Prospective isolation of human erythroid lineage-committed progenitors

Significance

We have identified the first step of erythrocyte lineage commitment in human bone marrow as a CD71^{intermediate/+} CD105⁺ cell fraction of a previously defined megakaryocyte/erythrocyte progenitor population. This purification could be a useful tool for studying physiological and pathological red blood cell development, and should be analyzed in patients suffering from anemia or erythrocytosis such as in myelodysplastic syndrome or polycythemia vera.

Abstract

Determining the developmental pathway leading to erythrocytes and being able to isolate their progenitors are crucial to understanding and treating disorders of red cell imbalance such as anemia, myelodysplastic syndrome, and polycythemia vera. Here we show that the human erythrocyte progenitor (hEP) can be prospectively isolated from adult bone marrow. We found three subfractions that possessed different expression patterns of CD105 and CD71 within the previously defined human megakaryocyte/erythrocyte progenitor (hMEP; Lineage⁻ CD34⁺ CD38⁺ IL-3Rα⁻ CD45RA⁻) population. Both CD71⁻ CD105⁻ and CD71⁺ CD105⁻ MEPs, at least in vitro, still retained bipotency for the megakaryocyte (MegK) and erythrocyte (E) lineages, although the latter subpopulation is skewed in differentiation toward the erythroid lineage. Notably, the proliferative and differentiation output of the CD71^{intermediate(int)/+} CD105⁺ subset of cells within the MEP population was completely restricted to the erythroid lineage with the loss of MegK potential. CD71⁺ CD105⁻ MEPs are erythrocyte-biased MEPs (E-MEPs) and CD71^int/+ CD105⁺ cells are EPs. These previously unclassified populations may facilitate further understanding of the molecular mechanisms governing human erythroid development and serve as potential therapeutic targets in disorders of the erythroid lineage.

It is now apparent that hematopoiesis derives throughout postnatal life from the constant input from a small fraction of hematopoietic stem cells (HSCs) (1, 2), themselves rare, undergoing cell divisions that include self-renewal to HSCs and differentiation. The differentiation largely to multipotent progenitors (MPPs) that undergo transient amplifying divisions to give rise to all blood cell types but do not undergo long-term self-renewal (3). In mice and humans, the steps between HSCs and mature blood cells are stepwise quantal genetic/epigenetic changes with bifurcations to produce progeny with more limited fates; these are the oligopotent and unipotent progenitors, now almost fully described in mouse hematopoiesis (4). Determining the differentiation pathway leading to erythrocytes and isolating erythroid-specific progenitors are crucial to understanding how fate determinations are made that result in homeostatic production of blood cells and elements. Such an understanding can also elucidate disorders of red cell imbalance, such as anemia, myelodysplastic syndrome (MDS), and polycythemia vera (PV) (5, 6). Here we sought to find cell-surface markers that can be used to isolate from human bone marrow each stage of differentiation after the fate determinations that lead MPPs to common myeloid progenitors (CMPs) to megakaryocyte/erythroid progenitors (MEPs) and allow the commitment to erythropoiesis. Certain aspects of erythrocyte development have been elucidated, such as the overall pathway from HSCs through CMPs and MEPs (7–9). However, the original human CMP and MEP populations still appear to be heterogeneous, and the subsequent stages of differentiation (i.e., from MEP to erythrocyte) remain unclear.

Given this background, we sought markers that could subdivide CMPs and/or MEPs into cells specifically destined to generate erythrocytes. Erythrocytes, like all blood lineages, develop through a series of differentiation stages that begin with HSCs, which have been prospectively isolated in humans (10). The surface markers of intermediate erythroid progenitors/precursors and the expression pattern of some pivotal transcription factors (TFs) during their differentiation from HSCs have been analyzed (11–19). As a result, the erythroid-committed progenitor (EP), which exists downstream of the bipotent MEP, has been isolated in mouse but not human (9).

To isolate the presumed human erythrocyte progenitor (hEP), we first looked at markers crucial to isolating the mouse EP. One such key marker is CD105 (endoglin), whereby mouse EPs are Lineage (Lin)⁻ stem cell antigen (Sca)-1⁻ receptor tyrosine kinase c-Kit⁺ CD16/32⁻ CD150⁺ CD105⁺ CD41⁻. In humans, CD105 and CD71 have been classified as early erythroid cell markers (14–19). However, CD105 and CD71 expression in oligopotent myeloid progenitors (8) [i.e., CMPs, granulocyte/macrophage progenitors (GMPs), and MEPs] has not been evaluated. Thus, we examined closely the expression of these markers in CMPs, GMPs, MEPs, and their progeny.

