Prospective Isolation of Human Erythroid Lineage-Committed Progenitors

Prospective isolation of human erythroid lineage-committed progenitors

Yasuo Moria,b, James Y. Chena,b, John V. Pluvinagea,b, Jun Seitaa,b,1,2, and Irving L. Weissmana,b,1,2

aInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; and bLudwig Center for Cancer Stem Cell Biology and Medicine at Stanford University, Stanford, CA 94305

Contributed by Irving L. Weissman, June 21, 2015 (sent for review February 17, 2015) Determining the developmental pathway leading to erythrocytes generate erythrocytes. Erythrocytes, like all blood lineages, de- and being able to isolate their progenitors are crucial to un- velop through a series of differentiation stages that begin with derstanding and treating disorders of red cell imbalance such as HSCs, which have been prospectively isolated in humans (10). The anemia, myelodysplastic syndrome, and polycythemia vera. Here surface markers of intermediate erythroid progenitors/precursors we show that the human erythrocyte progenitor (hEP) can be and the expression pattern of some pivotal transcription factors prospectively isolated from adult bone marrow. We found three (TFs) during their differentiation from HSCs have been analyzed subfractions that possessed different expression patterns of CD105 (11–19). As a result, the erythroid-committed progenitor (EP), and CD71 within the previously defined human megakaryocyte/ − + + − which exists downstream of the bipotent MEP, has been isolated erythrocyte progenitor (hMEP; Lineage CD34 CD38 IL-3Rα − − − + − in mouse but not human (9). CD45RA ) population. Both CD71 CD105 and CD71 CD105 To isolate the presumed human erythrocyte progenitor (hEP), MEPs, at least in vitro, still retained bipotency for the megakaryo- we first looked at markers crucial to isolating the mouse EP. One cyte (MegK) and erythrocyte (E) lineages, although the latter sub- such key marker is CD105 (endoglin), whereby mouse EPs are − − population is skewed in differentiation toward the erythroid Lineage (Lin) stem cell antigen (Sca)-1 receptor tyrosine kinase lineage. Notably, the proliferative and differentiation output of the + − + + − intermediate(int)/+ + c-Kit CD16/32 CD150 CD105 CD41 . In humans, CD105 CD71 CD105 subset of cells within the MEP popu- and CD71 have been classified as early erythroid cell markers lation was completely restricted to the erythroid lineage with the –

+ − (14 19). However, CD105 and CD71 expression in oligopotent CELL BIOLOGY loss of MegK potential. CD71 CD105 MEPs are erythrocyte- + + myeloid progenitors (8) [i.e., CMPs, granulocyte/macrophage pro- biased MEPs (E-MEPs) and CD71int/ CD105 cells are EPs. These genitors (GMPs), and MEPs] has not been evaluated. Thus, we previously unclassified populations may facilitate further under- examined closely the expression of these markers in CMPs, GMPs, standing of the molecular mechanisms governing human erythroid MEPs, and their progeny. development and serve as potential therapeutic targets in disorders of the erythroid lineage. Results CD71 and CD105 Expression Patterns Subfractionate Myeloid Progenitors. erythroid progenitor | endoglin | lineage commitment | hematopoiesis | First, CMPs, GMPs, and MEPs were purified from human bone transcription factor marrow (BM) according to the flow cytometric sorting scheme shown in Fig. 1A. The BM mononuclear cell fraction that is − t is now apparent that hematopoiesis derives throughout negative for lineage-affiliated antigens (Lin ) was subdivided + − − + Ipostnatal life from the constant input from a small fraction of into the CD34 CD38 CD45RA HSC/MPP and CD34 + − + + hematopoietic stem cells (HSCs) (1, 2), themselves rare, un- CD38 progenitor fractions (10, 20). The Lin CD34 CD38 dergoing cell divisions that include self-renewal to HSCs and progenitors were then fractionated into CMP, GMP, and MEP differentiation. The differentiation largely to multipotent pro- populations according to the expression patterns of IL-3Rα and genitors (MPPs) that undergo transient amplifying divisions to CD45RA, as previously reported (8): CMPs, GMPs, and MEPs + − + + give rise to all blood cell types but do not undergo long-term self- were defined as IL-3Rα CD45RA ,IL-3Rα CD45RA ,and − − renewal (3). In mice and humans, the steps between HSCs and IL-3Rα /lo CD45RA populations, respectively. 