines in serum-free medium on thekinetics of the first cell doubling and the resulting phenotype of eachof individual daughter cell. CD34⁺Thy-1⁺lincells were plated 1 cell per well in Terasaki plates inserum-free medium containing cytokines. Each well containing a singlecell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype ofthe daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with anundivided single cell, wells in which the cell had divided, and wellsin which the cell had died were scored. The number of doublets withconserved phenotype (CD34⁺lin) wascompared to those wells with one or more differentiated daughter cells(CD34⁺lin⁺). Over 7 days, cells cultured insingle factors showed that between 13% (interleukin-6 [IL-6]) and29% (thrombopoietin [TPO]) of the cells were undivided, between 13%(IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) andgreater than 60% (IL-11, IL-1, or hepatocyte growth factor[HGF]) died. When combinations of cytokines were usedover 7 days, between 5% (FLT-3 ligand [FLT-3L], stemcell factor [SCF], IL-3, IL-6, granulocytecolony-stimulating factor [G-CSF],  nerve growth factor[NGF]) and 22% (FLT-3L + HGF) of the cellsremained 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 withconserved phenotype (percent conserved doublets + percent with 1 cellconserved), followed by SCF + TPO, (50%) and the combination ofFLT-3L, SCF, IL-3, IL-6, G-CSF, NGF (53%). These combinations alsoproduced the highest yield of cells with conserved phenotype after onedivision (FLT-3L + TPO  81 cells/100 initial cells, SCF + TPO  68 cells/100 initial cells) (P = .01). Observation of thetime of the initial cell division and phenotype of the daughter cellsallowed us to identify candidate combinations of cytokines that promotemaintenance of lin cells (TPO), or recruit the primitivecells to divide and undergo phenotypic self-renewal (FLT-3L + TPO,SCF + TPO).