Figure 1. In vivo expansion of functional LT-HSCs in the absence of STAT5-regulated miR-193b.

(a) Differential miR expression in the presence and absence of STAT5 after stimulation with TPO. Expression fold change was calculated and normalized to the corresponding untreated (unstim.) cells. The data represent the mean expression of two independent experiments using LT-HSCs from 16 to 20 mice per group. (b) Percentage of LT-HSCs and MPPs in the LSK fraction of the BM from mice of the indicated age groups, N=4 miR-193b^+/+ mice and N=6 miR-193b^−/− mice at 2–3 months of age, N=6 mice/genotype at 6 months of age, N=8 miR-193b^+/+ mice and N=4 miR-193b^−/− mice at 12 months of age. Exemplary FACS plots display the CD150 and CD34 expression of LSK (Lineage⁻ c-Kit⁺ Sca1⁺) BM cells from 12-month-old mice, representing LT-HSCs (CD150⁺ CD34^lo) and MPPs (CD150⁻ CD34⁺). (c) Experimental scheme of the primary and secondary transplantation of LT-HSCs from miR-193b^−/−and miR-193b^+/+mice into NSG mice. (d) Donor cell engraftment in the peripheral blood of primary recipients after competitive transplantation of LT-HSCs from 12-month-old miR-193b^−/− and miR-193b^+/+ mice was assessed via FACS. Mann–Whitney test. (e) FACS plots gated for donor LT-HSCs (left panel) and absolute numbers (right panel) after 16 weeks in both the femurs and tibiae of each primary recipient. Mann–Whitney test. (f) Donor cell engraftment in the peripheral blood of secondary recipients after transplanting 1 × 10⁶ BM cells from primary transplanted mice. BM of one primary transplanted mouse was transplanted into two recipients. Mann–Whitney test. (g) FACS plots gated for donor LT-HSCs (left panel) and absolute numbers (right panel) after 24 weeks in both femurs and tibiae of each secondary recipient. Mann–Whitney test. (h,i) The distribution of donor stem and progenitor populations in primary (h) and secondary (i) recipient BM. Mann–Whitney test. The data are represented as the mean±s.d. *P<0.05; **P<0.01 and ***P<0.001.