Results

CD71 and CD105 Expression Patterns Subfractionate Myeloid Progenitors.

First, CMPs, GMPs, and MEPs were purified from human bone marrow (BM) according to the flow cytometric sorting scheme shown in Fig. 1A. The BM mononuclear cell fraction that is negative for lineage-affiliated antigens (Lin⁻) was subdivided into the CD34⁺ CD38⁻ CD45RA⁻ HSC/MPP and CD34⁺ CD38⁺ progenitor fractions (10, 20). The Lin⁻ CD34⁺ CD38⁺ progenitors were then fractionated into CMP, GMP, and MEP populations according to the expression patterns of IL-3Rα and CD45RA, as previously reported (8): CMPs, GMPs, and MEPs were defined as IL-3Rα⁺ CD45RA⁻, IL-3Rα⁺ CD45RA⁺, and IL-3Rα^−/lo CD45RA⁻ populations, respectively.

Fig. 1.

Flow cytometric analysis of human myeloid progenitors in the bone marrow. (A) CD71 and CD105 (endoglin) expression reveals phenotypic heterogeneity of myeloid progenitor cell compartments. (Upper) Gating strategy for HSCs/MPPs, CMPs, GMPs, and MEPs. (Lower) CD105 and CD71 expression for each population. The percentage of events in each gate is shown in the plot. PI, propidium iodide; FSC, forward scatter. (B) Evaluation of other erythroid-affiliated surface markers CD36 and GPA. The threshold between negative and positive was defined by the fluorescence minus one (FMO) method, and erythrocytes were used as a positive control for GPA expression.

We analyzed the expression pattern of putative erythroid markers for each of the progenitor populations. The expression of CD71 was detectable in the CMP (31.5 ± 11.1%, n = 8; Fig. 1A represents one of these experiments) and MEP fractions (82.3 ± 6.4%) but in very few cells in the CD34⁺ CD38⁻ HSC/MPP (5.8 ± 2.8%) and GMP fractions (5.3 ± 5.4%). Among these populations, only MEPs expressed CD105, and CD105-positive cells coexpress CD71 at intermediate to positive intensity [hereafter CD71^{intermediate(int)/+} CD105⁺ MEPs; 34.5 ± 6.9% of MEPs]. CD36, a known early marker of erythroid development (19, 21, 22), was expressed in some CD71⁺ CD105⁻ MEPs but almost all CD71^int/+ CD105⁺ MEPs. In contrast, glycophorin A (GPA/CD235), a late marker of erythroid development (11), was not detectable in any fractions examined (Fig. 1B). Subfractions based on CD71 and CD105 expression were also found in cord blood, although the CD71^int/+ CD105⁺ MEP fraction was much smaller in cord blood than in adult bone marrow (Fig. S1).

Fig. S1.

Flow cytometric analysis of human myeloid progenitors in cord blood. Analysis of CD71 and CD105 expression in CMPs and MEPs in human cord blood (CB). Two independent human cord blood samples in parallel with a BM sample (Upper). The percentage of events in each gate is shown in the plot.

CD71 Expression Initiated at the CMP Stage Represents Megakaryocyte/Erythrocyte-Biased Lineage Potential.

We functionally examined the differentiation potential of CD71⁻ CD105⁻ CMPs and CD71⁺ CD105⁻ CMPs (Fig. 2A). Methylcellulose colony assay revealed that colony-forming efficiencies are decreased from CD71⁻ CD105⁻ CMPs (38.2 ± 5.2%) to CD71⁺ CD105⁻ CMPs (33.0 ± 2.2%) (P = 0.047) (Fig. 2B) and the differentiation potential of CD71⁺ CD105⁻ CMPs skewed toward the megakaryocyte (MegK)/erythrocyte (E) (MegE) lineage, whereas CD71⁻ CD105⁻ CMPs generated a variety of myeloid colonies including colony-forming unit granulocyte/macrophage (CFU-GM) (Fig. 2 C and D). We further tested the MegK and erythroid potentials of each fraction by a serum-free liquid culture supplemented with IL-3, stem cell factor (SCF), erythropoietin (EPO), and thrombopoietin (TPO). As shown in Fig. 2 E and F, both CD71⁻ CD105⁻ CMPs and CD71⁺ CD105⁻ CMPs could give rise to CD41-expressing MegKs as well as GPA⁺ erythrocytes. However, CD71⁺ CD105⁻ CMPs gave rise to higher numbers of both CD41⁺ cells (P = 0.048) and GPA⁺ cells (P = 0.002) than did CD71⁻ CD105⁻ CMPs (Fig. 2G).

