Enrichment of phenotypic and functional LT-HSCs by Epcr expression. (A) Contribution of LT-HSCs (LSK, CD48⁻, CD150⁺) to LSK cells in the different gene groups in the primary recipients (Epcr vs negative control [##P < .0001]). (B) Total number of LT-HSCs of LSK cells in the different gene groups in the primary recipients (positive control, n = 10; negative control, n = 9; Pbx1, n = 7; Epcr, n = 6; Hoxa10 and Mycn, n = 3; Epcr vs negative control [##P = .004]). (C) Representative flow cytometry of a primary transplanted mouse from the Epcr group compared with the negative and positive controls. (D) The percentage of GFP⁺ cells was measured by flow cytometry in the LSK and LT-HSC compartment. Shown is the fold difference of GFP⁺ cells compared with total BM GFP⁺ cells. Epcr-transduced and -transplanted Mpl^−/− cells are significantly enriched in the LT-HSC compartment. (A-D) Mean plus or minus SD; 2-way ANOVA with Tukey multiple comparisons test (##P < .0001). Because coexpressed GFP from the Pbx1-encoding vector was very low, the analysis was not performed in this group. (E) Contribution of LT-HSCs to LSK cells in the different gene groups in the secondary recipients. (F) Number of LT-HSCs of LSK cells in the different gene groups in the secondary recipients. (G) Donor chimerism (CD45.2⁺) on LT-HSCs in the different gene groups in secondary recipients. (H) To measure an expansion of LT-HSCs after transplantation, the number of transplanted phenotypic LT-HSCs from 1° recipients was correlated to the number of phenotypic LT-HSCs detected in the 2° recipients. In case the ratio was 1 or below, not efficient engraftment and expansion of LT-HSCs took place. In case the ratio was higher than 1, LT-HSCs have expanded in number (dotted line at 1, the values for Hoxa10 were 0). (E-H) Positive control, n = 5; negative control, n = 6; Pbx1, n = 7; Epcr, n = 6; Hoxa10 and Mycn, n = 3; mean plus or minus SD; 2-tailed, unpaired Student t test with Welch correction (**P ≤ .01; *P ≤ .05).