American Society of Hematology

Figure 1

$Figure 1. Numeric expansion and differentiation following exposure of bone marrow (BM) to various factors. (A) Schematic of test culture system. (B) BM freshly harvested from C57BL/6 mice was cultured for 6 days (step 1 culture) in the specified factors (dosing in “Methods”), then counted. Each bar represents averaged 3 to 11 determinations plus or minus SD, each performed in synchronous comparison to at least five other groups. Asterisked bars indicate treatment combinations that displayed significant proliferative synergy (see “Methods”). (C) Fresh uncultured mouse BM cell suspensions were mAb-stained and FACS sorted to separate CD34+ and CD34− subpopulations. CD34+ cells initially represented 10.8% (±7%) of the total BM cells. The following groups were then subjected to 6-day step 1 culture either in Flt3L + SCF plus IL-6 (striped bars) or in Flt3L + IL-6 (solid bars): unfractionated BM (12-15 million per flask); FACS sorted CD34− cells (12-15 million per flask); FACS-sorted CD34+ cells (2-3 million per flask); or FACS-sorted, unirradiated CD34+ cells plus irradiated (3000 cGy) CD34− cells. Labels above bars indicate fold numeric expansion during the 6-day culture. All numeric expansion observed was attributable to proliferation of the CD34+ subpopulation, with no CD34− feeder layer requirement. (N.T. indicates condition not tested). (D) Surface expression profiles at end of 6-day step 1 culture in various conditioning treatments. Individual treatments are listed in far left column. Ten different treatments were synchronously compared (all groups shown in Figure S1A). Number within each histogram plot indicates the mean fluorescence specificity index for the molecule tested, defined as the geomean fluorescent intensity of all cells after staining with the specified mAb (filled histogram), divided by the geomean background staining intensity for isotype control mAb (unfilled histogram). Results shown are representative of three comprehensive comparisons. (E) Following the step 1 conditioning treatments listed in far left column, each group was replated for step 2 in fresh medium with GMCSF plus IL-4 for 24 hours, followed by overnight exposure to paired TLR agonists (CpG ODN 1826 and LPS). FACS analyses were then performed on day 2 of this step 2 culture. Ten different conditioning treatments were compared (selected groups shown in panel E; all groups shown in Figure S2A). Far right column shows gross numeric expansion during initial step 1 culture. Number within each histogram is the calculated mean fluorescence specificity index (see panel D). Representative of three comprehensive comparison experiments.$

Numeric expansion and differentiation following exposure of bone marrow (BM) to various factors. (A) Schematic of test culture system. (B) BM freshly harvested from C57BL/6 mice was cultured for 6 days (step 1 culture) in the specified factors (dosing in “Methods”), then counted. Each bar represents averaged 3 to 11 determinations plus or minus SD, each performed in synchronous comparison to at least five other groups. Asterisked bars indicate treatment combinations that displayed significant proliferative synergy (see “Methods”). (C) Fresh uncultured mouse BM cell suspensions were mAb-stained and FACS sorted to separate CD34⁺ and CD34⁻ subpopulations. CD34⁺ cells initially represented 10.8% (±7%) of the total BM cells. The following groups were then subjected to 6-day step 1 culture either in Flt3L + SCF plus IL-6 (striped bars) or in Flt3L + IL-6 (solid bars): unfractionated BM (12-15 million per flask); FACS sorted CD34⁻ cells (12-15 million per flask); FACS-sorted CD34⁺ cells (2-3 million per flask); or FACS-sorted, unirradiated CD34⁺ cells plus irradiated (3000 cGy) CD34⁻ cells. Labels above bars indicate fold numeric expansion during the 6-day culture. All numeric expansion observed was attributable to proliferation of the CD34⁺ subpopulation, with no CD34⁻ feeder layer requirement. (N.T. indicates condition not tested). (D) Surface expression profiles at end of 6-day step 1 culture in various conditioning treatments. Individual treatments are listed in far left column. Ten different treatments were synchronously compared (all groups shown in Figure S1A). Number within each histogram plot indicates the mean fluorescence specificity index for the molecule tested, defined as the geomean fluorescent intensity of all cells after staining with the specified mAb (filled histogram), divided by the geomean background staining intensity for isotype control mAb (unfilled histogram). Results shown are representative of three comprehensive comparisons. (E) Following the step 1 conditioning treatments listed in far left column, each group was replated for step 2 in fresh medium with GMCSF plus IL-4 for 24 hours, followed by overnight exposure to paired TLR agonists (CpG ODN 1826 and LPS). FACS analyses were then performed on day 2 of this step 2 culture. Ten different conditioning treatments were compared (selected groups shown in panel E; all groups shown in Figure S2A). Far right column shows gross numeric expansion during initial step 1 culture. Number within each histogram is the calculated mean fluorescence specificity index (see panel D). Representative of three comprehensive comparison experiments.

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