Fig. 2.

CD71⁺ fraction within the original human CMP showed differentiation potential skewed toward the MegE lineage. (A and B) The morphology (A) (May–Giemsa stain) and in vitro colony-forming potential (B) of FACS-purified CD71⁻ CD105⁻ or CD71⁺ CD105⁻ CMPs. The number of colony-forming units from 100 cells of each fraction is shown. Data presented are mean ± SD (n = 6); *P < 0.05. In this culture condition, unfractionated CMPs form 38.7 ± 4.0 colonies per 100 cells (n = 3). (Scale bars, 10 μm.) (C and D) Colonies were picked up, cytospun, and stained by the May–Giemsa method to determine the cell types included. (E) FACS-purified 2,000 cells of each fraction were cultured for 10 d under serum-free conditions and then analyzed. CD41 or GPA positivity was compared with an unstained control (Left). (F) Cytospin preparations (May–Giemsa staining) of sorted CD41⁺ MegKs (Upper) and GPA⁺ erythrocytes (Lower) are shown. The progeny from CD71⁺ CD105⁻ CMPs are shown. (G) Absolute number of CD41⁺ MegKs or GPA⁺ erythrocytes. Data shown are mean ± SD (n = 3); *P < 0.05, **P < 0.01.

Subsequent CD105 Up-Regulation Within MEPs Marks Complete Erythroid Lineage Commitment.

Among three subfractions of MEPs (shown in Fig. 3A), the colony-forming potential was higher in the CD71⁺ CD105⁻ fraction (37.0 ± 4.9%) than in the CD71^int/+ CD105⁺ fraction (29.2 ± 4.2%) (P = 0.014) and in the CD71⁻ CD105⁻ fraction (14.7 ± 3.4%) (P < 0.0001) (Fig. 2B). CD71⁺ CD105⁻ MEPs generated primarily large E colonies [defined as burst-forming unit-erythroid colony (BFU-E)–derived], although they still retained some MegK potential (Fig. 4A). In contrast, the CD71^int/+ CD105⁺ MEPs generated only the E lineage, and small-sized colonies from this fraction contained more mature (enucleated) erythrocytes [scored as colony-forming unit-erythroid (CFU-E)–derived] (Fig. 3 C and D). These results suggest that CD71^int/+ CD105⁺ MEPs exist at a stage downstream of CD71⁺ CD105⁻ MEPs. A similar result was observed in a serum-free liquid culture system: CD71^int/+ CD105⁺ MEPs lost the MegK lineage potential (Fig. 3 E and F). Based on these findings, we designate the Lin⁻ CD34⁺ CD38⁺ IL-3Rα^−/lo CD45RA⁻ CD71⁺ CD105⁻ as E-biased MEPs (E-MEPs) and the Lin⁻ CD34⁺ CD38⁺ IL-3Rα^−/lo CD45RA⁻ CD71^int/+ CD105⁺ fraction as E-committed progenitors.

Fig. 3.

CD71^int/+ CD105⁺ fraction within the original human MEP represents the human EP. (A and B) The morphology (A) (May–Giemsa stain) and in vitro colony-forming potential (B) of FACS-purified MEP subfractions. The number of CFUs from 100 cells of each fraction. Data shown are mean ± SD (n = 6); *P < 0.05, **P < 0.01. (Scale bars, 10 μm.) (C and D) Colonies were picked up, cytospun, and stained by the May–Giemsa method to determine the cell types included. (E) Representative flow cytometry plot of 10-d progeny of MEP subfractions. The percentage of events in each gate is shown. (F) Absolute numbers of CD41⁺ MegKs and GPA⁺ erythrocytes. Data shown are mean ± SD (n = 3).

Fig. 4.

Lineage relationship of subfractionated human myeloid progenitors. Lineage potential of FACS-purified progenitors from the culture of each primary fraction. After 60 h in liquid culture, cells were subfractionated by secondary sort and subjected to colony assay. CD71^int/+ CD105⁺ MEPs formed only CFU-E.

