American Society of Hematology

Fig. 2.

$Fig. 2. Functional progenitor capacity of primitive subsets isolated by lineage depletion and FACS sorting from G-CSF– or G-CSF plus SCF–mobilized PB. / (A-B) Primitive subsets were purified by depletion of mature cells by means of a cocktail of 9 lineage-specific antibodies (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and a magnetic column. The resulting Lin− fractions from both G-CSF– and G-CSF plus SCF–mobilized PB were stained with CD34 and CD38 antibodies conjugated with the fluorochromes indicated and sorted for CD34+CD38− cells. The data represent a characteristic dot plot illustrating the abundance of CD34- and CD38-expressing cells isolated from G-CSF–mobilized (A) or G-CSF plus SCF–mobilized (B) PB. R1 represents CD34+CD38−Lin− cells selected to be assayed for progenitor activity and for transplantation into NOD/SCID mice. Quadrant statistics represent the frequency (mean ± SEM) of expression of CD34 and CD38 isolated from 4 G-CSF– and 5 G-CSF plus SCF–mobilized PB Lin− samples. A significant increase in the frequency of primitive CD34+CD38−cells was observed when G-CSF plus SCF Lin− cells were compared with G-CSF Lin−cells (P < .05). (C) Purified CD34+CD38−Lin− cells were assayed for progenitor function by means of in vitro colony-forming cell assays by plating 1000 CD34+CD38−Lin−cells in methylcellulose and were scored at 12 to 14 days. CFUs were also assessed in additional experiments at days 21 and 28 and showed similar CFU number and type regardless of the mobilization regime used. (Ci) The data represent the number of colonies per 1000 CD34+CD38−Lin− cells plated (mean ± SEM) from 4 G-CSF– and 5 G-CSF plus SCF–mobilized PB samples. A significant increase in the progenitor content of CD34+CD38−Lin− cells was observed when G-CSF plus SCF–mobilized PB was compared with G-SCF–mobilized PB (***P < .001). (Cii) Summary of the proportion of total progenitor colony types produced by G-CSF–mobilized PB (open bars) and G-CSF plus SCF–mobilized PB (shaded bars) CD34+CD38−Lin− cells. G-CSF plus SCF–mobilized PB CD34+CD38−Lin−cells demonstrated a significant increase in the frequency of erythroid burst-forming units (BFU-E) (*P < .05) and a significant decrease in the frequency of granulocyte colony-forming units (CFUs-G) (**P < .01) when compared with CD34+CD38−Lin− cells from G-CSF mobilization alone.$

Functional progenitor capacity of primitive subsets isolated by lineage depletion and FACS sorting from G-CSF– or G-CSF plus SCF–mobilized PB.

(A-B) Primitive subsets were purified by depletion of mature cells by means of a cocktail of 9 lineage-specific antibodies (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and a magnetic column. The resulting Lin⁻ fractions from both G-CSF– and G-CSF plus SCF–mobilized PB were stained with CD34 and CD38 antibodies conjugated with the fluorochromes indicated and sorted for CD34⁺CD38⁻ cells. The data represent a characteristic dot plot illustrating the abundance of CD34- and CD38-expressing cells isolated from G-CSF–mobilized (A) or G-CSF plus SCF–mobilized (B) PB. R1 represents CD34⁺CD38⁻Lin⁻ cells selected to be assayed for progenitor activity and for transplantation into NOD/SCID mice. Quadrant statistics represent the frequency (mean ± SEM) of expression of CD34 and CD38 isolated from 4 G-CSF– and 5 G-CSF plus SCF–mobilized PB Lin⁻ samples. A significant increase in the frequency of primitive CD34⁺CD38⁻cells was observed when G-CSF plus SCF Lin⁻ cells were compared with G-CSF Lin⁻cells (P < .05). (C) Purified CD34⁺CD38⁻Lin⁻ cells were assayed for progenitor function by means of in vitro colony-forming cell assays by plating 1000 CD34⁺CD38⁻Lin⁻cells in methylcellulose and were scored at 12 to 14 days. CFUs were also assessed in additional experiments at days 21 and 28 and showed similar CFU number and type regardless of the mobilization regime used. (Ci) The data represent the number of colonies per 1000 CD34⁺CD38⁻Lin⁻ cells plated (mean ± SEM) from 4 G-CSF– and 5 G-CSF plus SCF–mobilized PB samples. A significant increase in the progenitor content of CD34⁺CD38⁻Lin⁻ cells was observed when G-CSF plus SCF–mobilized PB was compared with G-SCF–mobilized PB (***P < .001). (Cii) Summary of the proportion of total progenitor colony types produced by G-CSF–mobilized PB (open bars) and G-CSF plus SCF–mobilized PB (shaded bars) CD34⁺CD38⁻Lin⁻ cells. G-CSF plus SCF–mobilized PB CD34⁺CD38⁻Lin⁻cells demonstrated a significant increase in the frequency of erythroid burst-forming units (BFU-E) (*P < .05) and a significant decrease in the frequency of granulocyte colony-forming units (CFUs-G) (**P < .01) when compared with CD34⁺CD38⁻Lin⁻ cells from G-CSF mobilization alone.

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