Differential Amplification of Murine Bipotent Megakaryocytic/Erythroid ...

Tissue-Specific Stem Cells Differential Amplification of Murine Bipotent Megakaryocytic/Erythroid Progenitor and Precursor Cells During Recovery from Acute and Chronic Erythroid Stress MASSIMO SANCHEZ,a IRVING L. WEISSMAN,b MARIA PALLAVICINI,c MAURO VALERI,a PAOLA GUGLIELMELLI,d ALESSANDRO MARIA VANNUCCHI,d GIOVANNI MIGLIACCIO,a ANNA RITA MIGLIACCIOa,e a

Istituto Superiore di Sanita`, Rome, Italy; bDepartment of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA; cSchool of Natural Sciences, University of California, Merced, California, USA; dDepartment of Hematology, University of Florence, Florence, Italy; eDepartment of Pathology and Cancer Center, University of Illinois at Chicago, Chicago, Illinois, USA Key Words. Erythroid progenitors • Megakaryocytic progenitors • Erythroid stress • GATA-1 • Phenylhydrazine

ABSTRACT Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegckitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%–70% of the cells in BFU-E– derived colonies but are not present

among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%– 84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented. STEM CELLS 2006;24:337–348

INTRODUCTION

potential or by an orderly sequence of molecular switches regulated by specific cell interactions with a wide range of cytokines [3, 4]. Several lines of evidence suggest that stem cell commitment toward erythroid and megakaryocytic differentiation involves generation of bipotent erythroid/ megakaryocytic cells: megakaryocyte markers are expressed on most human [5– 8] and murine [9, 10] erythroleukemic

Red cells, platelets, and all other cellular elements in blood derive from stem cells through a complex cellular process that involves extensive proliferation, commitment, and maturation toward a particular lineage [1, 2]. It has been debated for some time whether commitment is determined by stochastic events that randomly restrict stem cell differentiation

Correspondence: Anna Rita Migliaccio, Ph.D., Istituto Superiore Sanita`, Viale Regina Elena 299, 00161 Rome, Italy. Telephone: 39-06-49902690; Fax: 39-06-49902530; e-mail: migliar@iss.it Received January 17, 2005; accepted for publication July 26, 2005; first published online in STEM CELLS EXPRESS September, 6, 2005. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/ stemcells.2005-0023

STEM CELLS 2006;24:337–348 www.StemCells.com

Dynamics of Cell Compartments in Erythroid Stress

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cells; erythropoietin (EPO) and thrombopoietin (TPO), which regulate erythroid [11] and megakaryocytic [12] differentiation, respectively, amplify progenitor cells in both lineages [13–18]; and the transcription complex formed by GATA-1 with its obligatory partner FOG-1 activates both erythroidand megakaryocytic-specific genes [19, 20]. Recently, two bipotent erythroid/megakaryocytic progenitors were identified prospectively in murine tissues. CD34 and CD16/CD32 discriminate subpopulations of linnegc-kitpos Sca-1neg marrow cells corresponding to common myeloid progenitors (CMPs) (CD16/CD32lowCD34high), granulocyte-monocyte progenitors (GMPs) (CD16/CD32highCD34high), and megakaryocytic-erythroid progenitors (MEPs) (CD16/ CD32lowCD34low), respectively [21]. CMP, GMP, and MEP are functionally defined by their capacity to differentiate in vivo and in vitro into granulocytes, monocytes, and erythroid and megakaryocytic cells, into granulocytes and monocytes, or into megakaryocytic and erythroid cells, respectively. On the other hand, a bipotent precursor for erythroid and megakaryocytic cells (PEM) was identified in the spleen of mice recovering from hemolytic anemia induced by phenylhydrazine [22]. PEM cells display the phenotype TER-119pos4A5pos (or 2D5pos) and, when stimulated by TPO and EPO, generate megakaryocytic or erythroid cells within 24 to 48 hours (i.e., after only one division) [22] (for a summary of the phenotypic and functional definitions of the hemopoietic cells studied here, see Table 1). The relationship between MEP and PEM in normal hematopoiesis, as well as their involvement in response to acute and chronic erythroid stress, is unknown. Several murine models have been developed to address phenotypic and functional cellular relationships during the recovery of acute and chronic erythroid stress. The hemolytic

anemia induced by phenylhydrazine represents a model of acute stress well known for its sensitivity to EPO. In fact, the amount of 3H-TdR incorporated by splenic erythroblasts produced in response to this stress has represented for a long time a biological assay for this growth factor [23]. However, there is extensive genetic evidence suggesting that, in addition to the EPO-dependent mechanism, the splenic recruitment of erythroid progenitors, required for the recovery from this hemolytic anemia, is controlled by a locus, Fv2, that encodes the Stk receptor and that confers susceptibility to the Friend leukemia virus [24]. Strains susceptible (DBA/2 and CD1) or not (C57BL) to the leukemia induced by this virus carry either the Fv2s or the Fv [2] allele. The Fv2s allele contains an internal GATA-1– dependent promoter from which is transcribed an alternative transcript that encodes sf-Stk, a truncated form of Stk [25], capable of forming a complex with the EPO receptor [26]. The sf-Stk/EPO receptor complex is exclusively required for progenitor cell recruitment in the spleen, both as part of the process of virusinduced leukemogenesis and as part of recovery from phenylhydrazine-induced hemolytic anemia [25]. On the other hand, experimentally induced mutations in genes involved in the regulation of erythroid differentiation, such as STAT-5null [27] or the hypomorphic GATA-1low mutation [28] (a deletion of the first enhancer, DNA hypersensitive site I, and distal promoter of the gene) induce chronic erythroid stress by increasing the rate of erythroblast apoptosis. Although the spleen is also recruited as an additional hemopoietic site in response to chronic erythroid stress [27, 29], the precise contribution of the erythroid output from this organ to the circulating red cell pool in these mice has yet to be established. Both the STAT-5null and the GATA-1low mutants