In contrast, CD71⁻ CD105⁻ MEPs frequently generated MegK-containing colonies but not CFU-GM. Serum-free liquid culture also showed this fraction gave rise most efficiently to CD41⁺ MegK cells, indicating that these are not just contaminated CD71⁻ CD105⁻ CMPs but might contain putative MegK progenitors.

Immunophenotypic Comparisons Among Subfractionated CMPs/MEPs.

We further characterized the cell-surface markers of the CMP and MEP subfractions. Flt3/flk2 is a tyrosine kinase receptor known for its heterogeneous expression in CMPs, high expression in GMPs, and down-regulation in MegE lineages (23, 24). Among CMPs, Flt3⁻ cells are considered to be in a transitional stage to MEPs. In accordance with these earlier findings, our results indicated that Flt3 is expressed on CD71⁻ CMPs, then gradually down-regulated from CD71⁺ CMPs to MEPs but highly expressed on GMPs (Fig. S2A).

Fig. S2.

Immunophenotypic comparison among subfractionated CMPs/MEPs. (A) Analysis of Flt3 expression pattern among myeloid progenitors. FMO, fluorescence minus one control. (B) CD41 expression in CMP/MEP subpopulations. The percentage of CD41⁺ cells is shown. (C) The percentage of CD41-, CD9-, CD226-, and CD42b-positive cells in CMP/MEP subpopulations. The mean ± SD (n = 2) is shown.

Consistent with the in vitro MegK potential, we found that MegK-associated molecules CD41 (13, 25) and CD9 (26–28), known to be expressed in the mouse MegK progenitor, were expressed at higher frequencies on the surface of human CD71⁺ CD105⁻ CMPs than on CD71⁻ CDS105⁻ CMPs, whereas CD226 and CD42b were not significantly expressed on CD71⁻ CD105⁻ CMPs (∼1%) or CD71⁺ CD105⁻ CMPs (∼5%). In contrast, the MegK-related markers analyzed were down-regulated or not expressed on the surface of CD71^int/+ CD105⁺ MEPs (Fig. S2 B and C).

Human EPs Develop from E-MEPs.

To test the lineage relationship of these subfractionated myeloerythroid progenitors directly, we reanalyzed cell-surface marker expression patterns after a short-term liquid culture. After culture for 60 h, CD71⁻ CD105⁻ CMPs gave rise to CD71⁺ CD105⁻ CMPs, GMPs, and MEPs (both CD71⁻ CD105⁻ and CD71⁺ CD105⁻), whereas CD71⁺ CD105⁻ CMPs differentiated toward MEPs but not GMPs (Fig. S3A). Furthermore, a minor fraction of progeny possessed the surface phenotype corresponding to EPs (Fig. S3A). These data suggested that the CD71⁻ CD105⁻ CMPs are the precursor of CD71⁺ CD105⁻ CMPs. However, CD71⁺ CD105⁻ CMPs generated a few CD71⁻ CD105⁻ CMPs, suggesting either CD71⁻ contamination or possible bidirectionality between them. In the same culture conditions, CD71⁺ CD105⁻ E-MEPs differentiated mainly into CD71^int/+ CD105⁺ EPs and a small number of CD71⁻ CD105⁻ MEPs (Fig. S3B), whereas CD71^int/+ CD105⁺ EPs did not generate E-MEPs or CD71⁻ CD105⁻ MEPs. These data clearly indicate that EPs exist at a stage downstream of E-MEPs. To perform a secondary analysis, cells were sorted and subjected to colony-forming assays (Fig. 4A). Such phenotypically defined secondary myeloid progenitors displayed differentiation activity consistent with their original phenotypic definition. CD71-expressing cells mainly gave rise to the MegE lineage, and CD105⁺ cells almost exclusively produced CFU-E–type erythroid cells.

Fig. S3.

Cell-surface marker expression analysis after short-term liquid culture of subpopulations. Cell-surface marker expression of cells after short-term liquid culture of CD71⁻ CD105⁻ CMPs (A) and CD71⁺ CD105⁻ CMPs (B). Cell-surface marker expression of cells after short-term liquid culture of CD71⁺ CD105⁻ MEPs (C) and CD71^int/+ CD105⁺ MEPs (D).

Suppressive Effect of TGF-β on Proliferation of EPs/E-MEPs Is Accompanied by Accelerated Terminal Differentiation.