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 Significance almost fully described in mouse hematopoiesis (4). Determining the differentiation pathway leading to erythrocytes and isolating We have identified the first step of erythrocyte lineage com- intermediate/+ + erythroid-specific progenitors are crucial to understanding how mitment in human bone marrow as a CD71 CD105 fate determinations are made that result in homeostatic pro- cell fraction of a previously defined megakaryocyte/erythro- duction of blood cells and elements. Such an understanding can cyte progenitor population. This purification could be a useful also elucidate disorders of red cell imbalance, such as anemia, tool for studying physiological and pathological red blood cell myelodysplastic syndrome (MDS), and polycythemia vera (PV) development, and should be analyzed in patients suffering (5, 6). Here we sought to find cell-surface markers that can be from anemia or erythrocytosis such as in myelodysplastic syn- used to isolate from human bone marrow each stage of differ- drome or polycythemia vera. entiation after the fate determinations that lead MPPs to com- Author contributions: Y.M., J.Y.C., J.V.P., J.S., and I.L.W. designed research; Y.M., J.Y.C., J.V.P., mon myeloid progenitors (CMPs) to megakaryocyte/erythroid and J.S. performed research; Y.M., J.Y.C., J.V.P., and J.S. analyzed data; and Y.M., J.Y.C., J.V.P., progenitors (MEPs) and allow the commitment to erythropoiesis. J.S., and I.L.W. wrote the paper. Certain aspects of erythrocyte development have been elucidated, The authors declare no conflict of interest. such as the overall pathway from HSCs through CMPs and MEPs Freely available online through the PNAS open access option. – (7 9). However, the original human CMP and MEP populations 1J.S. and I.L.W. contributed equally to this work. still appear to be heterogeneous, and the subsequent stages of 2To whom correspondence may be addressed. Email: [email protected] or irv@stanford. differentiation (i.e., from MEP to erythrocyte) remain unclear. edu. Given this background, we sought markers that could sub- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. divide CMPs and/or MEPs into cells specifically destined to 1073/pnas.1512076112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1512076112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 + P = − − Singlet PI- Lin- CD34+CD38- CD34+CD38+ 0.048) and GPA cells ( 0.002) than did CD71 CD105 A G CMP CMPs (Fig. 2 ). 2.95% 9.51% HSC/MPP 35.2% 80.2% GMP Subsequent CD105 Up-Regulation Within MEPs Marks Complete Erythroid FSC CD34 IL-3R α IL-3R α 26.3% Lineage Commitment. Among three subfractions of MEPs (shown in + 15.4% MEP31.0% Fig. 3A), the colony-forming potential was higher in the CD71 − + + Lineage CD38 CD45RA CD45RA CD105 fraction (37.0 ± 4.9%) than in the CD71int/ CD105 − − CD34+CD38- CMP MEP GMP fraction (29.2 ± 4.2%) (P = 0.014) and in the CD71 CD105 + − 0.2% 1.26% 32.9% 0% fraction (14.7 ± 3.4%) (P < 0.0001) (Fig. 2B). CD71 CD105 MEPs 96.8% 75.9% 16.4% 98.1% generated primarily large E colonies [defined as burst-forming unit- erythroid colony (BFU-E)–derived], although they still retained some CD105 CD105 CD105 CD105 A int/+ + 2.5% 21.0% 46.7% 1.48% MegK potential (Fig. 4 ). In contrast, the CD71 CD105 MEPs generated only the E lineage, and small-sized colonies from this CD71 CD71 CD71 CD71 B fraction contained more mature (enucleated) erythrocytes [scored as CD36 GPA colony-forming unit-erythroid (CFU-E)–derived] (Fig. 3 C and D). + + FMO FMO These results suggest that CD71int/ CD105 MEPsexistatastage - - CD71-CD105- CMP + − CD71 CD105 CMP CD71+CD105- CMP CD71+CD105- CMP - - downstream of CD71 CD105 MEPs. A similar result was observed - - CD71 CD105 MEP + + CD71 CD105 MEP CD71+CD105- MEP in a serum-free liquid culture system: CD71int/ CD105 MEPs CD71+CD105- MEP CD71int/+CD105+ MEP CD71int/+CD105+ MEP Erythrocyte lost the MegK lineage potential (Fig. 3 E and F). Based on these − + + − findings, we designate the Lin CD34 CD38 IL-3Rα /lo − + − Fig. 1. Flow cytometric analysis of human myeloid progenitors in the bone CD45RA CD71 CD105 as E-biased MEPs (E-MEPs) and the − + + − − + + marrow. (A) CD71 and CD105 (endoglin) expression reveals phenotypic Lin CD34 CD38 IL-3Rα /lo CD45RA CD71int/ CD105 frac- heterogeneity of myeloid progenitor cell compartments. (Upper) Gating tion as E-committed progenitors. 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 contrast, CD71 CD105 MEPs frequently generated MegK- containing colonies but not CFU-GM. Serum-free liquid culture in the plot. PI, propidium iodide; FSC, forward scatter. (B) Evaluation of other + erythroid-affiliated surface markers CD36 and GPA. The threshold between also showed this fraction gave rise most efficiently to CD41 − negative and positive was defined by the fluorescence minus one (FMO) MegK cells, indicating that these are not just contaminated CD71 − method, and erythrocytes were used as a positive control for GPA expression. CD105 CMPs but might contain putative MegK progenitors.

