Reduced c-Myb activity compromises HSCs and leads to a myeloproliferation with a novel stem cell basis

Abstract

Murine haematopoietic stem cells (HSCs) are contained in the Kit+Sca1+Lin(-) (KSL) population of bone marrow and are able to repopulate lethally irradiated mice. Myeloproliferative disorders (MPDs) are thought to be clonogenic diseases arising at the level of the HSC. Here, we show that mice expressing low levels of the transcription factor c-Myb, as the result of genetic knockdown, develop a transplantable myeloproliferative phenotype that closely resembles the human disease essential thrombocythaemia (ET). Unlike wild-type cells, the KSL population in c-myb knockdown bone marrow cannot repopulate irradiated mice and does not transfer the disease. Instead, cells positive for Kit and expressing low to medium levels of CD11b acquire self-renewing stem cell properties and are responsible for the perpetuation of the myeloproliferative phenotype.

Figure 1

The c-myb knockdown has profound effects on adult haematopoiesis. (A) Histograms comparing the numbers of red blood cells (RBC), platelets, and white blood cells (WBC) and the percentage of monocytes and neutrophils in the WBC fraction in wild-type (WT) and c-myb knockdown (KD) peripheral blood. (B) Diff-Quick staining from WT and KD blood smears. (C) Histogram comparing the total number of cells in WT and KD bone marrow after red cell lysis (mean±s.d., n=7). The micrographs on the right show hematoxylin and eosin-stained paraffin sections of WT and KD bone marrow, the arrows indicating megakaryocytes. (D) Comparative picture of the spleens from WT and KD mice. (E) Reticulin staining from WT and KD bone marrow.

Figure 2

The c-myb knockdown retains phenotypically defined HSCs, but these show changes in quiescence and stem cell phenotype and differentiation capacity. (A) Flow cytometric profiles of red-cell-depleted WT and KD bone marrow stem cells (KSL: Kit⁺Sca1⁺Lin⁻). The histogram shows a comparison of the number of KSL cells (right) and Kit⁺Sca1⁻Lin⁻ (left) in WT and KD as a percentage of the total number of bone marrow cells (mean±s.d., n=7). (B) Histogram representing a quantification of the relative proportions of KSL cells defined on the basis of their expression of Flt3 (mean±s.d., n=7). (C) Flow cytometric profiles comparing staining with Hoechst 33342 of WT and KD KSL cells. The side population cells are defined as regions R2 and R3. (D) Histograms representing a quantification of gated KSL cells in different cell-cycle phases staining with an antibody specific for the nuclear antigen Ki67 and with propidium iodide to determine the DNA content (mean±s.d., n=3). (E) WT and KD KSL cells subdivided for Flt3 expression were seeded in methylcellulose as described in Materials and methods. Colonies were counted and their morphology assessed after 6 days (mean±s.d., n=3). The May–Grünwald Giemsa-stained cells illustrate the phenotype of the predominant colony types seen in the KSL Flt3^high population isolated from WT (macrophage) and KD (megakaryocyte).

Figure 3

Short-term engraftment by c-myb knockdown bone marrow. Irradiated hosts were injected with 2.5 × 10⁶ test WT or KD total bone marrow cells together with 5 × 10⁵ competitor WT from CD45.1/CD45.2 animals (n=3). The percentages of the wild-type competitor and test donor populations are indicated. The ratios of wild-type competitor to test donor for all animals transplanted gave means and s.d. of 3±0.28, 6±1.4 and 3.2±0.15 for the peripheral blood (PB) from 4, 8 and 12 weeks, respectively. (A) Flow cytometric profiles of peripheral blood taken from primary engrafted animals 4, 8 and 12 weeks after transplantation with identifying host, competitor and KD donor cell populations based on CD45.1/CD45.2 staining. The competitor/donor ratio (C:D) is indicated in the top right corner of each histogram. (B) CD4, CD8, CD11b and B220 expression on peripheral blood cells of host, competitor and donor origin from primary engrafted animals 12 weeks after transplantation with WT or KD donor cells. (C) Blood smears from primary engrafted animals 6 months after transplantation with WT or KD donor cells. (D) Peripheral blood cells from primary engrafted animals 6 months after transplantation with WT or KD donor cells were cultured for 6 days. (E) Acetylcholine esterase staining (left panel) and analysis of CD41 and CD11b expression (right panel) in 6 day-cultured peripheral blood from a recipient transplanted with KD donor cells. (F) Methylcellulose culture of peripheral blood from a recipient transplanted with KD donor cells, showing a typical colony present after 2 weeks.

