|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 March 2005, Vol. 105, No. 5, pp. 1937-1945. Prepublished online as a Blood First Edition Paper on November 2, 2004; DOI 10.1182/blood-2004-09-3459.
HEMATOPOIESIS Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)From the Department for Immunology, University of Ulm, Ulm, Germany; Institute of Pathology, Charité, Benjamin Franklin University Hospital, Berlin, Germany; and Department of Molecular Genetics, Institute of Molecular Pharmacology, Berlin, Germany.
The erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the "nonstem," "nonlymphoid" compartment (lineage marker [lin]–c-Kit+Sca-1–IL-7R  –) into interleukin 3 receptor negative (IL-3R–) and IL-3R+ subsets. Within IL-3R– but not IL-3R+ cells we have identified TER119–CD41–CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)–deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin–c-Kit+Sca-1–IL-7R–IL-3R–CD41–CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.
Hematopoietic stem cells (HSCs) give rise to lineage (lin)–restricted, intermediate progenitors which, in turn, generate single-lineage committed progenitors.1,2 Multipotent cells might commit themselves stochastically into certain lineage fate,3 visible as single-lineage progeny. Therefore, it is difficult to assign, in retrospect, the origin of hematopoietic colonies to stem cells, intermediate, or single-lineage committed progenitors. Clonogenic developmental potential must be prospectively predictable. This requires the physical isolation of lineage-committed progenitors based on a unique cell surface phenotype.
Myeloid progenitors lack stem cell activity and lymphoid potential but possess erythrocyte, megakaryocyte, or granulocyte-monocyte (GM) potential. Several myeloid progenitors have been isolated, which satisfy the criteria of committed progenitors. These include mast cell progenitors (c-KithiThy-1lo),4 megakaryocyte progenitors (MPs; CD9+CD41+Fc
We have analyzed the expression pattern of a major stem/progenitor cell growth factor receptor, the interleukin 3 receptor
Mice, monoclonal antibodies, and cell sorting
C57Bl/6 mice were analyzed at 4 to 8 weeks of age. Interferon consensus sequence-binding protein, (ICSBP)+/+ and ICSBP–/–, mice10 were analyzed in age-matched groups between 3 and 6 months of age when the myeloproliferative syndrome was prominent. Lineage antibodies were B220 (clone RA3-6B2), CD3
In vitro colony assays Cell sorter-purified progenitors were placed in methylcellulose cultures as described.12,13 Briefly, cells were cultured in MethoCult M3231 (StemCell Technologies, Vancouver, BC, Canada) for BFU-E, CFU-GM, CFU-megakaryocyte (CFU-Meg), and mixed CFU (CFU-Mix) colonies, or in MethoCult M3334 with erythropoietin (Epo) for CFU-E colonies. Added growth factors were granulocyte-macrophage colony-stimulating factor (GM-CSF; 1 ng/mL), macrophage colony-stimulating factor (M-CSF; 10 U/mL), thrombopoietin (Tpo; 10 ng/mL), stem cell factor (SCF; 50 ng/mL); all of these were murine (R&D Systems, Minneapolis, MN); IL-3 (1% supernatant, expressed as reported14), and human Epo (3 U/mL; Erypo, FS2000, Janssen-Cilag, Neuss, Germany). CFU-E and BFU-E colonies were stained with 0.4% benzidine (Sigma, Taufkirchen, Germany) in 12% glacial acetic acid and 0.3% H2O2. Cytospins were stained by Diff-Quik (Dade Behring, Marburg, Germany) according to the manufacturer's recommendation. Cells were inspected with an Axioskop microscope (Zeiss, Oberkochen, Germany) using an objective with a 100 x magnification and a 1.3 aperture (Zeiss) with oil, and photomicrographs (Figure 1) were taken using the OM-11 color camera (Olympus, Hamburg, Germany). Adoptive cell transfers