Immunophenotypic Comparisons Among Subfractionated CMPs/MEPs. We analyzed the expression pattern of putative erythroid We further characterized the cell-surface markers of the CMP markers for each of the progenitor populations. The expression and MEP subfractions. Flt3/flk2 is a tyrosine kinase receptor 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/ AE ± ± CD71- CD105- CD71+ CD105- Unstained Stained MPP (5.8 2.8%) and GMP fractions (5.3 5.4%). Among control these populations, only MEPs expressed CD105, and CD105- 0% 1.74% - positive cells coexpress CD71 at intermediate to positive in- CD71 0% 81.3% + + - tensity [hereafter CD71intermediate(int)/ CD105 MEPs; 34.5 ± CD105 B 50 C 100 100% 14.1% 6.9% of MEPs]. CD36, a known early marker of erythroid de- * CFU-GM + − 40 80 velopment (19, 21, 22), was expressed in some CD71 CD105 CFU-E 0% 0.91% int/+ + 30 60 MEPs but almost all CD71 CD105 MEPs. In contrast, BFU-E CD71+ 0% 94.7% 20 40 ± MegK CD105- glycophorin A (GPA/CD235), a late marker of erythroid de- 10 20 100% 3.23% CD41 Proportion (%) velopment (11), was not detectable in any fractions examined CFUs/100 cells 0 0 (Fig. 1B). Subfractions based on CD71 and CD105 expression CD71 - +CD71- + GPA int/+ + CD105 -- CD105 -- were also found in cord blood, although the CD71 CD105 + + D CFU-GM BFU-E F G CD41 GPA CD41+ 1000 MEP fraction was much smaller in cord blood than in adult ** bone marrow (Fig. S1). )

3 100

CD71 Expression Initiated at the CMP Stage Represents Megakaryocyte/ (x10 GPA+ 10 Erythrocyte-Biased Lineage Potential. We functionally examined the * − − + Absolute number differentiation potential of CD71 CD105 CMPs and CD71 1 − CD105 CMPs (Fig. 2A). Methylcellulose colony assay revealed that CD71 - +-+ − − CD105 -- -- colony-forming efficiencies are decreased from CD71 CD105 + − + CMPs (38.2 ± 5.2%) to CD71 CD105 CMPs (33.0 ± 2.2%) Fig. 2. CD71 fraction within the original human CMP showed differenti- (P = 0.047) (Fig. 2B) and the differentiation potential of ation potential skewed toward the MegE lineage. (A and B) The morphology + − (A) (May–Giemsa stain) and in vitro colony-forming potential (B) of FACS- CD71 CD105 CMPs skewed toward the megakaryocyte − − + − − − purified CD71 CD105 or CD71 CD105 CMPs. The number of colony- (MegK)/erythrocyte (E) (MegE) lineage, whereas CD71 CD105 forming units from 100 cells of each fraction is shown. Data presented are CMPs generated a variety of myeloid colonies including colony- mean ± SD (n = 6); *P < 0.05. In this culture condition, unfractionated CMPs forming unit granulocyte/macrophage (CFU-GM) (Fig. 2 C and D). form 38.7 ± 4.0 colonies per 100 cells (n = 3). (Scale bars, 10 μm.) (C and D) We further tested the MegK and erythroid potentials of each Colonies were picked up, cytospun, and stained by the May–Giemsa method fraction by a serum-free liquid culture supplemented with IL-3, to determine the cell types included. (E) FACS-purified 2,000 cells of each stem cell factor (SCF), erythropoietin (EPO), and thrombopoietin fraction were cultured for 10 d under serum-free conditions and then ana- − − lyzed. CD41 or GPA positivity was compared with an unstained control (Left). (TPO). As shown in Fig. 2 E and F, both CD71 CD105 CMPs + + − (F) Cytospin preparations (May–Giemsa staining) of sorted CD41 MegKs and CD71 CD105 CMPs could give rise to CD41-expressing + + + + − (Upper) and GPA erythrocytes (Lower) are shown. The progeny from CD71 MegKs as well as GPA erythrocytes. However, CD71 CD105 CD105− CMPs are shown. (G) Absolute number of CD41+ MegKs or GPA+ + CMPs gave rise to higher numbers of both CD41 cells (P = erythrocytes. Data shown are mean ± SD (n = 3); *P < 0.05, **P < 0.01.