Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBPα
2006; Springer Nature; Volume: 25; Issue: 14 Linguagem: Inglês
10.1038/sj.emboj.7601199
ISSN1460-2075
AutoresYumi Fukuchi, Fumi Shibata, Miyuki Ito, Yuko Goto‐Koshino, Yusuke Sotomaru, Mamoru Ito, Toshio Kitamura, Hideaki Nakajima,
Tópico(s)Acute Myeloid Leukemia Research
ResumoArticle6 July 2006free access Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBPα Yumi Fukuchi Yumi Fukuchi Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Fumi Shibata Fumi Shibata Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Miyuki Ito Miyuki Ito Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuko Goto-Koshino Yuko Goto-Koshino Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yusuke Sotomaru Yusuke Sotomaru Central Institute for Experimental Animals, Kanagawa, Japan Search for more papers by this author Mamoru Ito Mamoru Ito Central Institute for Experimental Animals, Kanagawa, Japan Search for more papers by this author Toshio Kitamura Toshio Kitamura Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hideaki Nakajima Corresponding Author Hideaki Nakajima Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yumi Fukuchi Yumi Fukuchi Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Fumi Shibata Fumi Shibata Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Miyuki Ito Miyuki Ito Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuko Goto-Koshino Yuko Goto-Koshino Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yusuke Sotomaru Yusuke Sotomaru Central Institute for Experimental Animals, Kanagawa, Japan Search for more papers by this author Mamoru Ito Mamoru Ito Central Institute for Experimental Animals, Kanagawa, Japan Search for more papers by this author Toshio Kitamura Toshio Kitamura Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Hideaki Nakajima Corresponding Author Hideaki Nakajima Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Yumi Fukuchi1, Fumi Shibata1,‡, Miyuki Ito2,‡, Yuko Goto-Koshino1, Yusuke Sotomaru3, Mamoru Ito3, Toshio Kitamura1 and Hideaki Nakajima 2 1Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2Center of Excellence, Institute of Medical Science, University of Tokyo, Tokyo, Japan 3Central Institute for Experimental Animals, Kanagawa, Japan ‡These authors contributed equally to this work *Corresponding author. Center of Excellence, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5759; Fax: +81 3 5449 5453; E-mail: [email protected] The EMBO Journal (2006)25:3398-3410https://doi.org/10.1038/sj.emboj.7601199 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CCAAT/enhancer-binding protein (C/EBP) α is a critical regulator for early myeloid differentiation. Although C/EBPα has been shown to convert B cells into myeloid lineage, precise roles of C/EBPα in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPα activation in various progenitors and to address the underlying mechanism of lineage conversion in detail, we established transgenic mice expressing a conditional form of C/EBPα. Using these mice, we show that megakaryocyte/erythroid progenitors (MEPs) and common lymphoid progenitors (CLPs) could be redirected to functional macrophages in vitro by a short-term activation of C/EBPα, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPα in mice led to the increase of mature granulocytes and myeloid progenitors with a concomitant decrease of hematopoietic stem cells and nonmyeloid progenitors. Our study reveals that C/EBPα can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo. Introduction Sequential lineage specification initiating from hematopoietic stem cells (HSCs) is a fundamental characteristic of blood cell production. These processes, called commitment, are often dictated by the instructive action of lineage-specific transcription factors, which ultimately restricts their fate of differentiation (Shivdasani and Orkin, 1996). It has been thought that the commitment was an irreversible process, and cells differentiated into a certain lineage would not change their own