We tested the effect of TGF-β1 on MEP subfractions according to the following rationale. CD105 is an accessory molecule of the transforming growth factor beta (TGF-β) type III receptor complex, able to bind TGF-β1 and TGF-β3 but not TGF-β2 (29). TGF-β, as well as tumor necrosis factor alpha and IFN gamma, is a powerful inhibitor of erythropoiesis both in vivo and in vitro. The suppressive effect of TGF-β on E-lineage cell proliferation is accompanied by an acceleration of terminal maturation (30–34).

We found that TGF-β1 did not affect the number of colonies generated by all three MEP subfractions (Fig. 5A) but affected the types of colonies generated: The proportion of CFU-E–type colonies was increased at the expense of BFU-E–type colonies (Fig. 5 B and C). By using a serum-free suspension culture, we also monitored the effects of TGF-β1 at an earlier time point. Additional TGF-β1 yielded two- to threefold fewer cells after 4 d of culture (Fig. S4A), with higher expression levels of GPA (Fig. 5D) and morphological signs of maturation (Fig. 5E) in all subfractions. These findings suggested that TGF-β1 provided a signal that inhibited proliferation and promoted differentiation among CD71^int/+ CD105⁺ EPs as well as in CD105-expressing progenies of CD71⁻ CD105⁻ or CD71⁺ CD105⁻ MEPs (30–34).

Fig. 5.

TGF-β1 accelerates erythroid cell maturation from all subfractions of human MEPs. (A) FACS-purified MEP subfractions were cultured in methylcellulose-based medium for 10–12 d in the presence of a cytokine mixture with or without TGF-β1 (2 ng/mL). The number of colony-forming units from 100 cells of each fraction was assayed. The data shown are mean ± SD (n = 3); ns, not significant. (B) Proportion of colonies determined by microscopic analysis. (C) Representative photographs of BFU-E–type colonies (Upper; without TGF-β1) and CFU-E–type colonies (Lower; with TGF-β1) from CD71⁺ MEPs. (D) Each FACS-purified fraction was cultured for 4 d in serum-free medium supplemented with SCF, TPO, and EPO, ± TGF-β1 (2 ng/mL), and then assayed for GPA expression. Black, unstained control; blue, without TGF-β1; red, with TGF-β1. Similar results were reproduced in two independent experiments. (E) Representative photographs of GPA⁺ cells obtained from CD71⁻ CD105⁻ MEPs (Left) and CD71^int/+ CD105⁺ EPs (Right) in the absence (Upper) and presence (Lower) of TGF-β.

Fig. S4.

Effect of TGF-β on erythrocyte and megakaryocyte development. (A) Total cell number at day 4 of culture with or without TGF-β1. Data presented are mean ± SD (n = 2). (B) CD41 and GPA expression of MEP subpopulations on day 8 of culture with or without TGF-β1. The percentage of events in each gate is shown in the plot. (C) Absolute number of CD41⁺ cells on day 8 of culture with or without TGF-β1. Data presented are mean ± SD (n = 2).

On day 8 of the same culture, we analyzed the effect of TGF-β on MegK development (Fig. S4B). TGF-β negatively affected the production of CD41⁺ MegKs from CD71⁻ CD105⁻ or CD71⁺ CD105⁻ MEPs (50–65% reduction compared with controls; Fig. S4C). As a result, the frequency of CD41⁺ MegKs in the progeny of CD71⁻ CD105⁻ and CD71⁺ CD105⁻ MEPs was slightly elevated. Adding TGF-β did not change the E lineage–restricted potential of EPs.

Gene Expression Analysis of Human Myeloerythroid Progenitors.

We previously reported that various TFs play a pivotal role in lineage specification in both mouse (7, 35) and human (8, 36) hematopoiesis. Each purified population described above (CMP and MEP subpopulations and GMPs) was subjected to real-time PCR analysis to test the expression profiles of TFs and lineage-related cytokine receptor genes (shown in Fig. S5). GATA family TFs (GATA-1, GATA-2) and their cofactor Friend of GATA (FOG)-1 are required for MegE lineage development: Knockout of GATA-1 (37, 38) or FOG-1 (39) genes is embryonically lethal in mice due to severe anemia. These GATAs were up-regulated according to MegE lineage differentiation but down-regulated in the GMP stage. On the other hand, GM-affiliated TFs (i.e., C/EBPα, PU.1) were elevated along with GM commitment from CMPs to GMPs but suppressed in MEPs, consistent with previous reports (7, 8). Erythroid Krüppel-like factor (EKLF; KLF-1) is a critical TF for erythroid development (9, 40, 41); KLF-1 and a critical MegK activator, Friend leukemia integration (Fli)-1 (42), antagonize each other at the bifurcation of MegK versus erythroid lineages (40, 43). Consistent with their hematopoietic outcome, CD71^int/+ CD105⁺ EPs showed the highest KLF-1 but the lowest Fli-1 expression among the MEP subfractions (Fig. S5). Conversely, CD71⁻ CD105⁻ MEPs showed the lowest KLF-1 but the highest Fli-1 expression among the MEP subfractions, presumably reflecting its robust MegK potential.

