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Comparative Study
. 2017 Jul 20;130(3):372-376.
doi: 10.1182/blood-2017-03-776849. Epub 2017 Jun 6.

CD27 Marks Murine Embryonic Hematopoietic Stem Cells and Type II Prehematopoietic Stem Cells

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Comparative Study

CD27 Marks Murine Embryonic Hematopoietic Stem Cells and Type II Prehematopoietic Stem Cells

Yan Li et al. Blood. .
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Figures

Figure 1.
Figure 1.
RNA sequence analysis to identify markers of pre-HSCs and embryonic HSCs. (A) Schematic showing the 4 cell populations that were compared. (B) Venn diagram of differentially expressed genes (DEGs) from pairwise comparisons. DEGs were identified by using a false discovery rate (FDR) cutoff <0.05 and a fold change cutoff >2. (C) Expression clusters defined by using consensus clustering and DEGs. Color indicates row-wise normalized expression levels. Numbers in parentheses indicate the number of genes in each cluster. (D) Gene ontology (GO) terms associated with genes in cluster 3. Level 5 biological process terms were used for enrichment analysis. Enrichment P values were computed by using the hypergeometric distribution. Nominal P values were corrected for multiple testing by using the Benjamini-Hochberg method. (E) List of cell surface marker genes in cluster 3 with more than twofold changes in gene expression in Ly6a-GFP+ HCCs relative to Ly6a-GFP HCCs or Ly6a-GFP+/− endothelial cells. (F) Quantitative polymerase chain reaction analysis of Tnfrsf7 messenger RNA in all 4 populations. n = 6, whiskers represent the 5th to 95th percentile. Significance is by 1-way analysis of variance and Dunnett’s multiple comparison test with Ly6a-GFP+ HCCs as a comparator (#). **P < .01. E, endothelial cells; Endo, endothelial cells (CD31+CD144+ESAM+Kit); FC, fold change; H, hematopoietic clusters.
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. 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.

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