fate. However, recent evidence suggests that many immature progenitors still sustain latent differentiation programs to other lineages than their own, which can be initiated by ectopic activation of cytokine signals or transcription factors. It was reported that enforced signals from interleukin (IL)-2 or granulocyte macrophage-colony stimulating factor (GM-CSF) could induce myeloid conversion in common lymphoid progenitor (CLP) and pro-T cells (Kondo et al, 2000; King et al, 2002; Iwasaki-Arai et al, 2003). In addition, ectopic expression of GATA-1 redirected lymphoid and myeloid progenitors—including CLP, pro-B, common myeloid progenitor (CMP), and granulocyte/monocyte progenitor (GMP)—to the megakaryocyte/erythroid (Meg/E) lineage (Iwasaki et al, 2003). CD19+ B cells can also be reprogrammed into macrophages by enforced expression of CCAAT/enhancer binding protein α (C/EBPα) (Xie et al, 2004). These studies collectively revealed unexpected plasticity of primary lymphoid and myeloid progenitors, especially a latent multipotentiality of lymphoid cells. However, plasticity of megakaryocyte/ erythroid progenitor (MEP) has not yet been reported. C/EBPα is a member of the C/EBP family of transcription factors that contain a conserved leucine-zipper dimerization motif adjacent to a basic DNA-binding domain (Landschulz et al, 1988). Within the hematopoiesis, C/EBPα acts as a key factor for early granulopoiesis (Radomska et al, 1998) and regulates a number of myeloid genes (Tenen et al, 1997; Iwama et al, 1998). The loss of C/EBPα in mice leads to the complete absence of mature neutrophils and eosinophils in fetal liver (FL) and newborns because of the differentiation-arrest at an early myeloblast stage (Zhang et al, 1997). C/EBPα−/− FL progenitors were hyperproliferative and showed decreased differentiation potential by a block in the ability of multipotential progenitors to differentiate into bipotential granulocyte/monocyte (G/M) progenitors and their progeny (Heath et al, 2004). In addition, conditional disruption of C/EBPα revealed that it was necessary during the transition from CMP to GMP, but not beyond this stage, for terminal granulocyte maturation in adult bone marrow (BM) (Zhang et al, 2004). Enforced expression of transcription factors by retrovirus has been utilized to address the lineage plasticity of hematopoietic progenitors (Iwasaki et al, 2003). However, this system cannot control the activity of the transcription factors, which hampered the detailed examination of the conversion process. In addition, the conversion cannot be investigated directly in vivo, as ex vivo manipulation of the progenitor is inevitable for virus infection. For these reasons, a novel system that does not involve virus transduction and enables conditional regulation of transcription factor activity is warranted to analyze the molecular basis of lineage conversion in more detail. In this study, we established transgenic mice expressing a conditional form of C/EBPα whose activity can be regulated by 4-hydroxy tamoxifen (4-HT). Using these mice, we tested various progenitors, especially MEP and CLP, to determine if they could be redirected to myeloid lineage by C/EBPα activation in vitro and in vivo. We found that both MEP and CLP could be converted to the myeloid lineage by C/EBPα through biphenotypic intermediate cells by a clonal analysis, and surprisingly, this lineage conversion was accomplished by only a short-term activation of C/EBPα. Moreover, in vivo activation of C/EBPα induced an increase of mature granulocytes in peripheral blood and myeloid progenitors in bone marrow with dynamic compositional changes in HSC and nonmyeloid progenitor populations. These data establish a critical role of C/EBPα not only in the myeloid lineage but also in a whole hematopoietic system in vivo. Results Establishment of C/EBPα-ER transgenic mice To investigate the role of C/EBPα in the various stages of hematopoietic differentiation, we generated transgenic mice (Tg) expressing an inducible form of C/EBPα (C/EBPα-ER; CEBPα fused to the ligand-binding domain of estrogen receptor (ER)). This approach enabled us to analyze directly the impact of C/EBPα activation on cell-fate conversion without culturing cells for gene transduction in vitro, which might induce undesired phenotypic changes of the target cells. The H-2K promoter (Domen et al, 1998) was used to drive C/EBPα-ER for obtaining high levels of expression in hematopoietic tissues (Figure 1A). Real-time reverse transcription (RT)–PCR analysis revealed that the expression of C/EBPα-ER was achieved in most hematopoietic tissues including spleen, bone marrow (BM), thymus, and peripheral blood (PB) in these mice (Figure 1B). Western blot analysis confirmed that C/EBPα-ER was highly expressed in spleen and thymus (Figure 1C, upper panel). Relatively low but significant expression was also detected in BM and PB. Expression levels of C/EBPα-ER protein were approximately 1/2 to 1/4 of endogenous C/EBPα in hematopoietic tissues such as spleen, BM, and thymus (Figure 1C, lower panel). Next, we isolated various hematopoietic progenitors at key differentiation branch points such as CLP, CMP, GMP, and MEP, and checked the expression of C/EBPα-ER in comparison to endogenous C/EBPα by semiquantitative RT–PCR (Figure 1D). Expression of endogenous C/EBPα was observed in CLP, CMP, GMP, and MEP, with somewhat higher expression in CMP and GMP. There was no substantial difference between Tg and non-Tg littermates in endogenous C/EBPα expression, except that a slight increase and decrease were observed in transgenic GMP and transgenic CMP, respectively. High, constitutive expression of C/EBPα-ER was detected in all progenitors examined, with approximately 2–4 times higher expression compared with the endogenous C/EBPα. These results indicate that C/EBPα-ER was expressed at high levels in CLP, CMP, GMP, and MEP and that it did not significantly affect the endogenous C/EBPα expression. There were no notable abnormalities in the complete blood cell counts (CBC), the differentiation profile of leukocytes, or FACS profile of peripheral blood in the Tg mice (data not shown). Figure 1.Establishment of C/EBPα-ER transgenic mice. (A) Construction of H-2K-C/EBPα-ER transgene. Arrows, primer set used in PCR-based genotyping of C/EBPα-ER Tg mice. ER-LBD, mouse estrogen receptor ligand-binding domain. (B) Quantification of C/EBPα-ER transcript by real-time RT–PCR. The data were normalized against 18S rRNA. (C) Expression of C/EBPα-ER protein. (Upper panel) Whole-cell lysates from various tissues of Tg mice and cell lines were subjected to Western blot analysis. C/EBPα-ER protein was detected by anti-ER antibody. α-Tubulin was probed as a loading control. BaF3/α-ER is BaF3 cells expressing C/EBPα-ER. (Lower panel) Comparison of expression levels for endogenous C/EBPα and C/EBPα-ER proteins by blotting with anti-C/EBPα antibody. Asterisks indicate nonspecific bands. (D) Comparison of endogenous C/EBPα and C/EBPα-ER transgene mRNA in hematopoietic progenitors of Tg mice. mRNA from sorted progenitors were prepared and subjected to semiquantitative RT–PCR as described in Materials and methods. (E) Gel-shift analysis of C/EBPα-ER protein. (Left panel) Thymocytes were prepared from Tg or non-Tg littermate mice and treated by 1 μM of 4-HT for 2 h. Lanes 1, 5: no treatment. Lanes 2, 6: supershift (s.s.) with anti-C/EBPα antibody. Lanes 3, 7: supershift with control rabbit IgG. Lanes 4, 8: cold competition. (Right panel) Same extract from 4-HT-treated Tg thymocytes was subjected to supershift reaction by anti-C/EBPα or anti-ER antibodies. Download figure Download PowerPoint Next, we investigated whether C/EBPα-ER could be activated by 4-HT in these mice. Gel-shift analysis revealed that there was no band shift observed in control-treated transgenic thymocytes and that 4-HT treatment induced specific binding to C/EBP oligonucleotides, which was supershifted by anti-C/EBPα and anti-ER antibodies (Figure 1E). As little as 30 min of 4-HT treatment could achieve activation