American Society of Hematology

Figure 1

$NUP98-HOXA10hd promotes multilog expansion in vitro of individually purified adult bm HSCs. (A) Schematic of the experimental design followed to directly investigate the capacity of NUP98-HOXA10hd to enhance clonal HSC expansion in vitro. TNCs indicates total nucleated cells. (B) Average number of viable nucleated GFP- (gray) or NUP98-HOXA10hd–transduced (black) cells at the end of 14-day culture. Values shown are the means ± SD of 16 GFP- or 40 NUP98-HOXA10hd–transduced cell counts. (C) Proportion of peripheral blood leukocytes produced by a fraction (indicated as a transplantation dose) of NUP98-HOXA10hd–transduced and expanded CD45+lin−Rho−SP (top panel) and E-SLAM (bottom panel) clones transplanted into irradiated recipients 20 weeks previously. Each triangle represents an individual recipient/mouse. Solid triangles indicate recipients of the smallest fraction(s) tested of each clone, positive for the presence of transplanted cells. Open triangles indicate recipients of the highest fraction(s) tested of each clone, negative for the presence of transplanted cells. Symbol (†) indicates a dead mouse. (D) DNA extracted from NUP98-HOXA10hd–transduced bone marrow cells of representative long-term reconstituted (24-week) recipients (as shown in C top panel) was analyzed for proviral integrants by Southern blotting. Arrows indicate common integration pattern(s) among recipients of the cells from the same HSC-expanded clone (NUP98-HOXA10hd–transduced clones 16, 17, 18, 20, and 21). Transplantation dose is expressed in fraction of the culture. (E) Light gray bar indicates the maximal number of HSCs generated by day 14 in cultures of GFP-transduced cells, estimated by complete lack of reconstitution in recipients of 1/5 of 16 such cultures. Dark gray bar indicates the number of HSCs generated by day 14 in cultures of NUP98-HOXA10hd–transduced cells, as determined by limiting dilution CRU assay. Value shown is the means ± SEM of results from 40 NUP98-HOXA10hd–transduced clones tested. The presented result is the minimal number of HSCs estimated because the limiting dilution was not reached for the majority of NUP98-HOXA10hd–transduced HSC clones tested.$

NUP98-HOXA10hd promotes multilog expansion in vitro of individually purified adult bm HSCs. (A) Schematic of the experimental design followed to directly investigate the capacity of NUP98-HOXA10hd to enhance clonal HSC expansion in vitro. TNCs indicates total nucleated cells. (B) Average number of viable nucleated GFP- (gray) or NUP98-HOXA10hd–transduced (black) cells at the end of 14-day culture. Values shown are the means ± SD of 16 GFP- or 40 NUP98-HOXA10hd–transduced cell counts. (C) Proportion of peripheral blood leukocytes produced by a fraction (indicated as a transplantation dose) of NUP98-HOXA10hd–transduced and expanded CD45⁺lin⁻Rho⁻SP (top panel) and E-SLAM (bottom panel) clones transplanted into irradiated recipients 20 weeks previously. Each triangle represents an individual recipient/mouse. Solid triangles indicate recipients of the smallest fraction(s) tested of each clone, positive for the presence of transplanted cells. Open triangles indicate recipients of the highest fraction(s) tested of each clone, negative for the presence of transplanted cells. Symbol (†) indicates a dead mouse. (D) DNA extracted from NUP98-HOXA10hd–transduced bone marrow cells of representative long-term reconstituted (24-week) recipients (as shown in C top panel) was analyzed for proviral integrants by Southern blotting. Arrows indicate common integration pattern(s) among recipients of the cells from the same HSC-expanded clone (NUP98-HOXA10hd–transduced clones 16, 17, 18, 20, and 21). Transplantation dose is expressed in fraction of the culture. (E) Light gray bar indicates the maximal number of HSCs generated by day 14 in cultures of GFP-transduced cells, estimated by complete lack of reconstitution in recipients of 1/5 of 16 such cultures. Dark gray bar indicates the number of HSCs generated by day 14 in cultures of NUP98-HOXA10hd–transduced cells, as determined by limiting dilution CRU assay. Value shown is the means ± SEM of results from 40 NUP98-HOXA10hd–transduced clones tested. The presented result is the minimal number of HSCs estimated because the limiting dilution was not reached for the majority of NUP98-HOXA10hd–transduced HSC clones tested.

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