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1512076112 Mori et al. Downloaded by guest on September 29, 2021 colony-forming assays (Fig. 4A). Such phenotypically defined sec- A CD71- CD71+ CD71int/+ E Unstained Stained CD105- CD105- CD105+ control ondary myeloid progenitors displayed differentiation activity con- 0% 1.9% sistent with their original phenotypic definition. CD71-expressing + CD71- 0% 94.9% - cells mainly gave rise to the MegE lineage, and CD105 cells al- CD105 – 100% 2.01% most exclusively produced CFU-E type erythroid cells. B C 50 ** * 100 MegK ± E 0% 0.29% β 40 80 Suppressive Effect of TGF- on Proliferation of EPs/E-MEPs Is Accompanied CFU-E CD71+ 0% 98.5% 30 60 CD105- by Accelerated Terminal Differentiation. We tested the effect of TGF- 20 40 BFU-E β1 on MEP subfractions according to the following rationale. CD105 10 20 100% 0.35% Proportion(%)

CFUs/100 cells 0 0 0% 0.01% is an accessory molecule of the transforming growth factor beta CD71 - + int/+ CD71 - + int/+ int/+ β β CD71 0% 99.4% (TGF- ) type III receptor complex, able to bind TGF- 1andTGF- CD105 --+ CD105 --+ CD105+ β β β MegK CFU-E BFU-E 3 but not TGF- 2 (29). TGF- , as well as tumor necrosis factor 100% 0.29% D CD41 alpha and IFN gamma, is a powerful inhibitor of erythropoiesis both GPA in vivo and in vitro. The suppressive effect of TGF-β on E-lineage F CD41+ GPA+ 1000 cell proliferation is accompanied by an acceleration of terminal

) 100 3 maturation (30–34). 10 β (x10 1 We found that TGF- 1 did not affect the number of colonies CD71 - + int/+ - + int/+ generated by all three MEP subfractions (Fig. 5A) but affected Absolute number CD105 --+ --+ the types of colonies generated: The proportion of CFU-E–type + + – Fig. 3. CD71int/ CD105 fraction within the original human MEP represents colonies was increased at the expense of BFU-E type colonies the human EP. (A and B) The morphology (A) (May–Giemsa stain) and (Fig. 5 B and C). By using a serum-free suspension culture, we in vitro colony-forming potential (B) of FACS-purified MEP subfractions. The also monitored the effects of TGF-β1 at an earlier time point. number of CFUs from 100 cells of each fraction. Data shown are mean ± SD Additional TGF-β1 yielded two- to threefold fewer cells after 4 (n = 6); *P < 0.05, **P < 0.01. (Scale bars, 10 μm.) (C and D) Colonies were d of culture (Fig. S4A), with higher expression levels of GPA picked up, cytospun, and stained by the May–Giemsa method to determine (Fig. 5D) and morphological signs of maturation (Fig. 5E) in all the cell types included. (E) Representative flow cytometry plot of 10-d progeny subfractions. These findings suggested that TGF-β1 provided a of MEP subfractions. The percentage of events in each gate is shown. + + signal that inhibited proliferation and promoted differentiation (F) Absolute numbers of CD41 MegKs and GPA erythrocytes. Data shown + +