at a concentration as low as 0.04 μM (data not shown). The same results were also observed in bone marrow cells from Tg mice (data not shown). These results confirmed that activity of C/EBPα-ER could be tightly regulated by 4-HT in these mice, which made them an ideal system to analyze the consequences of C/EBPα activation in various hematopoietic cells in vitro and in vivo. Ectopic activation of C/EBPα in MEP induces myeloid differentiation We first investigated whether C/EBPα could induce myeloid conversion in MEPs. Sorted MEPs from C/EBPα-ER Tg mice were subjected to colony assay in the presence or absence of 4-HT. As shown in Figure 2A, day 3 CFU-E was dramatically decreased from 68 to 20% by 4-HT stimulation. Moreover, Meg/E colonies such as CFU-EM, BFU-E, and CFU-MK were markedly decreased to less than 1% by 4-HT treatment. In contrast, G/M colonies were dramatically increased from 3.5 to 28% by 4-HT treatment. Of note, there was no substantial difference between MEPs from wild-type mice and control-treated MEPs from Tg mice in terms of colony composition (data not shown). To confirm myeloid conversion of MEPs is indeed the consequence of C/EBPα activation, we performed the same experiments with MEPs from wild-type C57BL/6 and H-2K-ER Tg mice. H-2K-ER Tg mice express only ER ligand-binding domain, and therefore should serve as a perfect control for C/EBPα-ER Tg mice (Supplementary Figure 1A). The results showed that there was no difference between control and 4-HT-treated MEPs in colony assays (data not shown). Figure 2.Conversion of MEPs into myeloid lineage by C/EBPα. (A) Colony assay. Sorted MEPs were cultured in methylcellulose in the absence or presence of 4-HT, and the colony formation was assessed at day 3 for CFU-E and day 7 for other progenitors. MK, CFU-MK; EM, CFU-EM; GEM, CFU-GEM; GEMM, CFU-GEMM; G/M, CFU-GM+CFU-G+CFU-M. (B) Expression of lineage-specific genes. Cells were recovered from the colonies and RT–PCR was performed as described in Materials and methods. (C) Changes of surface-antigen expression and cellular morphology during myeloid conversion. Sorted MEPs were cocultured on an S17 stromal layer with SCF, IL-3, EPO, TPO, and G-CSF in the absence or presence of 4-HT. After 7 days, cells were analyzed for surface-antigen expression by FACS. Cells in the gates A, B, and C were sorted, cytospun onto glass slides, and stained with Wright–Giemsa solution (magnification × 400). For cells from gate C, phagocytic activity was examined as described in Materials and methods. Gate A, CD71+CD11b−; gate B, CD71+CD11b−; gate C, CD71+CD11b+. Download figure Download PowerPoint To analyze the expression of lineage-affiliated genes and to see their changes by 4-HT, cells were recovered from the colonies and examined by RT–PCR (Figure 2B). Meg/E-affiliated genes such as GATA-1, FOG-1, erythropoietin receptor (EpoR), and β-globin were clearly downregulated by 4-HT stimulation. In sharp contrast, the myeloid-associated genes such as granulocyte-colony-stimulating factor receptor (G-CSF R), granulocyte/macrophage-colony stimulating factor receptor α chain (GM-CSF Rα), and macrophage-colony stimulating factor receptor (M-CSF R) were upregulated by 4-HT treatment. To investigate the early molecular events during myeloid conversion by C/EBPα, we examined MEPs treated with or without 4-HT for 16 h by RT–PCR (Supplementary Figure 2A). The data revealed that FOG-1 is clearly downregulated at this time point, whereas GATA-1 remained relatively unchanged. This suggests that the downregulation of FOG-1 in MEP is one of the key initial events for limiting erythroid/megakaryocyte differentiation by C/EBPα. To rule out the possibility that the conversion of colony types by C/EBPα resulted from the selective expansion of contaminated myeloid progenitors, we next tried to trace the reprogrammed cell fate by surface markers in a liquid culture system (Figure 2C). Sorted MEPs were plated on an inactivated S17 stromal cell layer and cultured in the presence or absence of 4-HT. In the control