Comprehensive analysis of myeloid lineage conversion using mice expressing an inducible form of C/EBP alpha

Abstract

CCAAT/enhancer-binding protein (C/EBP) alpha is a critical regulator for early myeloid differentiation. Although C/EBPalpha has been shown to convert B cells into myeloid lineage, precise roles of C/EBPalpha in various hematopoietic progenitors and stem cells still remain obscure. To examine the consequence of C/EBPalpha 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/EBPalpha. 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/EBPalpha, and the conversion occurred clonally through biphenotypic intermediate cells. Moreover, in vivo activation of C/EBPalpha 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/EBPalpha can activate the latent myeloid differentiation program of MEP and CLP and shows that its global activation affects multilineage homeostasis in vivo.

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.

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⁺.

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.

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.

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.

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 shown in each figure. The staining and the gating protocols for FACS are described in Materials and methods. (E) FACS profiles of CMP/GMP/MEP in control- or 4-HT-treated mice. Percentages of each progenitor fraction against gated parental population are shown in each figure. Morphology and clonogenic activity of sorted cell population are also shown.

Figure 7

Lineage conversion of hematopoietic progenitors by C/EBPα. Lineage conversions by C/EBPα shown in this study are indicated by broken arrows.