Table 1. Abbreviations of the hematopoietic progenitors/precursor cells analyzed in the study Abbreviations CMP, common myeloid progenitor cell

MEP, common erythroid/megakaryocyte progenitor cell

GMP, common granulocyte/monocyte progenitor cell

PEM, common precursor for the erythroid and megakaryocyte lineage

Phenotype Lin⫺ cKit⫹Sca1⫺ CD16/CD32lowCD34high [21, 33]

Lin⫺ cKit⫹Sca1⫺ CD16/CD32lowCD34low [21, 33]

Lin⫺ cKit⫹Sca1⫺ CD16/CD32highCD34high [21, 33]

TER119pos/4A5pos2D5pos [22]

Function In vivo: mostly day-12 CFU-S

In vitro: generation of granulocytes, monocytes, erythroblasts, and megakaryocytes in cultures stimulated with SCF, FLT3 ligand, IL-11, TPO, and EPO In vivo: mostly day-8 CFU-S

In vitro: generation of erythroblasts and megakaryocytes in liquid cultures stimulated with SCF, FLT3 ligand, IL11, TPO, and EPO In vivo: mostly day-8 CFU-S

In vitro: generation of granulocytes and monocytes in liquid cultures stimulated with SCF, FLT3 ligand, IL-11, TPO and EPO In vitro: generation of erythroblasts and megakaryocytes within 24–48 hours of incubation with EPO and TPO alone

Abbreviations: EPO, erythropoietin; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin.

Sanchez, Weissman, Pallavicini et al. retain the capacity to respond to EPO and to recover from phenylhydrazine-induced anemia [27, 29]. Thus, the mechanism that compensates the erythroid deficiency of these mutants must be, at least partially, both EPO-independent and sf-Stk–independent. We used multivariate flow cytometric analysis, functional assays, and models of acute and chronic erythroid stress to explore the relationship between PEM, MEP, and CMP progenitors. To clarify the role of MEP and PEM in steady state, as well as in stress erythropoiesis, we compared the biological properties and the relative frequencies of these two cell populations purified from marrow and spleen of normal and phenylhydrazine-treated mice and GATA-1low mice. Our data suggest that MEP and PEM represent distinct cell populations that might be linked in an upstream-downstream differentiation relationship only under conditions of erythroid stress.

MATERIALS

AND

METHODS

Mice CBA mice (2 to 4 months old) were purchased from Charles River Laboratories (Calco, Italy, http://www.chriver.com). GATA-1low mice were bred in the CD1 background at the animal facilities of the Istituto Superiore Sanita` [29, 30]. These mutant mice have been provided to Jackson Laboratory (Bar Harbor, ME, http://www.jax.org; JAX@Mice DATAbaseSTOCK Gata1 ⬍tm2Sho⬎) and are available to other investigators. Littermates were genotyped at birth by polymerase chain reaction [29, 30], and those lacking the mutation were used as wild-type controls. All studies were performed with sex- and age-matched mice under protocols approved by the institutional animal care committee.

In Vivo Treatments Phenylhydrazine Treatment Anemia was induced with phenylhydrazine (60 mg/kg body weight, Sigma-Aldrich, St. Louis, http://www.sigmaaldrich. com) injected intraperitoneally for 2 consecutive days [23, 31]. On the first day after the second phenylhydrazine injection, mice were euthanized by cervical dislocation and bones and spleens were removed under sterile conditions for further analysis. Untreated mice were used as controls.

Surgical Removal of Spleen Mice were anesthetized with xylazine (10 mg/kg, Bayer, Milan, Italy, http://www.bayer.com) and ketamine (200 mg/kg, Gellini Farmaceutics, Latina, Italy), i.p. 1 day after food withdrawal. The spleen was removed after double ligation of the splenic artery and vein. The muscle, peritoneum, and skin were closed in separate layers using sterile 5– 0 absorbable suture. Animals received the analgesic butorphanol s.c. (5 mg/kg per day, Intervet Italia Srl, Milan, Italy, http://www.intervet.it) for 4 days after surgery.

Hematological Parameters Blood was collected from the retro-orbital plexus into ethylendiamino-tetracetic acid– coated microcapillary tubes (20 – 40 ␮L/sampling). Hematocrit (Hct), white cell, and platelet counts www.StemCells.com

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were determined manually. Reticulocytes were enumerated over a total of 1,000 red cells after methylene blue staining.

