Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E

Abstract

Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) and CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(+)CD36(-)CD71(low) cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45(+)GPA(-)IL-3R(-)CD34(-)CD36(+)CD71(high) cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.

Figure 1

Expression of surface proteins and colony forming ability of the cultured CD34⁺ cells during early erythropoiesis. (A) Immunoblot analysis. Blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cellular protein prepared from cells cultured from day 1 to day 7 were probed with antibodies against the indicated proteins. Note the decreased expression of CD34 and IL-3R, and the increased expression of CD36 and CD71. CD45 remained constant throughout and GPA was negative until day 6. Glyceraldehyde-3-phosphate dehydrogenase was used as loading control. (B) Flow cytometric analysis. The surface expression of indicated proteins was measured by flow cytometry. The representative profiles are shown. Gray line: autofluorescence control from unstained cells; black line: fluorescence from cells stained with indicated antibody. (C) Quantitation of colony forming ability. BFU-E colonies peak at day 4 then gradually decrease, whereas CFU-E colonies peak at day 5 and then decrease. The data are from 7 independent experiments.

Figure 2

Isolation of BFU-E and CFU-E cells by cell-sorting using CD34, CD36, and IL-3R as markers. (A) Colony forming ability of the sorted IL-3R⁺ and IL3R⁻ cells. (B) Colony forming ability of the sorted IL-3R⁻CD34⁺CD36⁻ in complete medium, EPO-only medium, and EPO+SCF+IL3 or EPO+SCF medium. (C) Colony forming ability of the sorted IL-3R⁻CD34⁻CD36⁺ cells in complete medium, EPO-only medium, and EPO+SCF+IL3 or EPO+SCF medium. (D) Expression of surface markers of sorted IL-3R⁻CD34⁺CD36⁻ and IL-3R⁻CD34⁻CD36⁺ cells. The surface expression of indicated proteins was measured by flow cytometry. The representative profiles are shown. Gray line: autofluorescence control from unstained cells; black line: fluorescence from cells stained with indicated antibody. Results from 3 independent experiments are shown.

Figure 3

PCA of expressed genes and pairwise comparisons of different stages of early human erythroid progenitors. (A) PCA plot of 3 biological replicates at 4 different stages of early erythroid differentiation. (B) Pairwise comparison of the gene expression at different stages of early erythroid progenitors. (Upper right) Pearson and Spearman coefficient values. (Lower left) Scatter density plot. PC, principle component; R_p, Pearson product-moment correlation coefficient; R_s, Spearman rank correlation coefficient.

Figure 4

Global gene expression at different stages of human early erythriod progenitor. Expression values of differentially expressed genes between the different stages are shown as a heat map. In each row, the red, white, and blue colors represent the expression level (from high to low) of a particular gene. The rows are organized by hierarchical clustering with complete linkage and Euclidean distance metric.

Figure 5

Clusters of gene expression across the different stages of early erythriod differentiation. Differentially expressed genes between adjacent stages with log fold >2 and fpkm >10 were clustered into 16 groups by the process of self-organized maps. The 4 major groups are illustrated with heat maps of the gene members of the cluster (left) and the expression profile is shown graphically (middle). GO analysis of the genes within the cluster identified the top 5 GO terms and their corresponding P-values (right).

Figure 6

Pairwise comparison of transcription factor genes in human early stage erythroid progenitors. Transcription factor genes with log twofold >1 and fpkm >10 in at least 1 stage for CD34 to BFU-E (A), BFU-E to CFU-E (B), and the CFU-E to Pro (C) transitions are shown. In each row, the red, white, and blue colors represent the expression level (from high to low) for each gene.

Figure 7

Isolation of and quantification of BFU-E and CFU-E cells from primary samples. (A) Representative plot of a combination of surface markers that isolates BFU-E and CFU-E populations from primary human BM samples. (i) Plot of SSC vs CD45 expression of all cells being analyzed; (ii) SSC vs CD3, CD4, CD14, CD19, and 7AAD of CD45+ population (P1), the resulting negative population designated as P2; (iii) SSC vs CD41 and GPA of the P2 population, namely P3; (iv) SSC vs IL3R expression of the P3 population; (v) SSC vs CD36 expression of IL3R⁻ population, resulting in CD36⁻ and CD36⁺ populations; (vi, top) CD34 vs SSC of the CD36⁻ population revealed CD34⁺, which gave rise to BFU-E colonies; (vi, bottom) CD34 vs CD71 of BFU-E population revealed that BFU-E cells were CD34+CD71low; (vii, top) CD71 vs SSC of the CD36⁺ cells revealed CD71⁺ population, which gave rise to CFU-E colonies; and (vii, bottom) CD34 vs CD71 of CFU-E population revealed that CFU-E cells are CD34⁻CD71^hi. (B) Colony forming ability of the sorted BFU-E and CFU-E cells from BM (i), cord blood (ii), and peripheral blood (iii). (C) Quantitative analysis of BFU-E and CFU-E populations in cord blood and peripheral blood.