culture, the majority of MEPs became CD71 (transferrin receptor; a marker of developing erythroid cells) positive and CD11b negative after 7 days of culture (gate A). Surprisingly, activation of C/EBPα converted the majority of the MEPs (50–70%, depending on the culture conditions) into CD71+CD11b+ biphenotypic cells, which were considered to be the differentiating intermediates to myeloid cells (gate C). However, the remaining MEPs (20–40%) sustained the CD71+ CD11b− erythroid phenotype (gate B). CD71+CD11b+ cells that appeared in the 4-HT culture were functionally mature macrophages, as revealed by their morphology and phagocytic activity against fluorescent beads, whereas cells with the CD71+CD11b− phenotype still retained immature erythroid morphology (Figure 2C; gate B). Taken together, these data suggest that the ectopic induction of C/EBPα-ER activity reprograms a large fraction of MEPs into macrophages through differentiating intermediates. C/EBPα converts CLP into the myeloid lineage Next, we investigated whether ectopic activation of C/EBPα activity in CLP could convert these cells into the myeloid lineage. CLPs from C/EBPα-ER Tg mice were subjected to colony assay in the presence or absence of 4-HT (Figure 3A). Although the plating efficiency was low, composition of the colonies was strikingly different between control and 4-HT cultures. Most colonies appearing in the 4-HT-treated plate were G/M colonies, whereas only lymphoid colonies were observed in the control culture. Cytospin preparation of the colonies showed morphological differentiation of CLP into macrophages by 4-HT stimulation, whereas cells from the control culture showed typical lymphocyte morphology (Figure 3A). Control experiments by treating CLPs from wild-type C57BL/6 or ER-Tg mice with 4-HT did not induce any myeloid conversion by colony assays (data not shown). RT–PCR analysis revealed downregulation of lymphoid-associated genes, such as EBF, Pax-5, PU.1, GATA-3, and Notch-1, and upregulation of myeloid-specific genes, such as M-CSF R and GM-CSF Rα, in 4-HT-treated cells (Figure 3B). In addition, clear induction of Id2 and Id3, well-known inhibitors for E2A proteins (Busslinger, 2004), with a slight increase of E2A were observed by 4-HT treatment (Figure 3B). These data suggest that inhibition of E2A activity by Id2 and Id3 combined with a decrease of EBF, Pax-5, PU.1, GATA-3, and Notch-1 expressions limit the differentiation capacity of CLP into B or T cells during myeloid conversion by C/EBPα. The analysis of early molecular events in C/EBPα-induced myeloid conversion of CLPs revealed that Id2 and Id3 genes are still not upregulated after 5 h of 4-HT stimulation. In contrast, Notch-1 is clearly downregulated at this time point, suggesting that downregulation of Notch-1 is one of the early events induced by C/EBPα in CLPs (Supplementary Figure 2B). Figure 3.C/EBPα converts CLPs into the myeloid lineage. (A) Colony assay. Day 8 colonies derived from C/EBPα-ER Tg CLPs in the absence or presence of 4-HT. Cytospin preparations of the colonies were stained with Wright–Giemsa solution (magnification × 400). (B) Expression of lineage-specific genes. Cells recovered from the colonies were subjected to RT–PCR as described in Materials and methods. (C) FACS analysis of surface-antigen expression during myeloid conversion. Sorted CLPs were cocultured on an S17 stromal layer with SCF, IL-3, IL-7, FL, and G-CSF in the absence or presence of 4-HT. After 4 days, cells were analyzed for B220, CD11b, and Gr-1 expressions by FACS. Cells were also sorted for analysis of morphology and phagocytic activity. Download figure Download PowerPoint Next, we performed liquid culture of CLP on S17 stromal cells, which allows B-lymphoid differentiation, and examined B220, CD11b, and Gr-1 expression by FACS (Figure 3C). Strikingly, cells differentiated into B220lowCD11b+Gr-1+ myeloid cells upon C/EBPα activation by 4-HT. These cells had macrophage-like morphology and exhibited phagocytic activity in vitro. In contrast, the