Figure 4

c-myb knockdown bone marrow is capable of long-term engraftment. (A) Flow cytometric profiles of CD45.1/CD45.2 expression in bone marrow (BM) and spleen (Sp) from primary engrafted animals described in Figures 3 and 6 months after transplantation. The percentages of the wild-type competitor and test donor populations are indicated. The ratios of wild-type competitor to test donor for all animals transplanted gave means and s.d. of 15±1.8, 12±0.45 and 0.45±0.2 for the KD BM, WT Sp and KD Sp, respectively. (B, C) Cells from donor origin (CD45.2) were gated and analysed for (B) Kit/CD41 or CD11b/Gr1, and (C) lineage antigens (Lin) versus cell size (FSC). Lin⁻ cells (indicated by the gated region in the upper panels) were further analysed for expression of c-Kit and Sca1, the KSL population being defined as those cells in the gated region (C, central panels). KSL cells were further analysed by staining with Hoechst 33342 (C, lower panels), side population cells being indicated by the gated region.

Figure 5

Primary and secondary transplanted animals receiving c-myb knockdown bone marrow cells develop a myeloproliferative disorder. Irradiated hosts were injected with 2 × 10⁶ (n=3) bone marrow cells derived from primary transplants that had received WT or KD total bone marrow cells. (A) Weight curve of secondary engrafted animals. (B) CD45.1/CD45.2 staining of peripheral blood taken from secondary engrafted animals 4 weeks after transplantation with donor cells from either a WT competitor/WT test (WT:WT primary) or WT competitor/KD test (WT:KD primary) primary transplant. The percentages of the wild-type competitor and test donor populations are indicated. The ratios of wild-type competitor to test donor for all animals transplanted gave means and s.d. of 12±6.3 and 29.6±7.2 WT:WT primary and WT:KD primary, respectively). (C, D) Blood smears (C) and blood cell counts (D) for secondary engrafted animals described in (B). (E) Staining for CD11b/Gr1 expression in bone marrow cells of test donor origin (CD45.2⁺) from secondary engrafted animals as described in (C). (F) Analysis of multipotent progenitors (GMP, CMP and MEP) of test donor (CD45.2⁺) origin, depicting FcγRII/III/CD34 expression on the gated Kit⁺Sca1⁻Lin⁻IL-7R⁻ population. Staining boundaries for the relevant isotype controls are indicated by the dashed lines.

Figure 6

Engrafting cells from c-myb knockdown bone marrow are not in the KSL population. Irradiated hosts were injected with 500 (n=6) or 2 × 10³ (n=6) WT or KD sorted KSL cells together with 1 × 10⁶ CD45.1/CD45.2 WT competitor cells. (A) Peripheral blood and (B) bone marrow taken from animals engrafted with KSL cells 12 weeks after transplantation, distinguishing host and donor cells based on CD45.1/CD45.2 staining as described in Figure 3. The percentages of the wild-type competitor and test donor populations are indicated. The ratios of wild-type competitor to test donor for all animals transplanted gave means and s.d. of 0.15±0.05 and 0.17+0.02 for WT in the peripheral blood and the bone marrow, respectively. (C) Semi-quantitative RT–PCR of KSL-cell RNA from WT versus KD animals using primers corresponding to the indicated gene (see Supplementary Table 2). Each reaction was sampled at three-cycle intervals in the exponential range of the amplification. (D) Histogram representing the results of quantitative RT–PCR of KSL-cell RNA from WT versus KD animals using primers corresponding to the indicated genes (see Supplementary Table 3). The data are normalised against β2 microglobulin expression and the wild-type values set as 1.