Cell Staining and Purification Myeloid progenitor cells (CMP and MEP) were purified according to the procedure described by Akashi et al. [21]. Briefly, mononuclear cell suspensions were labeled with a cocktail of biotinylated rat anti-mouse antibodies containing CD3, CD4, CD8, CD5, CD45R/B220, CD19, CD127 (interleukin [IL]-7R␣ chain), TER-119, IgM, Thy 1.1, and unconjugated Mac-1 and Gr-1 (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). These mature (Lin⫹) cells were partially removed by binding to sheep anti-rat immunoglobulin G (IgG)-conjugated magnetic beads (Dynabeads M-450, Dynal Biotech, Oslo, Norway, http://www.dynalbiotech.com). The recovered unbound cells (Lin-) were further stained with fluorescein isothiocyanate (FITC)-conjugated CD34, APC-conjugated CD117 (c-kit), Texas red– conjugated Sca-1, phycoerythrin (PE)-conjugated anti-FcR␥II/III (CD16/CD32, BD Pharmingen), and streptavidin-Cy5-PE (Caltag Laboratories, Burlingame, CA, http:// www.caltag.com). Cell analysis and sorting were performed using a FACS Vantage (Becton, Dickinson and Company, Foster City, CA, http://www.bd.com) with three lasers (488-nm argon laser, 599-nm dye laser, and ultraviolet laser). Erythroid/megakaryocyte precursors (PEM) were purified by labeling bone marrow cells with biotinylated rat anti-mouse CD3, CD4, CD8, CD45R/B220, Thy 1.1, and unconjugated Mac-1 and Gr-1, successively incubated with sheep anti-rat IgG-conjugated magnetic beads (Dynabeads M-450, Dynal Biotech) to partially remove Lin⫹ cells. Recovered Lin- cells were stained with PE-conjugated TER119, FITC-conjugated 2D5 or 4A5 [22, 32], and streptavidin-Cy5-PE and analyzed with the FACS Vantage or the Coulter Elite ESP Cell Sorter (Beckman Coulter, Miami, FL, http://www.beckmancoulter. com). Cells labeled with fluorophore-conjugated isotype antibodies (BD Pharmingen) were used to gate nonspecific fluorescence signals, whereas dead cells were excluded on the basis of propidium iodide (5 ␮g/ml, Sigma-Aldrich) fluorescence intensity.

Histochemical Analysis Spleens and bone marrow were routinely fixed in phosphatebuffered formalin (10%, vol/vol) and paraffin embedded, and sequential sections (2.5–3 ␮M) were stained either with hematoxylin-eosin or with fluorescein–terminal deoxynucleotidyl transferase to label apoptotic cells (TUNEL Assay In Situ Cell Death Detection Kit, Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com).

Liquid Cultures Purified PEM, MEP, and GMP cells were cultured for 3 to 6 days in Iscove’s modified Dulbecco’s medium supplemented with fetal calf serum (10% vol/vol), 7.5 ⫻ 10-5 M ␤-mercaptoethanol (Sigma-Aldrich), and 1% (vol/vol) antibiotic-antimycotic solution (penicillin, streptomycin, fungizone, Gibco, Grand Island, NY, http://www.invitrogen.com) and stem cell factor (SCF), FLT3 ligand, IL-3, IL-11, TPO, and EPO, as described previously [33].


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Progenitor Cell Counts

RESULTS

The frequency of progenitor cells in the light-density (p ⬍ 1.080) fractions (0.25–1.0 ⫻ 105 cells/plate) and in MEP and CMP (0.2–1 ⫻ 103 cells/plate) purified from marrow and spleen of normal, phenylhydrazine-treated, and GATA-1low mice was enumerated as described previously [22, 30] using standard methylcellulose culture (0.9% wt/vol) in the presence of fetal bovine serum (30% vol/vol, Sigma-Aldrich) and a combination of recombinant growth factors including rat SCF (100 ng/ml) and IL-3 (10 ng/ml) (both from Sigma-Aldrich) and human erythropoietin (EPO) (2 U/ml, Boehringer Mannheim) for BFU-E growth or mouse G-CSF and GM-CSF (50 ng/ml each; both from Sigma-Aldrich) for CFU-GM growth [34]. The growth of CFU-E-derived colonies was stimulated with EPO alone (2 U/ml) [35]. In addition, in selected experiments, a parallel set of cultures was stimulated with the cytokine combination used in liquid culture described above. The cultures were incubated at 37°C in a humidified incubator containing 5% CO2 in air and enumerated either 3 days (for CFU-E-derived colonies) or 7 days (for CFU-GM-derived and BFU-E-derived colonies) after initiation of culture.

CFU-S Determination Increasing numbers (500 –5,000) of purified PEM and MEP cells were injected into the tail vein of irradiated (9.0 Gy; Cstor, Atomic Energy of Canada, Ottawa, Cesium Irradiator Canada) syngeneic mice. The spleen was removed on days 8 to 12 after injection and fixed in Telleyesnicky’s solution for CFU-S determination.

Statistical Analysis Statistical analysis was performed by analysis of variance using Origin 3.5 software for Windows (Microcal Software Inc., Northampton, MA, http://www.originlab.com).

Comparison of the Frequency of MEP and PEM in Normal Mice The relationship between MEP and PEM along the erythroid differentiation pathway was initially examined by analysis of the two subpopulations in tissues from normal mice. Progenitor cells with the phenotype Linnegc-kitposSca-1neg comprise approximately 6% of bone marrow (Table 2) and are readily separated into CMP, GMP, and MEP on the basis of CD16/ CD32 and CD34 expression (Figs. 1, 2). CMP and GMP comprise approximately 30%– 40% of the Linnegc-kitposSca-1neg population, whereas the MEP frequency approximates 20%. However, because progenitor cells represent less than 0.01% of the cells in a normal spleen (Table 2 and [29]), categorization into individual progenitor subpopulations by flow cytometry is not possible in this organ. On the other hand, PEM comprise only 0.3%–1% and 0.01%– 0.2% of normal bone marrow and spleen cells, respectively (Table 2). Such a low frequency in hemopoietic tissues from normal mice precludes a direct comparison between PEM and MEP under steady-state conditions in vivo. The capacity of MEP to generate PEM in vitro was assessed by inducing differentiation of MEP purified from normal mice in liquid culture and by evaluating the phenotype of progeny in erythroid colonies. Under liquid culture conditions, MEPs generate cells that express either erythroid or megakaryocytic markers (R7 and R8 in the bottom panel of Figure 1) within 3 to 6 days of culture initiation. MEPs do not generate TER119/4A5 cells cultured under the same controlled conditions. On the other hand, most cells within the BFU-E colonies are TER119/2D5positive, and approximately 20% of the progeny in CFU-E colonies display the double-positive phenotype (Fig. 3). These