control-treated cells differentiated into B-lymphoid phenotype (B220+CD11blowGr-1low/−), and these cells retained lymphocyte morphology. These data clearly indicate that ectopic activation of C/EBPα reprograms CLPs into functionally mature macrophage-like cells. Conversion of MEPs and CLPs into G/M lineage occurs in cell-autonomous manner Given that ectopic activation of C/EBPα in MEP and CLP can redirect their colony-forming potential to the myeloid lineage, we next asked whether these changes could occur in a cell-autonomous manner or in a manner influenced by the coexisting cells. To answer this question, we performed a single-cell, 'clonal' colony assay. MEPs and CLPs were clonally deposited into methylcellulose in a 96-well plate by single-cell FACS sorting, and cultured in the presence or absence of 4-HT. As shown in Figure 4A, the plating efficiency and the composition of the colony types in both control and 4-HT-treated plates were similar to those from regular 'bulk' colony assays for both MEPs and CLPs. These results strongly suggest that C/EBPα-induced conversion of MEPs and CLPs into the G/M lineage occurs in a cell-autonomous manner, but not by the secondary effect such as cytokine secretion from the contaminated cells (i.e., T cells or monocytes). Figure 4.Clonal analysis of cell-fate conversion. (A) Single-cell colony assay. MEPs and CLPs were clonally sorted and deposited into methylcellulose containing vehicle or 4-HT in 96-well plates. Five plates for MEP and six plates for CLP for each culture condition (as numbered from 1 though 6) were analyzed. The colonies were evaluated at day 5 for MEP and at day 6 for CLP. Colony composition and the plating efficiency for each plate were plotted individually in the figure. (B) Paired daughter-cell colony assay. Clonally sorted MEPs were cultured in the presence of SCF, IL-11, IL-6, and IL-3. When a single MEP is divided to generate two daughter cells, cells were separately deposited into methylcellulose containing vehicle control or 4-HT by micromanipulation. Colonies from each daughter-cell pair were evaluated at day 5 to compare the fate of differentiation. Data from two experiments (Experiment 1 and Experiment 2) and the total numbers of colony pairs are shown. Download figure Download PowerPoint Cell-fate conversion of daughter-cell pairs from MEP by C/EBPα Although the colony data presented above strongly indicate that C/EBPα can redirect MEP and CLP to myeloid lineage, it is still possible that C/EBPα or 4-HT itself permits the survival of the contaminated myeloid progenitors that otherwise do not survive to form colonies in the control culture. To further confirm the cell-fate conversion at clonal levels, we performed a paired daughter-cell colony assay using MEPs. In brief, sorted MEPs were clonally deposited into 96-well plates and allowed to divide once to generate a daughter-cell pair in the liquid culture. Cells were then separated by micromanipulation and transplanted into control or 4-HT containing methylcellulose, respectively, and the fate of differentiation of each cell was compared (Figure 4B). A total of 768 MEPs successfully gave rise to 230 daughter-cell pairs, which were separated and transferred either into control- or 4-HT-methylcellulose. Out of these, 26 assessable colony pairs were obtained from two consecutive experiments (Figure 4B). Interestingly, the pairs could be classified clearly into two populations with regard to their response to 4-HT. In 16 daughter-cell pairs, one daughter cell in 4-HT chose to follow the myeloid path, whereas the other in the control culture formed GEM or MK colonies. In contrast, in the remaining 10 pairs, 4-HT did not induce myeloid differentiation, and both of the daughter cells followed the same differentiation pathways either in control or 4-HT. Of note, BFU-E was exclusively found in the latter group, indicating that BFU-E no longer retains plasticity having once gone through a cell division. In contrast, all MK colonies were found in the former group, indicating