Fig. S5.

Comparison of lineage-affiliated gene expression between human EPs and other myeloid progenitors. Quantitative real-time PCR analysis of lineage-instructive transcription factors and cytokine receptors. GMP, granulocyte/macrophage progenitor. Samples were monitored in triplicate; values were normalized to the expression level of CD71⁻ CD105⁻ CMPs. GAPDH was used as internal control. Data presented are mean ± SD.

EPO signaling is essential for erythroid cell development, especially following the progenitor stage, where EPO mainly blocks apoptosis (44, 45). We found that EPOR, a gene encoding the EPO receptor, was expressed at divergent levels among subpopulations: low in CMPs, higher in bipotent MegEs, and higher still in CD71^int/+ CD105⁺ EPs (Fig. S5). TPO is the most potent cytokine that physiologically regulates MegK and subsequent platelet production (46). Its receptor (TPOR; c-Mpl) expression was lowest in CD71^int/+ CD105⁺ EPs among the various MEP subfractions.

These findings suggest that changes in expression patterns of lineage-instructive TFs or lineage-related cytokine receptors in MEPs are similar between human and mouse (9), although the exact identity of human MegK progenitors remains unclear.

Discussion

We report in the present study that hEPs are prospectively isolatable in adult steady-state bone marrow as a subset of cells among the originally defined hMEPs, showing that the conventional hMEP population is heterogeneous. Moreover, truly bipotent hMEPs reside only in the CD105⁻ fraction of conventional hMEPs (Fig. 6).

Fig. 6.

Proposed model of the developmental pathway in human myeloid progenitors. The originally defined human CMP contained the unbiased (or slightly GM-biased) CD71⁻ CD105⁻ fraction and MegE-biased CD71⁺ CD105⁻ fraction (pre-MEPs; yellow). The hMEP downstream of these fractions contained a CD71⁺ CD105⁻ fraction of E-MEPs (yellow) and a CD71^int/+ CD105⁺ fraction of EPs (orange), respectively. NK, natural killer.

CD71, the transferrin receptor, has been well-established as one of the early E-lineage markers; the CD34⁺ CD71⁺ GPA⁻ fraction, defined as unipotent E-lineage progenitors/precursors, exists at a stage downstream of MEPs, and was used for functional and/or gene expression analysis (11, 13, 30, 47). However, we found CD71 expression at a much earlier stage of differentiation in a subset of CMPs. A fraction (∼30%) of CMPs is CD71-positive but still retains both GM potential and MegK potential (Fig. 2). In addition, even CD71⁺ CD105⁻ MEPs (E-MEPs) show MegK potential; thus, CD71 alone may be useful for enrichment but is insufficient for the purification of E-lineage progenitors.

Combining CD71 with CD105 (endoglin) staining enables us to identify hEPs. Most colonies that originate from CD71^int/+ CD105⁺ hEPs are of the CFU-E type (containing a low number of fully matured erythrocytes; Fig. 3), indicating that this isolated fraction corresponds to mouse pre–CFU-Es (9). Importantly, hEPs never generated MegK cells even under serum-free culture conditions, which allow optimal growth and differentiation of MegK progenitors (48). CD105 is part of the TGF-β receptor complex (29); several studies have revealed an inhibitory effect of TGF-β on E-lineage cell proliferation, causing accelerated terminal maturation by blocking the cell cycle of immature cells (30–34). However, these studies targeted CD34⁺ CD71⁺ or CD36⁺ “erythroid progenitors” and not CD105⁺ cells. We found a similar effect of TGF-β on the more specific CD71^int/+ CD105⁺ EPs (Fig. 5). Therefore, the effect of TGF-β observed in previous studies may be largely due to this subset, because CD71^int/+ CD105⁺ EPs expressed both CD36 and CD71 on the cell surface (shown in Fig. 1B). TGF-β is commonly present in FCS and is known to impose negative effects not only on erythroid but also on MegK production (48). In addition, TGF-β showed a less inhibitory effect on MegK production than that on erythrocyte production (Fig. 5), and thus it may reflect the absence of CD105 expression in putative MegK progenitors.