Table 2. Progenitors and precursors in marrow and spleen from normal mice and from mice under either acute (phenylhydrazinetreated) or chronic (GATA-1low) erythroid stress Total nucleated cells (X10 关6兴)

Treatment

PEM

Linneg Sca-1neg c-kitpos

CMP

MEP

GMP

a

Normal mice Femur Spleen Phenylhydrazine-treated mice Femur Spleen GATA-1low mice Femur Spleen

22.3 ⫾ 1.8 162 ⫾ 23

0.7 ⫾ 0.4 0.1 ⫾ 0.1

6.0 ⫾ 2.5 ⬍ 0.01

34.2 ⫾ 2.6 b.d.

20.1 ⫾ 1.0 b.d.

39.8 ⫾ 4.6 b.d.

23.4 ⫾ 2.4 430 ⫾ 40

4.5 ⫾ 1.0b 2.2 ⫾ 0.9b

4.3 ⫾ 1.5 2.6 ⫾ 0.8

21.3 ⫾ 1.1 10.6 ⫾ 0.7

46.2 ⫾ 1.0b 83.7 ⫾ 0.1

16.8 ⫾ 1.0b 2.5 ⫾ 0.1

7.6 ⫾ 2.1 560 ⫾ 162

0.6 ⫾ 0.4c 6.3 ⫾ 0.5b,c

6.6 ⫾ 0.7 1.2–0.4

39.3 ⫾ 0.1 29.9 ⫾ 4.8c

14.1 ⫾ 0.35 28.6 ⫾ 5.2c

36.1 ⫾ 5.1 24.2 ⫾ 4.0c

Control animals for phenylhydrazine-treated and GATA-1low mice are represented by untreated mice and by wild-type littermates, respectively. Since no difference is ever observed between the values obtained in the two groups, the data from the two controls are pooled and presented as normal mice. b Values statistically different (p ⬍ .05–.001) from those observed in normal mice. c Values statistically different (p ⬍ .05–.001) from those observed in phenylhydrazine-treated animals. Progenitor cells are defined as Linnegc-kitposSca-1neg, whereas precursor cells display the TER-119pos4A5pos phenotype. The frequency of these two populations is expressed as percent of total nucleated cells. Progenitor cells were further divided into CMP, MEP, and GMP, according to the phenotype CD16/CD32lowCD34high, CD16/CD32lowCD34low, and CD16/CD32highCD34high defined in Figure 1 (see also Table 1). In this case, the frequency is expressed as percent of the total progenitor population. The results represent the mean of two independent determinations in which the marrow and spleen from three mice were pooled before cytofluorimetric determination. Abbreviations: b.d., below detection; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyticerythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

a

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Figure 1. Flow cytometric analysis of MEP and CMP from bone marrow of normal mice. The panels in the top row show the gates to select c-kitpos-Sca-1neg cells with the phenotype CD34lowCD16/CD32low (MEP) and CD34highCD16/CD32low (CMP). Panels in the middle row show the phenotypic reanalyses of the sorted cells, as a control for purity. MEP and CMP were cultured for 3 days under conditions described in Materials and Methods. The two left panels in the bottom row show the expression of E (TER-119) and Mk (4A5) markers by cultured MEP, and the two bottom right panels show marker expression on cultured CMP. Abbreviations: CMP, common myeloid progenitor; MEP, megakaryocytic-erythroid progenitor.

double-positive cells were not detected among the progeny of CFU-GM analyzed in comparison as control. These data demonstrate that MEP can generate PEM in colony-forming assays but not in liquid culture.

1.2% (Fig. 4). These data confirm the major contribution of the spleen in chronic erythroid stress recovery. Furthermore, the elevated cellularity of the spleen is suggestive of enhanced progenitor activity during erythroid stress recovery.

Role of the Spleen in the Recovery from Chronic Erythroid Stress

Contributions of MEP and PEM to Recovery from Acute and Chronic Erythroid Stress

Two mouse models of acute and chronic erythroid stress were used to evaluate the relative contributions of MEP and PEM to erythroid stress recovery. Phenylhydrazine treatment induces rapid hemolytic anemia (24% and 28%, Hct and reticulocytes, respectively) (Table 3), thereby providing a murine model of acute erythroid stress. On the other hand, GATA-1low mice provide a model of chronic erythroid stress because they display a normal Hct in spite of the high degree of constitutive erythroblast apoptosis (20%–30% of TUNELpos cells) (Table 3). The spleens from both phenylhydrazine-treated mice and from GATA-1low mice are considerably larger than control spleens and contain twice as many cells (Table 2 and [22, 29]), and numerous foci of erythroid differentiation spread throughout the parenchyma [22, 29]. Whereas the contribution of the spleen to the recovery from the anemia induced by phenylhydrazine treatment is well established [23, 31], it is not clear how much of the increased erythroid output from the spleen contributes to the normal Hct in GATA-1low mice [29]. To clarify this point, we analyzed the Hct of the GATA-1low mice after splenectomy. Although splenectomy does not affect the Hct of normal mice, splenectomized GATA-1low mice become anemic (Hct, 34%– 36%) and die within 2 months, with a Hct as low as 6.6% ⫾