Mice were irradiated with 850 rad (split dose) and given intravenous injections of HSCs (2500 cells/mouse), total BM (2 x 105), CMP IL-3R Phenylhydrazine-induced anemia Mice were treated with a single intraperitoneal injection of phenylhydrazine (PHZ; 60 mg/kg body weight) as described.15 Frequencies of CFU-E colonies and EPs were determined in BM 3 days after the injection. DNA microarray gene expression analyses and bioinformatics Total RNA was isolated from purified populations from 10- to 16-week-old mice using the RNeasy kit including DNase digestion (Qiagen, Hilden, Germany). Total RNA (5 µg) was used to generate cDNA according to the technical manual (Affymetrix, Santa Clara, CA). cRNA was generated with the BioArray high-yield transcript labeling kit (ENZO, Farmingdale, NY), and 15 µg cRNA was hybridized to Affymetrix mouse expression 430A arrays at 45°C for 16 hours. DNA chips were stained, washed, and scanned according to the manufacturer's protocol. Scanned GeneChip DAT files were analyzed by the GeneChip Analysis Suite Software (Affymetrix) with global scaling to 500. Further analysis of data output was carried out using Data Mining Tool and NetAffx Web-Based Database (Affymetrix) as well as Microsoft Excel. The data have been submitted to the GEO Web site (http://www.ncbi.nlm.nih.gov/geo; submission no. GSE1584 [NCBI GEO] ).
Division of BM populations based on IL-3R chain expression identifies 6 distinct progenitor populations
Downstream from HSCs (lin–c-Kit+Sca-1+), progenitor potential for erythrocyte, megakaryocyte, monocyte, and granulocyte lineages is contained in the lin–c-Kit+Sca-1–IL-7R
Based on the suspected erythrocyte potential within the c-Kit+IL-3R
All 6 populations were isolated at high purity (> 98%) by FACS. Based on their developmental potential (Figure 1, Table 1), we designated the 3 c-Kit+IL-3R
Phenotype of c-Kit+IL-3R
Lin–c-Kit+Sca-1–IL-7R
EPs generate CFU-E colonies at about 70% cloning efficiency CFU-E colonies are small aggregates of approximately 16 to 32 globin-positive cells scored after 48 hours of culture in Epo.23-25 A clonogenic, phenotypically defined CFU-E has not been purified from BM. In 2 experiments, 105 total BM cells contained about 235 and 260 CFU-E (Table 1) colonies, a frequency similar to published data.23-25 Interestingly, 105 purified EPs gave rise to 76 000 (experiment 1), 69 000 (experiment 2), and 81 000 (experiment 3) CFU-E colonies. This corresponds to a cloning efficiency of at least 70% (Table 1). To estimate whether or not most CFU-E activity in BM was quantitatively retrieved within the EP fraction, the EP population was removed from BM by cell sorter depletion. In BM depleted of EPs by a factor of 8 (see "Materials and methods"), the CFU-E colony frequency dropped 10-fold (Table 1). Moreover, none of the other isolated progenitors (Figure 1A) had CFU-E potential (not shown). These data show that the vast majority of CFU-Es were quantitatively recovered from BM by isolating EPs. EPs are CFU-E committed
To analyze whether EPs were CFU-E committed, EPs and all other progenitors depicted in Figure 1A were analyzed for BFU-E, megakaryocyte, and GM colony formation (Figure 1B). BFU-E colonies were measured as large colonies of globin-positive cells, visualized by dark blue benzidine staining, scored after 8 days of culture in SCF (or Kit ligand), Epo, and IL-3. BFU-E activity was present in both CMP IL-3R
The potential to form CFU-Megs (Figure 1Bii) was examined by culture in Tpo and SCF. CFU-Meg potential was highly enriched in one of the c-Kit+IL-3R
CFU-GM potential was analyzed in the presence of IL-3, SCF, GM-CSF, and M-CSF (Figure 1Biii). High frequencies of CFU-GMs were detected in the c-Kit+IL-3R Next, all progenitor populations were assayed for their potential to form day 8 (CFU-S day 8) and day 12 (CFU-S day 12) spleen colonies in lethally irradiated mice.26 As positive controls, 2500 HSCs, or 2 x 105 total BM cells, were injected. Both for day 8 (Figure 1Biv) and for day 12 (Figure 1Bv), CFU-S potential was restricted to CMP subsets. In marked contrast, EPs as well as MP1, MP2, and GMPs lacked CFU-S potential. Erythrocyte potential in vivo