int/ CELL BIOLOGY ± = among CD71 CD105 EPs as well as in CD105-expressing are mean SD (n 3). − − + − progenies of CD71 CD105 or CD71 CD105 MEPs (30–34). On day 8 of the same culture, we analyzed the effect of TGF-β known for its heterogeneous expression in CMPs, high expres- on MegK development (Fig. S4B). TGF-β negatively affected the + − − + sion in GMPs, and down-regulation in MegE lineages (23, 24). production of CD41 MegKs from CD71 CD105 or CD71 − − CD105 MEPs (50–65% reduction compared with controls; Fig. Among CMPs, Flt3 cells are considered to be in a transitional + stage to MEPs. In accordance with these earlier findings, our re- S4C). As a result, the frequency of CD41 MegKs in the progeny − − − + − sults indicated that Flt3 is expressed on CD71 CMPs, then of CD71 CD105 and CD71 CD105 MEPs was slightly ele- + gradually down-regulated from CD71 CMPs to MEPs but highly vated. Adding TGF-β did not change the E lineage–restricted expressed on GMPs (Fig. S2A). potential of EPs. Consistent with the in vitro MegK potential, we found that MegK-associated molecules CD41 (13, 25) and CD9 (26–28), Gene Expression Analysis of Human Myeloerythroid Progenitors. We known to be expressed in the mouse MegK progenitor, were previously reported that various TFs play a pivotal role in lineage + expressed at higher frequencies on the surface of human CD71 specification in both mouse (7, 35) and human (8, 36) hemato- − − − CD105 CMPs than on CD71 CDS105 CMPs, whereas CD226 poiesis. Each purified population described above (CMP and − − and CD42b were not significantly expressed on CD71 CD105 MEP subpopulations and GMPs) was subjected to real-time + − PCR analysis to test the expression profiles of TFs and lineage- CMPs (∼1%) or CD71 CD105 CMPs (∼5%). In contrast, the related cytokine receptor genes (shown in Fig. S5). GATA family MegK-related markers analyzed were down-regulated or not + + TFs (GATA-1, GATA-2) and their cofactor Friend of GATA expressed on the surface of CD71int/ CD105 MEPs (Fig. S2 B (FOG)-1 are required for MegE lineage development: Knockout and C). of GATA-1 (37, 38) or FOG-1 (39) genes is embryonically lethal Human EPs Develop from E-MEPs. To test the lineage relationship in mice due to severe anemia. These GATAs were up-regulated of these subfractionated myeloerythroid progenitors directly, we according to MegE lineage differentiation but down-regulated in 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 Primary Sort Secondary Sort CFU-E MegK-containing − − + − + − BFU-E CFU-GM CD71 CD105 and CD71 CD105 ), whereas CD71 CD105 CD71- CD105- CMP CMPs differentiated toward MEPs but not GMPs (Fig. S3A). CD71- CD105- CMP CD71+ CD105- CMP Furthermore, a minor fraction of progeny possessed the surface CD71+ CD105- CMP/MEP phenotype corresponding to EPs (Fig. S3A). These data suggested CD71+ CD105- CMP − − + CD71int/+ CD105+ CMP/MEP that the CD71 CD105 CMPs are the precursor of CD71 + - − + − + - CD71 CD105 MEP CD105 CMPs. However, CD71 CD105 CMPs generated a few CD71 CD105 MEP int/+ + − − − CD71 CD105 MEP CD71 CD105 CMPs, suggesting either CD71 contamination or int/+ + int/+ + possible bidirectionality between them. In the same culture con- CD71 CD105 MEP CD71 CD105 MEP + − 01020 3040 ditions, CD71 CD105 E-MEPs differentiated mainly into CFUs/100 cells + + − − CD71int/ CD105 EPs and a small number of CD71 CD105 + + Fig. 4. Lineage relationship of subfractionated human myeloid progenitors. MEPs (Fig. S3B), whereas CD71int/ CD105 EPs did not − − Lineage potential of FACS-purified progenitors from the culture of each generate E-MEPs or CD71 CD105 MEPs. These data clearly primary fraction. After 60 h in liquid culture, cells were subfractionated + + indicate that EPs exist at a stage downstream of E-MEPs. To by secondary sort and subjected to colony assay. CD71int/ CD105 MEPs perform a secondary analysis, cells were sorted and subjected to formed only CFU-E.