To confirm whether the progenitor cell activity in the spleen is enhanced during stress hemopoiesis, we analyzed the frequency and biological properties of MEP and PEM in the bone marrow and the spleen in acute and chronic stress erythropoiesis. The frequency of progenitor cells in the marrow of mice treated with phenylhydrazine or carrying the GATA-1low mutation is similar to control animals. However, there is a striking increase (⬍ 0.01% up to 3% of the total spleen population) of progenitor cells in the spleen in both animal models of erythroid stress (Table 2). Of note, there is a selective increase (up to 46%– 84%) in the relative frequency of MEP both in the bone marrow and spleen of phenylhydrazine-treated animals (Fig. 2A and Table 2), whereas the relative proportion of CMP, MEP, and GMP in the marrow and spleen of GATA-1low mice is similar to that found in the bone marrow of normal mice (Fig. 2A, Table 2). These flow-cytometric data confirm previous colony-based estimates of progenitor cell frequency in hemopoietic tissues from our laboratory [22, 29, 30]. In phenylhydrazine-treated animals, the frequency of PEM rises to 4.5% and 2.2% of the total cell population of the marrow and spleen, respectively (Fig. 2, Table 2). On the other hand, in GATA-1low mice, the frequency of PEM remains comparable to

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Figure 2. Flow cytometric analysis of MEP and PEM in the marrow and spleen of normal, phenylhydrazine (PHZ)-treated, and GATA-1low mice, as indicated. (A, B): Frequencies of MEP and PEM, respectively, in the marrow and spleen from single mouse. Of note, the bone marrow from phenylhydrazine-treated mice contains a cell population that expresses 4A5 and TER-119 with intermediate fluorescence intensity and that is not present either in the spleen from the same animals or in the marrow and spleen from the two other groups of mice investigated. To ensure that similar populations were being analyzed in all the cases, this population was excluded by further gating the bone marrow cells from phenylhydrazine- treated mice, as indicated. Means (⫾ standard deviations) of results obtained in additional experiments for a total of at least three mice per experimental groups are shown in Table 2. Abbreviations: b.d., below detection; MEP, megakaryocytic-erythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

that of normal littermates in the marrow but increases to 6% in the spleen. Therefore, PEM are amplified in different hematopoietic tissues under acute (in the marrow and in the spleen) and chronic (only in the spleen) erythroid stress. Overall, these results indicate that although the frequency of both MEP and PEM increases in the hematopoietic tissues of mice recovering from erythroid stress, the anatomic site (marrow and/or spleen) and modality (both populations or only PEM) of amplification are clearly distinct under acute and chronic conditions.

Comparison of the Biological Properties of MEP Purified from Mice Recovering from Acute and Chronic Erythroid Stress It is possible that expression of specific antigen markers on the cell surface may not predict the real differentiation potential involved in recovery from acute erythroid stress. Alternatively, altered levels of GATA-1 expression at the progenitor cell level might have altered the phenotype of these cells in the mutant mice. In addition, differences in marrow and spleen microenvironment might also result in loss of phenotype/function correlations in these cells. To address these possibilities, we compared the biological functions of CMP and MEP purified from the bone marrow and the spleen of normal mice as well as from mice recovering from acute or chronic stress erythropoiesis. The ability of the cells to differentiate in liquid culture under defined growth factor combinations and the ability of the cells to gen-

erate colonies in semisolid assays were also investigated. The results are summarized in Figure 3 and Table 4. Thirty percent of the CMP purified from normal mice form colonies at day 8 of semisolid culture and proliferate in liquid culture, with a 30-fold cell increase by days 6 through 8. As expected, CMPs generate, under both culture conditions, granulocytic, monocytic, and, at low frequency, erythroid and megakaryocytic cells by day 8 (Table 4 and data not shown). On the other hand, 12% of the MEPs purified from normal mice generate colonies at day 8 and proliferate in liquid culture, yielding a 10-fold cell increase by days 6 through 8. In liquid culture, the progeny of MEPs is represented by single TER-119pos (25% by days 6 through 8) or 2D5pos (2.2% by days 6 through 8). The 2D5pos cells acquire the morphology of mature megakaryocytes by days 6 through 8 (Fig. 3). In semisolid cultures, the cells present within the day-8 colonies generated from these MEPs are represented by single TER-119pos erythroblasts and by double TER-119pos2D5pos blastlike cells (Fig. 3 and data not shown). CMP and MEP from normal and chronically stressed mice express similar biological activity in semisolid and liquid assays (Table 4). However, CMP and MEP purified from acutely stressed animals display functional differences compared with controls. CMPs from acutely stressed animals proliferate more quickly in liquid culture (28- and 112-fold increase in total cell numbers after 2 to 3 and 6 to 8 days, respectively) and generate colonies in semisolid media not only at day 8 (25%), but also at day 2 (30% of cloning efficiency) (Table 4). Erythroid and