In vitro, CMP subsets and EPs revealed erythrocyte potential indicative of early (BFU-E+CFU-E–) and late (BFU-E–CFU-E+) stages of erythropoiesis, respectively. We next asked whether progenitors with this in vitro potential could also contribute to erythropoiesis in vivo (Figure 3). Mice were myeloablated by irradiation with 850 rad, which led to the absence of reticulocytes in the circulation due to interruption of their de novo production. In PBS-injected mice, reticulocytes did not reappear in the peripheral blood for at least 2 weeks following irradiation (Figure 3A). Transfer of total BM caused an increase in the percentage of reticulocytes, which appeared as early as on day 10. Percentages increased to approximately 30% by day 14 (Figure 3B). HSCs and both CMP progenitors also reconstituted reticulocytes in vivo (Figure 3C-D,G). Reconstitution kinetics suggested that reticulocytes appeared earlier from CMP IL-3R
Interestingly, injection of EPs led to a marked increase in reticulocytes (Figure 3E). Unexpectedly, reticulocytes appeared later from EPs compared to CMPs. None of the remaining populations, that is, MP1 (Figure 3F), MP2 (Figure 3I), and GMP (Figure 3H), had reticulocyte potential. These experiments demonstrate that BFU-E and CFU-E assays correlated perfectly with the in vivo function of the respective progenitors. Frequencies of EPs and CFU-Es following PHZ-induced anemia If the EP phenotype represents the majority of the CFU-Es in BM, one should expect to measure parallel changes in both the frequencies of EP and the frequencies of CFU-E colonies. We tested this idea directly in a model of experimentally induced erythropoietic stress. Treatment of mice with PHZ (60 mg/kg body weight; single injection) leads to a drop in the hematocrit from about 45% to about 30%. This hemolytic anemia is accompanied by an increase in the frequency of CFU-E colonies in the BM15 (Table 2). We measured 245 (experiment 1) and 255 (experiment 2) CFU-E colonies/105 BM cells in untreated mice. Numbers rose to 520 (experiment 1) and 610 (experiment 2) CFU-E colonies/105 BM cells in mice 3 days after PHZ treatment. Parallel measurements of the abundance of cells with the EP phenotype revealed an increase from 400 (experiment 1) and 378 (experiment 2) EP/105 BM cells in untreated mice to 912 (experiment 1) and 887 (experiment 2) EP cells/105 BM cells in treated mice (Table 2). The major stress erythropoiesis response occurs usually in the spleen.15 To analyze whether the EP phenotype also reflects stress erythropoiesis in this organ, we analyzed the spleen for the EP phenotype and followed this phenotype after PHZ treatment. Spleen EPs had CFU-E colony activity in vitro, and 5 days after a single injection of PHZ, the EP phenotype in spleen was increased from 0.03% in untreated mice to 0.53% in treated mice (not shown). Collectively, an increase in the frequency of the EP phenotype faithfully reflects the changes of CFU-E colonies in this model of experimentally induced anemia. Reduced frequencies of both CFU-E colonies and EPs in mice lacking the transcription factor ICSBP We next asked whether numbers of EPs also correlated with numbers of CFU-E colonies in a chronic, genetically based erythropoietic defect. To this end, we determined the frequencies of EP and CFU-E colonies in mice lacking ICSBP. ICSBP is an important regulator of hematopoiesis. In vivo, ICSBP–/– BM is characterized by expansion of myeloid progenitors and concomitant suppression of erythropoiesis.10 With age, ICSBP–/– mice develop a myeloproliferative syndrome reminiscent of human chronic myelogenous leukemia (CML). We analyzed ICSBP+/+ and ICSBP–/– BM at 3 to 6 months of age, a time when the suppression of erythropoiesis was evident.