Mori et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 − bipotent hMEPs reside only in the CD105 fraction of conven- A ns B 100 50 MegK ± E CFU-E BFU-E tional hMEPs (Fig. 6). ns CD71, the transferrin receptor, has been well-established as 40 80 + + − C CD71- CD71int/+ one of the early E-lineage markers; the CD34 CD71 GPA CD105- CD105+ 30 ns 60 fraction, defined as unipotent E-lineage progenitors/precursors,

20 40 TGF-β - exists at a stage downstream of MEPs, and was used for functional Proportion(%) and/or gene expression analysis (11, 13, 30, 47). However, we CFUs/100 cells 20 10 found CD71 expression at a much earlier stage of differentiation 0 0 TGF-β + in a subset of CMPs. A fraction (∼30%) of CMPs is CD71-positive β β TGF- -+ -+ -+ TGF- -+ -+ -+ but still retains both GM potential and MegK potential (Fig. 2). CD71 - + + CD71 - + + + − CD105 --int/+ CD105 --int/+ In addition, even CD71 CD105 MEPs (E-MEPs) show MegK E CD71- CD71int/+ D CD71- CD71+ CD71int/+ CD105- CD105+ potential; thus, CD71 alone may be useful for enrichment but is CD105- CD105- CD105+ insufficient for the purification of E-lineage progenitors. TGF-β - Combining CD71 with CD105 (endoglin) staining enables + us to identify hEPs. Most colonies that originate from CD71int/ + CD105 hEPs are of the CFU-E type (containing a low number TGF-β + GPA of fully matured erythrocytes; Fig. 3), indicating that this isolated unstain control TGF-β - TGF-β + fraction corresponds to mouse pre–CFU-Es (9). Importantly, hEPs Fig. 5. TGF-β1 accelerates erythroid cell maturation from all subfractions of never generated MegK cells even under serum-free culture condi- human MEPs. (A) FACS-purified MEP subfractions were cultured in methyl- tions, which allow optimal growth and differentiation of MegK cellulose-based medium for 10–12 d in the presence of a cytokine mixture progenitors (48). CD105 is part of the TGF-β receptor complex with or without TGF-β1 (2 ng/mL). The number of colony-forming units from (29); several studies have revealed an inhibitory effect of TGF-β on 100 cells of each fraction was assayed. The data shown are mean ± SD (n = E-lineage cell proliferation, causing accelerated terminal matura- 3); ns, not significant. (B) Proportion of colonies determined by microscopic tion by blocking the cell cycle of immature cells (30–34). However, + + + analysis. (C) Representative photographs of BFU-E–type colonies (Upper; these studies targeted CD34 CD71 or CD36 “erythroid pro- β – β + + without TGF- 1) and CFU-E type colonies (Lower; with TGF- 1) from CD71 genitors” and not CD105 cells. We found a similar effect of TGF-β + + MEPs. (D) Each FACS-purified fraction was cultured for 4 d in serum-free on the more specific CD71int/ CD105 EPs(Fig.5).Therefore,the medium supplemented with SCF, TPO, and EPO, ± TGF-β1 (2 ng/mL), and then β effect of TGF-β observed in previous studies may be largely due to assayed for GPA expression. Black, unstained control; blue, without TGF- 1; int/+ + red, with TGF-β1. Similar results were reproduced in two independent ex- this subset, because CD71 CD105 EPs expressed both CD36 periments. (E) Representative photographs of GPA+ cells obtained from and CD71 on the cell surface (shown in Fig. 1B). TGF-β is com- CD71− CD105− MEPs (Left) and CD71int/+ CD105+ EPs (Right) in the absence monly present in FCS and is known to impose negative effects not (Upper) and presence (Lower) of TGF-β. 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 the GMP stage. On the other hand, GM-affiliated TFs (i.e., absence of CD105 expression in putative MegK progenitors. C/EBPα, PU.1) were elevated along with GM commitment from We sought to trace the developmental pathway in human − CMPs to GMPs but suppressed in MEPs, consistent with previous CMPs and MEPs in vitro (Fig. 4). After a 60-h culture, CD71 − + − reports (7, 8). Erythroid Krüppel-like factor (EKLF; KLF-1) is a CD105 CMPs could differentiate into CD71 CD105 CMPs as + − critical TF for erythroid development (9, 40, 41); KLF-1 and a well as MEPs/GMPs. CD71 CD105 CMPs generated more cells − critical MegK activator, Friend leukemia integration (Fli)-1 (42), corresponding to E-MEPs and EPs in addition to some CD71 − + antagonize each other at the bifurcation of MegK versus erythroid CD105 CMPs/MEPs at the same time point. Furthermore, CD71 lineages (40, 43). Consistent with their hematopoietic outcome, + + CD71int/ CD105 EPs showed the highest KLF-1 but the lowest Fli-1 expression among the MEP subfractions (Fig. S5). Con- − − versely, CD71 CD105 MEPs showed the lowest KLF-1 but the HSC highest Fli-1 expression among the MEP subfractions, presumably reflecting its robust MegK potential. MPP EPO signaling is essential for erythroid cell development, es- Original pecially following the