Figure 7

Engrafting cells from c-myb knockdown bone marrow are in a lineage⁺ fraction that is expanded relative to the wild type. Irradiated hosts were injected with WT or KD subpopulations sorted on the basis of Kit and Lin expression (see Materials and Methods for the numbers used) together with 1 × 10⁶ CD45.1/CD45.2 WT competitor cells (for each cell type n=3). (A) Flow cytometric profiles of bone marrow taken from animals engrafted with Kit⁺Lin⁺ cells seven weeks after transplantation identifying host, competitor and donor cell populations based on CD45.1/CD45.2 expression. The percentages of the wild-type competitor and test donor populations are indicated. The ratios of wild-type competitor to test donor for all animals transplanted gave means and s.d. of 0.89±0.55 and 27±13 for WT and KD test donors, respectively. Staining for CD11b/Gr1 (B) and KSL cells (C) in bone marrow of test donor origin (CD45.2⁺) from animals engrafted with Kit⁺Lin⁺ cells 7 weeks after transplantation.

Figure 8

Engrafting cells from c-myb knockdown bone marrow express low to medium levels of CD11b. Irradiated hosts were injected with 500 WT or KD Kit⁺CD11b^low/med cell subpopulations that had been sorted on the basis of Lin (CD5, CD8a, B220, Gr-1, Ter119) and Sca1 expression together with 1 × 10⁶ CD45.1/CD45.2 WT competitor cells (for each cell type n=3). (A) Flow cytometric profiles of bone marrow taken from animals engrafted with Kit⁺CD11^low/med cells 8 weeks after transplantation identifying host, competitor and donor cell populations based on CD45.1/CD45.2 expression. The percentages of the wild-type competitor and test donor populations are indicated. The means and s.d. of the ratios of wild-type competitor to test donor for all animals transplanted are indicated in the top left-hand corner of each profile (note: only one animal receiving transplantation of WT Kit⁺CD11b^low/medLin^lowSca1⁺ cells survived to be analysed). Staining for CD4/CD8 (B) and CD11b/Gr1 (C) in bone marrow of test donor origin (CD45.2⁺) from animals engrafted with Kit⁺CD11^low/med cells 8 weeks after transplantation. (D) Schematic representation of the engraftment achieved with fractionated WT and KD populations. The antigens expressed are indicated and the shading depicts whether transplanted cells gave full reconstitution (black), no reconstitution (white) or reconstitution of the myeloproliferative phenotype with either a limited myelomonocytic profile (white dotted) or the characteristic KD phenotype involving co-expression of lymphoid and myelomonocytic markers (black dotted).

Figure 9

Correct control of c-Myb levels is required to maintain the haematopoietic hierarchy. The diagram summarises the ways in which disrupted loss of normal (A) control of c-Myb expression has been shown to lead to expansion of cells with stem-cell characteristics at different points in the haematopoietic hierarchy, resulting in (B) myeloid and (C) lymphoid leukaemia or, as described here, myeloproliferation (D). The highly simplified representation of haematopoiesis is divided on the basis of cells being in the KSL stem cell compartment (purple) or committed towards myeloid (green) or lymphoid (blue) differentiation. The approximate range of expression of crucial markers is indicated at the bottom (SP, side population). Circular arrows are used to depict self-renewal, the extent of which is related to the size. Vertical bars indicate blocks to further differentiation. The dotted arrow and cell in (D) represent a putative new pathway of dedifferentiation seen in the c-myb knockdown.