Numbers of CFU-E colonies and EPs were compared between ICSBP+/+ and ICSBP–/– BM. In wild-type mice, 27% and 9% of lin–IL-7R–Sca-1–c-Kit+ cells were IL-3R Frequencies of both EPs (Figure 4G) and CFU-E colonies (Figure 4H) were 10- to 20-fold reduced comparing ICSBP+/+ (Figure 4C-D) and ICSBP–/– (Figure 4G-H) BM cells. These experiments demonstrate that the frequency of EPs is a very good indicator of CFU-E frequencies in this anemic BM. Global gene expression profiles in EPs and GMPs reveal EP-specific gene expression To characterize the RNA expression profile of the CFU-Es, we compared RNA expression in EPs and GMPs by array analysis. We chose this comparison because EPs and GMPs are both hematopoietic progenitors but located "closely" downstream from the erythroid versus GM branch. We identified 14 080 transcripts that were present in EPs or GMPs or both populations; 7795 of those were not differentially expressed. Using a signal ratio cutoff of more than 2.5, we identified 627 genes that were expressed higher in EPs than in GMPs; 136 genes were expressed at least 10-fold stronger in EPs compared to GMPs. Among those were hemoglobin A (Hba-a1), Rhesus blood group-associated A glycoprotein (Rhag), carboanhydrase 1 (Car1), glycophorin A (Gypa), aquaphorin (Aqp) 1 and 9, Rhesus blood group CE and D (Rhced), erythroid Kruppel-like factor (Klf1), CD36, Kell blood group (Kel), Gata-1, and transferrin receptor 2 (Trfr2) as shown in Figure 5A.
Using the same cutoff, we found 1255 genes whose expression was higher in GMPs than EPs; 330 genes were expressed at least 10-fold higher. Among those were histidine decarboxylase (Hdc), complement component 3 (C3), GM-CSFR (CSF2rb2), M-CSFR (Csf1r), high-affinity IgE receptor Next, we analyzed our array data for transcription factors expression. A total of 1317 transcripts involved in gene regulation were identified that were expressed in one or both of these populations. Among those, 48 and 103 were differentially expressed (2.5-fold or higher) in EPs and GMPs, respectively. Among the CCAAT/enhancer-binding proteins, C/ebpa, C/ebpb, and C/ebpd were expressed in GMPs but not in EPs. The 430A chip lacks C/ebpe probe sets. From the Ets family Elk3, Etv6/Tel, Fli1, and Pu.1 were expressed higher in GMPs than in EPs. Within the Gata family, Gata1 was expressed exclusively in EPs and Gata-2 specifically but weakly in GMPs. Friend of Gata (Fog)1 (also termed Zfpm1) and Tal1 expressions were very strong in EPs and absent (Fog1) and low (Tal1) in GMPs. Within the homeobox domain gene family, only HoxA9 was expressed, with lower expression in EPs and higher expression in GMPs. Among the interferon-regulated factors, Irf1 was expressed in both populations but was much stronger in GMPs. Irf3 was strongly expressed in EPs and GMPs. Icsbp was exclusively and strongly expressed in GMPs. Within the Kruppel-like factors, Klf1 and Klf3 were predominantly expressed in EPs. In contrast, Klf4 expression was exclusive for GMPs. Finally, within the STAT family, Stat3 and Stat4 expressions were stronger in GMPs, whereas Stat5a and Stat5b were predominantly expressed in EPs. The relative expression of these genes is shown in Figure 5B, and the full list of array data are available at http://www.ncbi.nlm.nih.gov/geo. This analysis should be taken as a systematic starting point to study and dissect the physiologic network of genes regulating and determining the developmental potential of undifferentiated, yet lineage-committed erythroid progenitors.27
Prospective isolation of a clonogenic EP
Distinct stages of erythrocyte development have been measured for a long time by BFU-E and CFU-E assays.23-25 Despite the wide usage of CFU-E assays to characterize erythropoiesis (for examples, see Wu et al,28 Zang et al,29 and Jegalian et al30), the colony-forming "unit" had not been purified to homogeneity by cell surface phenotype. We have now prospectively identified CFU-Es as lin–c-Kit+Sca-1–IL-7R In transgenic mice expressing a Gata-1 promoter-driven GFP transgene, GFP+CD71+ progenitors had only CFU-E potential32 indicating that Gata-1, together with CD71, can be a useful intracellular marker of CFU-Es. However, GFP+CD71+ cells gave rise to CFU-E colonies with