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Figure 3. Flow cytometric analysis for the expression of TER-119 and of 2D5 in the progeny obtained after 3 and 6 days of liquid culture from megakaryocytic-erythroid progenitor (MEP) purified from the spleen of normal mice (middle panels) and from those purified from the spleen of phenylhydrazine (PHZ)-treated animals (bottom panels), as indicated. The cells present in erythroid colonies harvested after 3 (CFU-E-derived) or 6 (BFU-E-derived) days of semisolid culture of normal MEP are also presented as comparison (top panels). Cells from the semisolid culture, but not those from the liquid culture, express the precursor for erythyroid and megakaryocytic cells (PEM) phenotype (i.e., coexpression of erythroid, TER119, and megakaryocytic, 2D5, markers). The insert within the quadrants presents May-Grunwald staining of representative 2D5pos cells isolated from the cultures. 2D5pos cells reach the stage of mature megakaryocytes by days 3 and 6 in liquid culture of MEPs purified from the phenylhydrazine-treated and normal mice, respectively. In contrast, the 2D5pos cells from the semisolid cultures have an immature blast cells morphology identical to that of the TER-119pos4A5pos PEM population already published [22].

megakaryocytic cells represent a high proportion of the cells formed from these CMPs at day 2 to 3 and 6 to 8 of liquid culture. Even larger differences are observed between the in www.StemCells.com

vitro functional properties of the MEPs purified from normal mice and those purified from phenylhydrazine-treated animals. In liquid culture, MEPs from acutely stressed animals proliferate


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Table 3. Hematologic and apoptotic parameters in acute and chronic erythroid stress Blood

Normal mice Phenylhydrazine-treated mice GATA-1low mice

Spleen

Hct

Reticulocytes (%)

48 ⫾ 3 24 ⫾ 5 48 ⫾ 4

1.3 ⫾ 0.02 28 ⫾ 5 2.1 ⫾ 1.1

pos

TUNEL

erythroblasts (%)

1–2 1–2 20–30

The values are the mean (⫾ standard error of the mean) of 5 to 8 mice per experimental group. The hematocrit (Hct) and reticulocyte counts are measured 24 hours after phenylhydrazine-treatment and in GATA-1low mice (4 months old) and age-matched controls.

Figure 4. Splenic erythropoiesis significantly contributes to the number of circulating red cells in GATA-1low mice. Hematocrit (Hct) of a GATA-1low mice 2 months after being splenectomized. The Hct of an age- and sex-matched GATA-1low littermate and of a splenectomized wild-type mouse is also shown.

quickly (11- and 117-fold increase in total cell numbers after 2 to 3 and 6 to 8 days, respectively) and generate many single positive TER-119pos (erythroid) and 2D5pos (megakaryocytic) cells. The 2D5pos cells have a distinct megakaryocyte morphology already by days 2 to 3 (Fig. 3, Table 4). Double TERpos2D5pos cells (PEM) are undetectable in these cultures. In semisolid media, MEPs from the marrow of phenylhydrazinetreated mice form colonies at both day 8 (13% efficiency) and day 2 (CFU-E-like colonies, 91% cloning efficiency), but BFU-E are never detected in cultures of MEP purified from the spleen of the same animals (up to 2,000 cells per plate) (Table 4). PEMs are clearly detectable among the cells harvested from the methylcellulose culture of these cells (data not shown).

Comparison of the Biological Properties of MEP and PEM Purified from Mice Recovering from Acute Erythroid Stress We next compared the biological properties of MEP and PEM generated in vivo in the spleen and bone marrow of phenylhy-

drazine-treated mice (Fig. 5). MEPs purified from phenylhydrazine-treated mice express c-kit, whereas this marker is lacking on purified PEMs (Fig. 6), reflecting the different surface phenotypes of these two subpopulations in vivo. The in vitro proliferative/differentiation potential of PEM and MEP isolated from phenylhydrazine-treated mice was compared by analyzing the progeny of the two cell populations in liquid and semisolid cultures (Table 4). As mentioned above, the MEPs isolated from the tissues of phenylhydrazine-treated mice display different in vitro properties than exhibited by cells with the same phenotype isolated from the tissues of normal animals (Table 4). The MEPs from phenylhydrazine-treated mice proliferate more (117- vs. 10-fold increase at days 6 through 8 in the two cases) and generate erythroid and megakaryocytic cells (Table 4) more rapidly (mature megakaryocytes are already recognized at day 3 of culture; Fig. 3) than MEPs from normal mice. The proliferative properties of PEM are different than MEP isolated both from normal and phenylhydrazine-treated mice. PEMs do not form colonies in semisolid media, nor proliferate in liquid culture. They do, however, differentiate into mature erythroid and megakaryocytic cells by 24 to 48 hours (Table 4 and results not shown). Similar differences in in vitro proliferation activity between MEP and PEM have previously been reported [21, 22]. Finally, the in vivo proliferative potential of PEM and MEP isolated from phenylhydrazine-treated mice was directly compared using spleen colony formation. Transplanted MEPs (500 cells/ mouse) from phenylhydrazine-treated animals generate both day-8 and day-12 CFU-S at a frequency of 1/15 cells and 1/53 cells, respectively. This activity is similar to the spleen colony-forming activity expressed by MEP purified form normal marrow [33]. In contrast, no spleen colonies are detected when up to 5 ⫻ 103 PEMs are injected into sublethally irradiated animals.