We sought to trace the developmental pathway in human CMPs and MEPs in vitro (Fig. 4). After a 60-h culture, CD71⁻ CD105⁻ CMPs could differentiate into CD71⁺ CD105⁻ CMPs as well as MEPs/GMPs. CD71⁺ CD105⁻ CMPs generated more cells corresponding to E-MEPs and EPs in addition to some CD71⁻ CD105⁻ CMPs/MEPs at the same time point. Furthermore, CD71⁺ CD105⁻ E-MEPs gave rise to EPs but fewer CD71⁻ CD105⁻ MEPs, whereas CD71^int/+ CD105⁺ EPs never generated CD105⁻ MEPs. These findings indicate that the main stream of E-lineage development continues from CD71⁻ CD105⁻ CMPs, via CD71⁺ CD105⁻ CMPs and CD71⁺ CD105⁻ MEPs, to EPs (Fig. 6). MegK progenitors may be isolatable somewhere in the latter pathway.

There are some unclarified issues at this point. (i) Is the CD71^int/+ CD105⁺ hEP the earliest stage of unipotent E-lineage cells? The expression of CD36 precedes that of CD105 (Fig. 1); thus, it could be that CD71⁺ CD36⁺ CD105⁻ cells within the original CMP and/or MEP fractions are earlier committed E progenitors (e.g., pre–BFU-E), although CD36 is reported to be expressed also on MegKs, monocytes (49), and/or a part of CD13⁺ CD133⁺ bipotent myeloerythroid progenitors (50). Moreover, as shown in Fig. 1B, the expression level of CD36 in CMP subpopulations is quite low, and thus additional cell-surface markers may be required to further investigate this issue. (ii) Is the E-lineage developmental pathway common between fetal and adult hematopoiesis? We found that far fewer EPs existed in cord blood (CB) than in BM (Fig. S1). This finding may suggest the immaturity of CB erythroid cells, and could partially explain the delayed recovery of red blood cells and platelets resulting in the increased frequency of blood transfusions frequently seen among patients who received CB transplants compared with allogeneic mobilized peripheral blood stem cell transplants. That being said, both HSC numbers and HSC engraftment potential are other potential contributing variables (51). CB might possess unknown E-lineage developmental pathways different from adult hematopoiesis. (iii) Are hEPs involved in physiological erythropoiesis in vivo? We have not tested the in vivo lineage potential of isolated hEPs in a xenotransplantation model (52) because of their small numbers and limited proliferation capacity. These subsets of hMEP and hEP populations in patients with unexplained anemia, MDS, or PV will need to be analyzed in comparison with healthy individuals. (iv) How can one isolate MegK lineage-committed progenitors? We found that the expression level of CD41, a well-established marker for MegK progenitors both in mouse (9) and human (13, 25), was up-regulated with MegE differentiation and down-regulated in CD105⁺ EPs (Fig. S2 B and C). To isolate MegK-committed progenitors at the CMP or MEP level, a marker other than or in addition to CD41 is necessary. Recently, MegK-biased HSCs (53) and MegK-committed progenitors with long-term repopulating capacity (54) within the mouse HSC compartment have been reported. In humans as well, a portion of MegK progenitors might develop directly from earlier stem cells/progenitors such as HSCs and MPPs, bypassing the CMP or MEP stage.

Although the MegK developmental pathway remains undetermined, the use of key TFs, at least in the E lineage, appears to be well-preserved between human and mouse (Fig. S5). The CD71⁻ CD105⁻ CMPs express low levels of both MegE-related (GATAs, KLF-1, Fli-1) and GM-related (C/EBPα, PU.1) TFs, with the up-regulation of the former and down-regulation of the latter as the CMP differentiates downstream toward the MEP via an intermediate CD71⁺ CD105⁻ CMP stage. Subsequent KLF-1 up-regulation accompanied by Fli-1 down-regulation may be critical for the E-lineage fate determination at the branch-point of the MegK versus E lineage.