considerably lower frequencies (1/10) compared to EPs described here. Zhang and colleagues,43 using day 14.5 fetal liver (FL) cells, enriched CFU-Es prospectively by phenotype from 7400 CFU-Es/105 total FL cells to 41 200/105 total FL cells by isolation of CD71lowTER119– FL cells. Thus, CD71lowTER119– FL cells are at least 41% pure CFU-Es. Taking the overall frequency of CD71lowTER119– cells in FL into consideration, the CD71lowTER119– phenotype can quantitatively account for about 30% (2000 of 7400 CFU-Es/105 total FL cells) of all CFU-Es in FL. Very recently, introduction of GFP as a reporter into the EpoR locus showed that CFU-Es are included in the lin–Sca-1– c-Kithi population.33
We have now identified CFU-Es among lin–Sca-1–c-Kithi BM cells as IL-3R Global gene expression pattern in EPs The isolation of EPs has opened access for global gene expression profiling of the CFU-E stage. Many genes specifically expressed in EPs, when compared to GMPs, are known to be erythroid, prototypically Gata-1, Klf1, and blood group-related genes (Figure 5). It was not known, however, which genes are expressed in a committed erythroid progenitor. The earliest erythroid commitment may take place prior to the CFU-E stage, at the BFU-E stage; however, up to now, BFU-E potential has not been prospectively separated from nonerythroid potential because, at the population level, all progenitors that contain BFU-Es also contain non-BFU-E potential. Hence, EPs are the only pure erythroid-committed progenitors available for analysis at present. This study now offers information on a large fraction of those genes that are expressed specifically at the CFU-E stage. Future experiments could determine which of these genes are crucial for the development of erythroid cells from EPs. Relationship of BFU-Es and CFU-Es It has been established a long time ago that BFU-Es and CFU-Es represent erythroid precursors at sequential stages of differentiation.24 BFU-Es and CFU-Es differ in cell size, sensitivity to cycle-active agents, response to plethora, and effects of the W/Wv24 and W/W13 genotypes. Based on these data, BFU-E and CFU-E colonies were thought to arise from committed progenitors. We now demonstrate the erythroid commitment of CFU-Es directly by showing prospectively that these cells give rise to erythroid colonies but not to any other lineage assayed for. Our data also show that BFU-Es and CFU-Es can be prospectively separated because EPs lack BFU-E potential (Figure 1B). BFU-Es arise from the CMP compartments. The generation of mixed BFU-E/CFU-GM colonies demonstrates that a single CMP can express "true" common myeloid potential. However, CMPs also generate pure BFU-E colonies. Until the prospective isolation of a phenotypically defined BFU-E will be achieved, it remains open whether a committed BFU-E exists, or whether single BFU-E colonies arise from CMPs stochastically. The identification of EPs as the CFU-Es, as well as the assignment of BFU-E activity to the multipotent CMP stage, places EPs downstream from HSCs and BFU-Es (Figure 6). Transplanted EPs also have significant potential in vivo as shown by a burst of reticulocytes following injection of EPs into myelosuppressed mice (Figure 3). Selective substitution of erythropoiesis may be clinically relevant under myelosuppressive conditions. We noted that reticulocytes did not arise earlier from EPs when compared to CMPs. Although the basis for this kinetic difference is unknown, it is possible that the irradiated host does not immediately support reticulocyte development from EPs. An example of an irradiation-induced developmental delay has been noted when pro–T cells were injected into an irradiated but not into a nonirradiated thymus.34 In this regard it is noteworthy that, on day 3, reticulocytes were absent even in mice injected with total BM, which should contain all CFU-E colony activity, regardless of any purification method or progenitor subset. Yet, at the typical time when the CFU-E colony arises in vitro, that is 2 to 3 days, we see no reticulocytes in vivo (Figure 3). We conclude that, in irradiated mice in vivo, it takes up to 10 days before reticulocytes are detectable from any cell type in the BM, including EPs.
