Figure 4. Pharmacological targeting of Cdc42 activity rejuvenates LT-HSCs function.
(A), Schematic representation of the experimental setup. 200 aged and young donor (Ly5.2+) LT-HSCs were cultured for 16hrs as indicated and subsequently transplanted into recipient (Ly5.1+) mice along with 3–105 BM competitor (Ly5.1+) cells. 24 wks post transplant recipient mice were sacrificed and secondary transplants were performed. (B and D), Percentage of donor contribution (Ly5.2+ cells) to total WBC in PB 8, 16 and 24 wks post transplant in primary (B) and secondary (D) transplants. Mice were considered as engrafted when the percentage of Ly5.2+ cells in PB was higher than 1.0 in primary transplants and than 0.5 in secondary transplants and contribution was detected for all PB lineages. Shown are mean values +1 S.E.; ** p < 0.01 and * p < 0.05 vs young control in B; *** p < 0.001, ** p < 0.01, * p < 0.05 vs aged control in D. (C and E), Percentage of B220+, CD3+ and myeloid cells among donor-derived Ly5.2+ cells in PB 24 weeks after primary (C) and secondary (E) transplants. * p < 0.05, ** p < 0.01, *** p < 0.001; shown are mean values +1 S.E. (F–G), Percentage of LT-HSCs, ST-HSCs and LMPPs cells in BM among donor-derived LSKs cells 24 weeks after primary (F) and secondary (G) transplants. ** p < 0.01; shown are mean values +1 S.E. Data is based on five (primary transplants) and four (secondary transplants) experimental repeats with 5 recipient mice per group (e.g. n=25 for primaries and n=20 for secondary transplants). (H), Representative distribution of Cdc42, tubulin and Per-2 in donor-derived LT-HSCs sorted from young, aged and aged CASIN treated LT-HSC recipient mice 24 weeks post transplant. Shown are overlaps with the phase contrast picture (panels i–iii) or cells on a dark background (panels iv–xv). Bar = 5µm. (I), Percentage of donor-derived LT-HSCs polarized for Cdc42 and tubulin sorted 24 weeks post transplant from recipient animals competitively reconstituted with young, aged and aged CASIN treated LT-HSC. Shown are mean values +1 SEM, n=3, ~50 cells scored per sample in total. ** p < 0.01, * p < 0.05. See also Figure S4.