In summary, we have identified hEPs as a CD71^int/+ CD105⁺ fraction of previously defined hMEPs. This population could be a useful tool for studying physiological and pathological erythroid cell development, and should be analyzed in patients suffering from anemia or erythrocytosis such as in MDS or PV.

Materials and Methods

Antibodies, Cell Staining, and Sorting.

The sorting procedures for HSCs and myeloid progenitor populations that we previously reported (20) were slightly modified. In brief, bone marrow mononuclear cells, purchased from AllCells, were first stained with phycoerythrin (PE)-Cy5–conjugated lineage antibodies, including anti-CD3, -CD4, -CD8, -CD10, -CD19, -CD20, -CD11b, -CD14, and -CD56. Cells were then stained with allophycocyanin (APC)-Cy7–conjugated anti-CD34 (BioLegend), APC-conjugated anti-CD38 (BD Pharmingen), PE-conjugated anti–IL-3Rα (BD Pharmingen), and Pacific blue (PaB)- or FITC-conjugated anti-CD45RA (BioLegend or eBioscience) antibodies. CMPs, GMPs, and MEPs were isolated as Lin⁻ CD34⁺ CD38⁺ IL-3Rα⁺ CD45RA⁻, Lin⁻ CD34⁺ CD38⁺ IL-3Rα⁺ CD45RA⁺, and Lin⁻ CD34⁺ CD38⁺ IL-3Rα^−/lo CD45RA⁻ populations, respectively. To sort EPs and MegK progenitors, FITC-conjugated anti-CD71 (BioLegend) and biotinylated anti-CD105 (endoglin; BioLegend) antibodies followed by streptavidin-PE-Cy7 (BD) were added. Pre-EMPs, E-MEPs, and EPs were purified as Lin⁻ CD34⁺ CD38⁺ IL-3Rα⁺ CD45RA⁻ CD71⁺ CD105⁻, Lin⁻ CD34⁺ CD38⁺ IL-3Rα^−/lo CD45RA⁻ CD71⁺ CD105⁻, and Lin⁻ CD34⁺ CD38⁺ IL-3Rα⁻ CD45RA⁻ CD71⁺ CD105⁺ populations, respectively. MegK progenitors were enriched within the Lin⁻ CD34⁺ CD38⁺ IL-3Rα^−/lo CD45RA⁻ CD71⁻ CD105⁻ fraction. To evaluate Flt3 expression, costaining of CD105 was omitted due to technical difficulties. Dead cells were excluded by propidium iodide staining. All sorting and analyses were performed on a three laser-equipped FACSAria II (BD Biosciences). The automatic cell-deposition system was used for single-cell assays. FACS data were analyzed with FlowJo software (Tree Star).

Cell Culture.

For short-term liquid cultures, purified populations were suspended on 12-well plates with the following medium: Iscove’s modified Dulbecco’s medium (Life Technologies) supplemented with 20% (vol/vol) FCS, antibiotics, 20 ng/mL human recombinant SCF, 20 ng/mL GM-CSF, 4 U/mL EPO, and 20 ng/mL TPO (R&D Systems). IL-3 (20 ng/mL) was added when CMPs were cultured. For clonogenic analysis of myeloid progenitors, cells were cultured for 14 d in Iscove's Modified Dulbecco's Medium (IMDM)-based methylcellulose medium (MethoCult GF H4434; StemCell Technologies), which contained FCS, BSA, 2-mercaptoethanol, recombinant SCF, IL-3, GM-CSF, and EPO, with an additional 20 ng/mL TPO. For the MegK assay, serum-free medium (StemSpanTM SFEM II; StemCell Technologies) was used.

All cultures were incubated in a humidified chamber in 5% CO₂. Colonies were scored and picked up for making cytospin preparations to define cell components.

Gene Expression Analysis.

Total RNA was extracted from purified progenitor populations using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. All RNA samples were reverse-transcribed with Oligo(dT) primers using the SuperScript III First-Strand Synthesis System (Invitrogen/Life Technologies). Quantitative real-time PCR assays were performed with the 7900HT Fast Real-Time PCR System (Life Technologies). All specific primers and probes were purchased from inventoried stocks of TaqMan Gene Expression Assays (Applied Biosystems/Life Technologies). GAPDH transcripts were simultaneously amplified as an internal standard for quantification. All samples were analyzed in triplicate.

Statistical Analysis.

The unpaired two-tailed Student t test was applied to all pairwise comparisons of mean values after F-test evaluation of variance. All statistical analyses were performed with Prism 5 software (GraphPad).