American Society of Hematology

Figure 2.

$Figure 2. Embryonic HSCs in the major arteries, FL HSCs, and type II pre-HSCs are CD27+. (A) Representative scatter plots of CD27 expression on HCCs (CD31+Kit+CD144+ESAM+) and on the Ly6a-GFP+ and Ly6a-GFP– fractions of HCCs. Data are from the A+U+V of E11.5 Tg(Ly6a-GFP) embryos. (B) The average percentage (± SD) of Ly6a-GFP+ cells and Ly6a-GFP+CD27+ cells in the CD31+CD144+ESAM+Kit+ hematopoietic clusters (HCCs) (n = 7). Unpaired 2-tailed Student t test. ***P < .001. (C) The average number of cells in hematopoietic clusters with the indicated markers per embryo (mean ± SD, analysis of variance, Tukey’s multiple comparison test). ****P < .0001. (D) Representative sort plots for CD31+CD27+ and CD31+CD27– cells used in transplantation assays. Cells were sorted from the A+U+V of E11.5 embryos. (E) Left, average percentage (± SD) of donor-derived cells in the recipient PB 16 weeks posttransplantation. The number of repopulated animals/total number of recipients is indicated above each column. Right, average contribution to BM SLAM (CD48– CD150+) LSK cells. (F) Representative scatter plots of FL SLAM LSK cells from E14.5 fetuses separated based on CD27 expression. Lineage, Ter119, CD3e, B220, Gr1. Bottom left scatter plot shows the location of CD27+ and CD27– cells within the SLAM LSK population. (G) Limiting dilution transplantation of CD27+ and CD27– SLAM LSK cells. Reconstitution (>1% donor-derived BM SLAM LSK cells) was assessed at 16 weeks posttransplantation (n = 4-8 recipients per dose). The frequencies of HSCs were determined by extreme limiting dilution analysis.25 P = 2.9e-32. (H) Mean fluorescence intensity (MFI) of CD150 in the CD27+ and CD27– populations of SLAM LSK cells (n = 4). Unpaired 2-tailed Student t test. **P < .01. (I) Schematic for assessment of pre-HSCs. A+U+V were isolated, dissociated, cells sorted, cultured on Akt-EC for 7 days, and transplanted into irradiated recipients. (J) Representative sort plots of CD27+ and CD27– type I and II pre-HSCs. (K) The average percentage (± SD) of donor-derived PB cells 16 weeks posttransplantation into primary recipient mice. (L) Percentage of donor-derived SLAM LSK cells in recipient BM 16 weeks posttransplantation. (M) Percentage of donor-derived SLAM LSK cells in the BM of secondary recipients. A total of 500 000 BM cells from each primary recipient were injected into secondary recipients. FMO, fluorescence minus 1 control for CD27.$

Embryonic HSCs in the major arteries, FL HSCs, and type II pre-HSCs are CD27⁺. (A) Representative scatter plots of CD27 expression on HCCs (CD31⁺Kit⁺CD144⁺ESAM⁺) and on the Ly6a-GFP⁺ and Ly6a-GFP^– fractions of HCCs. Data are from the A+U+V of E11.5 Tg(Ly6a-GFP) embryos. (B) The average percentage (± SD) of Ly6a-GFP⁺ cells and Ly6a-GFP⁺CD27⁺ cells in the CD31⁺CD144⁺ESAM⁺Kit⁺ hematopoietic clusters (HCCs) (n = 7). Unpaired 2-tailed Student t test. ***P < .001. (C) The average number of cells in hematopoietic clusters with the indicated markers per embryo (mean ± SD, analysis of variance, Tukey’s multiple comparison test). ****P < .0001. (D) Representative sort plots for CD31⁺CD27⁺ and CD31⁺CD27^– cells used in transplantation assays. Cells were sorted from the A+U+V of E11.5 embryos. (E) Left, average percentage (± SD) of donor-derived cells in the recipient PB 16 weeks posttransplantation. The number of repopulated animals/total number of recipients is indicated above each column. Right, average contribution to BM SLAM (CD48^– CD150⁺) LSK cells. (F) Representative scatter plots of FL SLAM LSK cells from E14.5 fetuses separated based on CD27 expression. Lineage, Ter119, CD3e, B220, Gr1. Bottom left scatter plot shows the location of CD27⁺ and CD27^– cells within the SLAM LSK population. (G) Limiting dilution transplantation of CD27⁺ and CD27^– SLAM LSK cells. Reconstitution (>1% donor-derived BM SLAM LSK cells) was assessed at 16 weeks posttransplantation (n = 4-8 recipients per dose). The frequencies of HSCs were determined by extreme limiting dilution analysis.²⁵P = 2.9e-32. (H) Mean fluorescence intensity (MFI) of CD150 in the CD27⁺ and CD27^– populations of SLAM LSK cells (n = 4). Unpaired 2-tailed Student t test. **P < .01. (I) Schematic for assessment of pre-HSCs. A+U+V were isolated, dissociated, cells sorted, cultured on Akt-EC for 7 days, and transplanted into irradiated recipients. (J) Representative sort plots of CD27⁺ and CD27^– type I and II pre-HSCs. (K) The average percentage (± SD) of donor-derived PB cells 16 weeks posttransplantation into primary recipient mice. (L) Percentage of donor-derived SLAM LSK cells in recipient BM 16 weeks posttransplantation. (M) Percentage of donor-derived SLAM LSK cells in the BM of secondary recipients. A total of 500 000 BM cells from each primary recipient were injected into secondary recipients. FMO, fluorescence minus 1 control for CD27.

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