progenitor stage, where EPO mainly blocks CMP - CD71 CLP apoptosis (44, 45). We found that EPOR, a gene encoding the CD71 CD105- EPO receptor, was expressed at divergent levels among sub- Flt3 Pre- populations: low in CMPs, higher in bipotent MegEs, and higher MEP Flt3 int/+ + IL-3Rα T, B, NK-cells still in CD71 CD105 EPs (Fig. S5). TPO is the most potent Original cytokine that physiologically regulates MegK and subsequent MEP E-MEP GMP platelet production (46). Its receptor (TPOR; c-Mpl) expression + + CD105 was lowest in CD71int/ CD105 EPs among the various MEP subfractions. EP Granulocytes Monocytes These findings suggest that changes in expression patterns of MkP lineage-instructive TFs or lineage-related cytokine receptors in MEPs are similar between human and mouse (9), although the Erythrocytes exact identity of human MegK progenitors remains unclear. Platelets Discussion Fig. 6. Proposed model of the developmental pathway in human myeloid progenitors. The originally defined human CMP contained the unbiased (or We report in the present study that hEPs are prospectively iso- − − + − slightly GM-biased) CD71 CD105 fraction and MegE-biased CD71 CD105 latable in adult steady-state bone marrow as a subset of cells fraction (pre-MEPs; yellow). The hMEP downstream of these fractions con- + − + + among the originally defined hMEPs, showing that the contained a CD71 CD105 fraction of E-MEPs (yellow) and a CD71int/ CD105 ventional hMEP population is heterogeneous. Moreover, truly fraction of EPs (orange), respectively. NK, natural killer.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1512076112 Mori et al. Downloaded by guest on September 29, 2021 − − − CD105 E-MEPs gave rise to EPs but fewer CD71 CD105 cell development, and should be analyzed in patients suffering + + − MEPs, whereas CD71int/ CD105 EPs never generated CD105 from anemia or erythrocytosis such as in MDS or PV. MEPs. These findings indicate that the main stream of E-lineage − − + development continues from CD71 CD105 CMPs, via CD71 Materials and Methods − + − CD105 CMPs and CD71 CD105 MEPs,toEPs(Fig.6).MegK Antibodies, Cell Staining, and Sorting. The sorting procedures for HSCs and progenitors may be isolatable somewhere in the latter pathway. myeloid progenitor populations that we previously reported (20) were There are some unclarified issues at this point. (i) Is the slightly modified. In brief, bone marrow mononuclear cells, purchased from + + – CD71int/ CD105 hEP the earliest stage of unipotent E-lineage AllCells, were first stained with phycoerythrin (PE)-Cy5 conjugated lineage cells? The expression of CD36 precedes that of CD105 (Fig. 1); thus, antibodies, including anti-CD3, -CD4, -CD8, -CD10, -CD19, -CD20, -CD11b, -CD14, + + − and -CD56. Cells were then stained with allophycocyanin (APC)-Cy7–conju- it could be that CD71 CD36 CD105 cells within the original gated anti-CD34 (BioLegend), APC-conjugated anti-CD38 (BD Pharmingen), CMP and/or MEP fractions are earlier committed E progenitors PE-conjugated anti–IL-3Rα (BD Pharmingen), and Pacific blue (PaB)- or FITC- (e.g., pre–BFU-E), although CD36 is reported to be expressed also + + conjugated anti-CD45RA (BioLegend or eBioscience) antibodies. CMPs, GMPs, on MegKs, monocytes (49), and/or a part of CD13 CD133 bipo- and MEPs were isolated as Lin− CD34+ CD38+ IL-3Rα+ CD45RA−,Lin− CD34+ + + + − + + − − tent myeloerythroid progenitors (50). Moreover, as shown in Fig. 1B, CD38 IL-3Rα CD45RA ,andLin CD34 CD38 IL-3Rα /lo CD45RA pop- the expression level of CD36 in CMP subpopulations is quite low, ulations, respectively. To sort EPs and MegK progenitors, FITC-conjugated and thus additional cell-surface markers may be required to further anti-CD71 (BioLegend) and biotinylated anti-CD105 (endoglin; BioLegend) ii antibodies followed by streptavidin-PE-Cy7 (BD) were added. Pre-EMPs, E-MEPs, investigate this issue. ( ) Is the E-lineage developmental pathway − + + + − + − and EPs were purified as Lin CD34 CD38 IL-3Rα CD45RA CD71 CD105 , common between fetal and adult hematopoiesis? We found that far − + + − − + − − + + Lin CD34 CD38 IL-3Rα /lo CD45RA CD71 CD105 , and Lin CD34 CD38 fewer EPs existed in cord blood (CB) than in BM (Fig. S1). This − − + + IL-3Rα CD45RA CD71 CD105 populations, respectively. MegK progenitors − + + − − − − finding may suggest the immaturity of CB erythroid cells, and could were enriched within the Lin CD34 CD38 IL-3Rα /lo CD45RA CD71 CD105 partially explain the delayed recovery of red blood cells and platelets fraction. To evaluate Flt3 expression, costaining of CD105 was omitted due to resulting in the increased frequency of blood transfusions frequently technical difficulties. Dead cells were excluded by propidium iodide staining. seen among patients who received CB transplants