DISCUSSION The commitment of stem cells toward erythroid and megakaryocytic differentiation has been suggested to involve one or more bipotent erythroid/megakaryocytic progenitors [36]. MEPs and PEMs have been identified as prospective bipotent progenitor and precursor cells in erythroid and megakaryocytic commitment from the tissues of normal mice and phenylhydrazinetreated animals, respectively [21, 22]. However, their functional location in the hemopoietic hierarchy of progenitor/clonogenic cells has not been clearly established. Elucidation of the functional relationships of these cells with each other and their relative contributions to recovery from erythroid stress has been complicated by the low frequency of these cells in steady-state hemopoiesis. We used models of acute and chronic erythroid


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Table 4. Functional comparison of MEP and CMP purified from normal mice and from phenylhydrazine-treated and GATA-1low mice Liquid culture

Semisolid culture

Days 2–3 FIa Normal mice CMP MEP Phenylhydrazine mice CMP MEP PEMb GATA-1low mice CMP MEP

TER119pos

Day 2 (CFU-E)

Days 6–8

2D5pos TER119pos/2D5pos FI

TER 119pos

2D5pos

TER119pos /2D5pos

0.1 25 ⫾ 3.1

6.7 2.2 ⫾ 0.1

⬍0.01 ⬍0.01

0 0

Day 8 (BFU-E/ CFU-GM)

Colonies/100 cells

1–3 1–3

0.3 13 ⫾ 7.1

1.8 7.1 ⫾ 0.4

⬍0.01 ⬍0.01

30 10

28 11 0.5

0.5 ⫾ 0.1 23 ⫾ 17 68 ⫾ 13

5.4 ⫾ 0.8 14 ⫾ 3 9⫾3

⬍0.01 ⬍0.01 9⫾3

112 117 b.d.

⬍0.01 14 ⫾ 7 ⫺

21 ⫾ 3 32 ⫾ 8 ⫺

⬍0.01 ⬍0.01 ⫺

30 91 ⫾ 1 0

1–3 1–3

⬍0.01 ⬍0.01

⬍0.01 ⬍0.01

⬍0.01 ⬍0.01

24 14

⬍0.01 0.4 ⫾ 0.1

18 27 ⫾ 1

⬍0.01 ⬍0.01

0 0

31 ⫾ 2 12 ⫾ 1 25 ⫾ 8 13 ⫾ 2 (0) 0 10 ⫾ 1 4⫾1

Values represent the mean (⫾ standard deviation) of two experiments performed in duplicate. Each data set was independently repeated, with identical results, with cells purified from the marrow and spleen of the same animal group (three animals per experimental point). The only exception is that day-8 colonies were detected in cultures of MEP purified from the bone marrow but not among those purified from the spleen of phenylhydrazine-treated animals (up to 2,000 cells/plate, as indicated by the 0 in parentheses). Representative FACS analysis and morphology of the cells obtained in liquid and semisolid cultures of MEP cells is shown in Figure 3. a FI, fold increase, with respect to day 0 of culture. bData on PEM are from [22] and are reported here only for direct comparison. Abbreviations: b.d., below detection; CMP, common myeloid progenitor; MEP, megakaryocytic-erythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

Figure 5. Purification of MEP and PEM from the spleen of phenylhydrazine-treated mice. The spleen from one phenylhydrazine-treated mouse contains both MEP (top row) and PEM precursors (bottom row) (see also Table 1). The sequential gating used to purify the MEP is indicated with R1, R2, and R3 in the top panel. In stead, in the bottom panels, R1 and R4 indicate single TER-119pos (erythroblasts) and 4A5pos (megakaryocytes) cells, whereas the double TER-119pos 4A5pos cells (PEM) are indicate by R2. Abbreviations: MEP, megakaryocytic-erythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

stress to investigate the functional characteristics of these two bipotent populations. Our data demonstrate that MEP and PEM are indeed distinct populations. MEPs, but not PEMs, express c-kit (Fig. 6) and respond to SCF (Fig. 1 and [21, 22, 33]) in vitro and form erythroid/megakaryocytic colonies in vitro and spleen colonies in vivo ([21, 22, 33] and this manuscript). Furthermore, the two www.StemCells.com

compartments are differentially amplified in the hemopoietic tissues under stress erythropoiesis; in fact, whereas the frequency of both MEP and PEM increases in the marrow and spleen of phenylhydrazine-treated animals, only PEM increase in the spleen of GATA-1low mice (Fig. 2, Table 1). Placement of MEP and PEM in the hemopoietic hierarchy is important to establish the relationship between these two sub-

346

Figure 6. Sca-1 and c-kit expression on MEP and PEM subpopulations. MEP and PEM were purified from the spleen of phenylhydrazinetreated mice as described in Figure 5, and subsequently c-kit (on the Y axes) and Sca-1 (on the X axes) expression was quantified. Similar results were obtained with MEP and PEM purified from the spleen of GATA-1low mice (not shown). Abbreviations: MEP, megakaryocyticerythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

populations and to gain insight into the commitment of stem cells to erythropoiesis. Our data exclude a simple hierarchical model in which MEPs precede PEMs, which then mature to erythroid and megakaryocytic cells (Fig. 7, model 1). Since c-kit expression and cell proliferative potential decline during hemopoiesis [37], the antigenic profile and colony-forming potential of MEP and PEM suggest that these subpopulations should be located in an upstream-downstream relationship. However, our data demonstrate that whereas PEMs comprise up to 50%–70%

Figure 7. Schematic diagram of two possible models for the relationship between MEP and PEM in normal and stress erythropoieis. Model 1 proposes that MEP might consistently generate PEM, as part of the steady-state differentiation process. Model 2, instead, suggests that under conditions of steady state, MEPs undergo the orderly unilineage differentiation pathway, as suggested by Akashi et al. [21], but that under conditions of stress, MEP and/or common myeloid progenitor may be forced to generate PEM as a short cut to a rapid recovery. See the text for further details. Abbreviations: CMP, common myeloid progenitors; MEP, megakaryocytic-erythroid progenitor; PEM, precursor for erythroid and megakaryocytic cells.