Identification of 2 late MPs
Our phenotypic BM dissection has uncovered 2 megakaryocyte progenitors, termed MP1 and MP2 (Figure 6). Both progenitors are committed to the megakaryocyte lineage, but they cannot be recognized by their morphology as megakaryocytes when isolated ex vivo. We propose that MP1 and MP2 represent subsequent, late stages of megakaryocyte development. MP1 are probably earlier than MP2. It remains to be determined whether MP1 and MP2 share a direct precursor-product relationship. IL-3 is a growth factor for the megakaryocyte lineage,35 and our data suggest that the effect of IL-3 on this lineage may occur at the level of the IL-3R Relationship of CFU-Es and MPs
The CFU-E colony is composed of 16 to 32 erythroid cells. Thus, EPs undergo 4 to 5 cell divisions to form a CFU-E colony. Interestingly, MPs have a very similar limited expansion potential. One MP often differentiates into only one megakaryocyte, and this megakaryocyte gives rise to 16 to 32 platelets. Therefore, we propose that EP and MP populations represent correspondingly late stages in erythrocyte and megakaryocyte development, respectively. The expansion potential of both types of progenitors is similar, the difference being "extracellular" colony formation from CFU-E versus "intracellular" colony formation from MPs. Collectively, MPs represent a later stage in megakaryocyte development when compared to CMPs, or the megakaryocyte-committed progenitors defined by Nakorn et al (lin–c-Kit+Sca-1–IL7R Assignment of key hematopoietic properties to common versus committed myeloid progenitors Analyses of isolated BM fractions revealed a clear correlation between key hematopoietic properties (CFU-S, BFU-E, CFU-E) and phenotypically defined stages of commitment from stem cells to myeloid lineages (Figure 6). BFU-E and CFU-S potential is restricted to CMPs. HSCs and CMPs both have CFU-S potential. Benzidine-positive BFU-E colonies originate from CMPs but not from HSCs within 8 days. Thus, from a practical viewpoint, CFU-Ss measure HSCs and CMPs, whereas benzidine-positive BFU-E colonies reflect CMP activity. CFU-E assays measure EPs because CFU-E potential is absent from HSCs, CMPs, and committed progenitors other than EPs. Because CFU-S activity is present in a common MEP37 but absent from MPs or EPs (Figure 6), it is likely that CFU-S potential is lost at the MEP-to-EP transition.
IL-3R
Cytokine receptor expression can be associated with lineage commitment,2,38 but cytokine receptor expression has not been widely used to purify progenitors. We have now divided myeloid progenitors into functionally distinct IL-3R | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
RloCD34+CD38+),
(clones 145-2C11 or 17A2), Gr-1 (clone RB6-8C5), Mac1 (clone M1/70), and TER119 (all labeled with fluorescein isothiocyanate [FITC]). Further antibodies were phycoerythrin (PE)–labeled anti-IL-3R
200-300 CFU-Es/105 total BM cells) reported by others