Figure 5.
RUX treatment improves serial replating ability and self-renewal capacity of cultured HSCs. (A) GSVA score of the Kyoto Encyclopedia of Genes and Genomes (KEGG) JAK/STAT signaling pathway gene-set (same as Figure 2H). The GSVA score was calculated per single cell with the line indicating the median and the upper and lower whiskers indicating the 25th and 75th percentile of expression. (B-C) Serial replating of human mPB HSC/MPPs (CD34+CD38−CD45RA−) cultured for 72 hours in EXPER conditions (B) or for 62 hours in GT conditions (C). Graphs (upper panels) show the fold change in the colony number in comparison with DMSO from primary (2 week), secondary (4 week), and tertiary (6 week) plating. There are n = 3 mPB biological replicates in (B) and n = 4 mPB biological replicates in (C); the individual donors are indicated by shapes. The mean ± SD are shown. Tables (lower panels) report the statistics from a generalized linear mixed-effects model (glmer) analysis performed with raw colony counts. Tukey corrected P values for pairwise comparisons with DMSO with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (D) Representative image of wells from tertiary replating experiment of mPB HSC/MPPs cultured for 72 hours in EXPER conditions (upper panel) or for 62 hours in GT conditions (lower panel) treated with either DMSO (left) or 10 nM RUX (right). Circles indicate manually scored colonies. Images brightened by 17%. (E-G) Serial replating of human mPB HSC/MPPs cultured for 62 hours in GT conditions. Graphs (upper panels) show fold change in colony number in comparison with DMSO from tertiary (6 week) plating in the conditions indicated as follows: (E-G) RUX (10 nM); (E) 0 hours fresh HSC/MPPs; (F) UM171 (35 nM), low TPO (20 ng/mL); (G) the pan-caspase inhibitor (CASi) Z-VAD(OH)-FMK (100 nM); (E,G) n = 3 mPB biological replicates; (F) n = 5 mPB biological replicates. Individual donors indicated by shapes and matching across (E-G). The mean ± SD are shown. Tables (lower panels) report the statistics from the glmer analysis with fitting of raw colony counts. Tukey corrected P values for pairwise comparisons of EM means with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (H) Workflow of in vivo transplantation of mPB CD34+CD38− cells cultured in GT conditions for 62 hours with LV transduction and RUX (10 nM) or DMSO. Secondary transplantations were performed from whole BM of engrafted mice. (I) Graft size (percentage of human CD45++ and GlyA+) in the BM at 18 weeks post transplantation of mPB CD34+CD38− cells cultured for 62 hours in GT conditions with RUX (10 nM) or DMSO. n = 6 biological replicates; the graph shows representative of n = 68 engrafted mice (n = 34 DMSO; n = 34 RUX). Two-way ANOVA with Sidak multiple comparisons was performed (30 000 cells DMSO vs 30 000 cells RUX; P = .005; all other doses DMSO vs RUX; P > .9). (J) The log-fraction plot of the limiting dilution model fitted to data in supplemental Table 14. Indicates the percentage of LTRC estimates from secondary transplantation experiments. Whole BM of engrafted mice from primary transplants was transplanted in NSG-SGM3 mice and analyzed 8 weeks posttransplantation (n = 1 experiment; n = 35 mice). Slope indicates the log-active fraction. The dotted line shows the 95% CI. Zero negative response indicated by triangle. Panel H was created with BioRender.com (license agreement UR26QKHL5H).

RUX treatment improves serial replating ability and self-renewal capacity of cultured HSCs. (A) GSVA score of the Kyoto Encyclopedia of Genes and Genomes (KEGG) JAK/STAT signaling pathway gene-set (same as Figure 2H). The GSVA score was calculated per single cell with the line indicating the median and the upper and lower whiskers indicating the 25th and 75th percentile of expression. (B-C) Serial replating of human mPB HSC/MPPs (CD34+CD38−CD45RA−) cultured for 72 hours in EXPER conditions (B) or for 62 hours in GT conditions (C). Graphs (upper panels) show the fold change in the colony number in comparison with DMSO from primary (2 week), secondary (4 week), and tertiary (6 week) plating. There are n = 3 mPB biological replicates in (B) and n = 4 mPB biological replicates in (C); the individual donors are indicated by shapes. The mean ± SD are shown. Tables (lower panels) report the statistics from a generalized linear mixed-effects model (glmer) analysis performed with raw colony counts. Tukey corrected P values for pairwise comparisons with DMSO with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (D) Representative image of wells from tertiary replating experiment of mPB HSC/MPPs cultured for 72 hours in EXPER conditions (upper panel) or for 62 hours in GT conditions (lower panel) treated with either DMSO (left) or 10 nM RUX (right). Circles indicate manually scored colonies. Images brightened by 17%. (E-G) Serial replating of human mPB HSC/MPPs cultured for 62 hours in GT conditions. Graphs (upper panels) show fold change in colony number in comparison with DMSO from tertiary (6 week) plating in the conditions indicated as follows: (E-G) RUX (10 nM); (E) 0 hours fresh HSC/MPPs; (F) UM171 (35 nM), low TPO (20 ng/mL); (G) the pan-caspase inhibitor (CASi) Z-VAD(OH)-FMK (100 nM); (E,G) n = 3 mPB biological replicates; (F) n = 5 mPB biological replicates. Individual donors indicated by shapes and matching across (E-G). The mean ± SD are shown. Tables (lower panels) report the statistics from the glmer analysis with fitting of raw colony counts. Tukey corrected P values for pairwise comparisons of EM means with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (H) Workflow of in vivo transplantation of mPB CD34+CD38− cells cultured in GT conditions for 62 hours with LV transduction and RUX (10 nM) or DMSO. Secondary transplantations were performed from whole BM of engrafted mice. (I) Graft size (percentage of human CD45++ and GlyA+) in the BM at 18 weeks post transplantation of mPB CD34+CD38− cells cultured for 62 hours in GT conditions with RUX (10 nM) or DMSO. n = 6 biological replicates; the graph shows representative of n = 68 engrafted mice (n = 34 DMSO; n = 34 RUX). Two-way ANOVA with Sidak multiple comparisons was performed (30 000 cells DMSO vs 30 000 cells RUX; P = .005; all other doses DMSO vs RUX; P > .9). (J) The log-fraction plot of the limiting dilution model fitted to data in supplemental Table 14. Indicates the percentage of LTRC estimates from secondary transplantation experiments. Whole BM of engrafted mice from primary transplants was transplanted in NSG-SGM3 mice and analyzed 8 weeks posttransplantation (n = 1 experiment; n = 35 mice). Slope indicates the log-active fraction. The dotted line shows the 95% CI. Zero negative response indicated by triangle. Panel H was created with BioRender.com (license agreement UR26QKHL5H).

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