Dynamics of Cell Compartments in Erythroid Stress of the cells in BFU-E-derived and CFU-E-derived colonies, they are not present among the progeny of MEP induced to differentiate in vitro in the presence of EPO and TPO. Thus, these findings suggest that if MEPs consistently generate PEM, they do so only under certain conditions, such as those conducive to formation of tight cellular contacts (i.e., colonies or the in vivo environment of tissues from animals recovering from erythroid stress) (Fig. 7, model 2). It is also possible that CMP can generate, in addition to MEP, PEM, as a short cut to rapidly increase the erythroid output in emergency conditions (Fig. 7, model 2). In this case, the orderly unilineage differentiation pathway suggested by Akashi et al. [21], in which CMPs generate MEPs, which differentiate to erythroid and megakaryocytic cells, would be limited to steady-state hemopoiesis. Under conditions of stress, bipotent progenitors and precursors would be generated by at least two alternative mechanisms. Our observation that under acute stress MEPs increase selectively in the marrow and then proliferate in the spleen suggests that MEP amplification occurs within the compartment itself (i.e., increased proliferation potential) in this condition. Furthermore, unlike MEPs purified from normal mice, those obtained from the spleen of phenylhydrazine-treated mice gave rise, with almost 100% efficiency, to CFU-E-derived colonies without losing megakaryocytic differentiation potential, as demonstrated by the high numbers of megakaryocytes generated in liquid cultures (Fig. 3, Table 4). In contrast, in chronic stress, where multiple hemopoietic progenitors colonize and proliferate in the spleen, the amplification of the progenitor cell compartment begins at the CMP level (either by acquisition of proliferative potential at this level or by increased differentiation from the stem cell pool) and is then followed by a cascade, resulting in increases in the MEP compartment. In this case, the in vitro proliferation/differentiation potential of CMP and MEP is similar to corresponding cells purified from normal mice. On the other hand, PEM, which are barely detectable under steady-state hemopoiesis, increase in numbers in response to acute and chronic stress (significantly larger increase in chronic compared with acute stress conditions). These data suggest that PEMs are generated at low frequency and provide limited contribution, if any, to the circulating red cell pool in normal mice and are generated at high frequency, from MEP and/or CMP, under conditions of stress. The modulation of progenitor cell amplification and the level in the hierarchy at which these cells lose erythroid differentiation potential are thought to play a major role in increasing the erythroid output in response to stress [11]. The fact that the amplification of the progenitor and precursor cell compartments occurred in two very distinct fashions in acute and chronic erythroid stress has further implications for the molecular control of the response to stress. One implication is that it is possible to specifically modulate the amplification potential of MEPs under certain conditions. In fact, whereas very few MEPs are present in the spleen under steady-state hematopoiesis, this cell population becomes the prevalent (46%– 84%) progenitor cell population in the marrow and spleens from phenylhydrazine-treated animals. In contrast, the ratio between MEP and the other progenitor cell populations remains normal in the marrow of GATA-1low mice, and all types of progenitor cells increase in the spleens of these mutants. Although we failed to modulate the in vitro proliferation of the MEP with growth

Sanchez, Weissman, Pallavicini et al. factor combinations, it is possible that two different types of extrinsic factors mediate the animal response to the two stress conditions. In fact, the multilineage progenitor amplification observed in mice under chronic erythroid stress resembles that observed in mice stimulated with lineage-specific hemopoietic growth factors, such as EPO, whose biological action becomes erythroid-restricted only at the precursor level [15]. On the other hand, the response to phenylhydrazine treatment in mice is abrogated by targeted deletion of the glucocorticoid receptor [38] but not by deletion of STAT-5 [27], a key element of the EPO receptor signal transduction pathway [39]. It is, therefore, possible that the bipotent amplification observed in acute stress might be specifically sustained by glucocorticoids. Another implication is that the rate at which erythroid and megakaryocytic differentiation potentials are lost during differentiation in vivo can be modulated. Under steady-state hemopoiesis, the final choice between these two differentiation options is lost early and the two pathways commit to distinctive progenitor cell characteristics before the proliferation potential is lost. Under stress hemopoiesis, the bipotent differentiation potential is lost late in differentiation, giving the system the plasticity to switch the final mature cell output toward the erythroid or megakaryocytic lineage as needed. In this regard, it is noteworthy that MEP purified from the spleen of phenylhydrazine-treated mice gave rise to CFU-E-derived colonies with 100% cloning efficiency; nevertheless, these MEPs are still

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capable of generating many (14%) mature megakaryocytes within 72 hours of liquid culture. The erythroid and megakaryocytic differentiation lineages share most of the intrinsic control machinery so that the cell fate is decided through subtle changes in the concentration in few transcription factors [40, 41]. As mentioned earlier, GATA-1, in association with its obligatory partner FOG-1 [42], plays an essential role in both lineages. Recently, plasticity in hemopoietic differentiation has been experimentally induced by ectopically increasing the concentration of GATA-1 in prospectively isolated myelo-monocytic progenitors [43]. Our data suggest that these alterations of expression might occur also in vivo under specific stimulation as part of the machinery to respond to erythroid stress.

ACKNOWLEDGMENTS This study was supported by Progetti di Ricerca di Interesse Nazionale 2002 and 2003 from the Ministry of Health, Associazione Italiana Ricerca sul Cancro, PRIN 2003/064888, National Project on Stem Cells, institutional funds from Istituto Superiore Sanita`, and NIH (to M.P. and I.L.W.). Dr. Thanyaphong Na Nakorn is gratefully appreciated for help with the initial cytofluorimetric characterization of the MEP cells and for performing the CFU-S assay.

DISCLOSURES The authors indicate no potential conflicts of interest.

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