compared with All sorting and analyses were performed on a three laser-equipped FACSAria II allogeneic mobilized peripheral blood stem cell transplants. That (BD Biosciences). The automatic cell-deposition system was used for single-cell being said, both HSC numbers and HSC engraftment potential are assays. FACS data were analyzed with FlowJo software (Tree Star). other potential contributing variables (51). CB might possess un- known E-lineage developmental pathways different from adult he- Cell Culture. For short-term liquid cultures, purified populations were suspended ’ ’ matopoiesis. (iii) Are hEPs involved in physiological erythropoiesis on 12-well plates with the following medium: Iscove s modified Dulbecco s CELL BIOLOGY invivo?Wehavenottestedtheinvivo lineage potential of isolated 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 hEPs in a xenotransplantation model (52) because of their small TPO (R&D Systems). IL-3 (20 ng/mL) was added when CMPs were cultured. numbers and limited proliferation capacity. These subsets of hMEP For clonogenic analysis of myeloid progenitors, cells were cultured for 14 d in and hEP populations in patients with unexplained anemia, MDS, or Iscove’s Modified Dulbecco’s Medium (IMDM)-based methylcellulose medium PV will need to be analyzed in comparison with healthy individuals. (MethoCult GF H4434; StemCell Technologies), which contained FCS, BSA, (iv) How can one isolate MegK lineage-committed progenitors? 2-mercaptoethanol, recombinant SCF, IL-3, GM-CSF, and EPO, with an addi- We found that the expression level of CD41, a well-established tional 20 ng/mL TPO. For the MegK assay, serum-free medium (StemSpanTM marker for MegK progenitors both in mouse (9) and human SFEM II; StemCell Technologies) was used. (13, 25), was up-regulated with MegE differentiation and down- All cultures were incubated in a humidified chamber in 5% CO2. Colonies were + regulatedinCD105 EPs (Fig. S2 B and C). To isolate MegK- scored and picked up for making cytospin preparations to define cell components. committed progenitors at the CMP or MEP level, a marker other than or in addition to CD41 is necessary. Recently, MegK-biased Gene Expression Analysis. Total RNA was extracted from purified progenitor HSCs (53) and MegK-committed progenitors with long-term repo- populations using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. All RNA samples were reverse-transcribed with pulating capacity (54) within the mouse HSC compartment have Oligo(dT) primers using the SuperScript III First-Strand Synthesis System been reported. In humans as well, a portion of MegK progenitors (Invitrogen/Life Technologies). Quantitative real-time PCR assays were per- might develop directly from earlier stem cells/progenitors such as formed with the 7900HT Fast Real-Time PCR System (Life Technologies). All HSCs and MPPs, bypassing the CMP or MEP stage. specific primers and probes were purchased from inventoried stocks of Although the MegK developmental pathway remains unde- TaqMan Gene Expression Assays (Applied Biosystems/Life Technologies). termined, the use of key TFs, at least in the E lineage, appears GAPDH transcripts were simultaneously amplified as an internal standard for to be well-preserved between human and mouse (Fig. S5). The quantification. All samples were analyzed in triplicate. − − CD71 CD105 CMPs express low levels of both MegE-related (GATAs, KLF-1, Fli-1) and GM-related (C/EBPα, PU.1) TFs, Statistical Analysis. The unpaired two-tailed Student t test was applied to all with the up-regulation of the former and down-regulation of the pairwise comparisons of mean values after F-test evaluation of variance. All statistical analyses were performed with Prism 5 software (GraphPad). latter as the CMP differentiates downstream toward the MEP via + − an intermediate CD71 CD105 CMP stage. Subsequent KLF-1 ACKNOWLEDGMENTS. We thank T. Storm and L. Jerabek for laboratory up-regulation accompanied by Fli-1 down-regulation may be management and T. J. Naik for technical assistance. This study was sup- critical for the E-lineage fate determination at the branch-point ported by fellowships from the Japan Society for the Promotion of Science of the MegK versus E lineage. (to Y.M.) and grants from the National Cancer Institute and National Heart, int/+ + Lung, and Blood Institute of the National Institutes of Health (R01 In summary, we have identified hEPs as a CD71 CD105 CA086065 and U01 HL099999; to I.L.W.), California Institute for Regenerative fraction of previously defined hMEPs. This population could be a Medicine (RT2-02060; to I.L.W.), and Leukemia & Lymphoma Society (700709; useful tool for studying physiological and pathological erythroid to I.L.W.).

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