that MK-progenitor was sensitive to myeloid-converting stimuli. These data clearly demonstrate that C/EBPα converts approximately 60% of MEPs to myeloid lineage at the clonal level. However, the remaining population was not sensitive for myeloid conversion by C/EBPα, and thus MEPs could be divided into two subpopulations with regard to the plasticity for myeloid lineage. Short-term activation of C/EBPα is sufficient to convert MEP and CLP into myeloid lineage We next attempted to determine the minimum time required for C/EBPα to convert MEPs and CLPs into myeloid lineage. Sorted MEPs or CLPs were first placed in the initial culture with 4-HT, and then 4-HT was washed off at various time points to assess further differentiation (Figure 5). As for MEPs, conversion seemed to occur gradually from 12 h through day 2, but there was a clear window between day 2 and day 4 in which most (80–90%) of the cells converted to the myeloid lineage with concomitant loss of their Meg/E fate (Figure 5A). In contrast, CLPs required only 12–24 h to lose lymphoid potential and fully switch to the myeloid lineage (Figure 5B and C). It is noteworthy that, like MEP, CLP converts into myeloid lineage through differentiating intermediates that have both B-cell and myeloid markers (B220+CD11b+Gr-1+). Figure 5.Time-course analysis of myeloid conversion of MEP and CLP. (A, B, C) Sorted MEPs (A) and CLPs (B, C) were cultured on S17 stromal cells with 4-HT, as described in Materials and methods. At the indicated time points, cells were recovered and washed with PBS twice. Washed cells were replated on new S17 stromal cell layer and further cultured without 4-HT. Cells were harvested at day 6 for MEP or day 4 for CLP, and expression of cell-surface antigens was analyzed by FACS. Download figure Download PowerPoint Collectively, both MEP and CLP could be converted into myeloid lineage by a short, limited-term activation of C/EBPα (2–4 days for MEP and 12–24 h for CLP). In addition, CLP is clearly more sensitive to converting stimuli than MEP, indicating that the threshold for lineage conversion varies depending on the cell types. Systemic activation of C/EBPα-ER induces myeloid expansion with a concomitant decrease of hematopoietic stem and nonmyeloid progenitor cells in vivo We have shown that induction of C/EBPα activity induced myeloid conversion of MEP and CLP in vitro. To confirm this result and to test the impact of C/EBPα activation on the whole hematopoietic differentiation system in vivo, we next performed serial systemic administration of 4-HT to C/EBPα-ER Tg mice. 4-HT was given intraperitoneally every other day for 4 weeks, and then mice were killed to examine the stem or progenitor cell fractions in BM and thymus (Figure 6A). As expected, mature granulocytes (seg+stab) in peripheral blood clearly increased from 27 to 41%, and lymphocytes decreased from 72 to 58% by 4-HT treatment (Figure 6B), while CBC did not show significant changes (data not shown). Similarly, the numbers of G/M progenitors increased by approximately two-fold in 4-HT-treated bone marrow and spleen (Figure 6C). Of note, a slight increase (about 1.5-fold) of Meg/E progenitors (CFU-EM, BFU-E, and CFU-MK) and CFU-GEM was also observed in spleen and bone marrow, respectively. Figure 6.Systemic activation of C/EBPα induces myeloid conversion in vivo. (A) Administration schedule of 4-HT. A vehicle control and 4-HT were intraperitoneally injected according to the schedule shown in the figure. Mice were killed at day 30 and hematopoietic stem cells/progenitors were analyzed. (B) Differential count of white blood cells in peripheral blood (%). Seg: segmented neutrophil, Stab; band neutrophil, Ly; lymphocyte, Mono; monocyte. (C) Colony assays of spleen and bone marrow cells from control- or 4-HT-treated mice. (D) FACS analysis of hematopoietic stem/progenitor cells after 4 weeks of treatment. Percentages of each stem cell/progenitor fraction against gated parental cell population are
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