Establishment of an ES Cell-Derived Murine Megakaryocytic Cell Line, MKD1, with Features of Primary Megakaryocyte Progenitors

Because of the scarcity of megakaryocytes in hematopoietic tissues, studying megakaryopoiesis heavily relies on the availability of appropriate cellular models. Here, we report the establishment of a new mouse embryonic stem (ES) cell-derived megakaryocytic cell line, MKD1. The cells are factor-dependent, their cell surface immunophenotype and gene expression profile closely resemble that of primary megakaryocyte progenitors (MkPs) and they further differentiate along the megakaryocyte lineage upon valproic acid treatment. At a functional level, we show that ablation of SCL expression, a transcription factor critical for MkP maturation, leads to gene expression alterations similar to that observed in primary, Scl-excised MkPs. Moreover, the cell line is amenable to biochemical and transcriptional analyses, as we report for GpVI, a direct target of SCL. Thus, the MKD1 cell line offers a pertinent experimental model to study the cellular and molecular mechanisms underlying MkP biology and more broadly megakaryopoiesis

Functional and transcriptional analyses in MKD1 cells.

<p>(<b>A</b>) <i>Scl</i> excision induces apoptosis in MKD1 cells. Annexin V and 7AAD staining of MKD1 cells (<i>Scl^fl/fl</i>) and upon Cre-mediated excision of the <i>Scl</i> floxed alleles (<i>Scl^ex/ex</i>). (Left) Left panels, FSC and SSC parameters analysis showing the gate of viable cells. Rights panels, Annexin V/7AAD analysis. The percentages of necrotic (AnnexinV⁺ 7AAD⁺) and apoptotic cells (Annexin V⁺ 7AAD⁻) are shown. The data show one representative experiment out of three. (Right) PCR showing amplification of the floxed (fl) and excised (Δ) alleles in MKD1 cells (<i>Scl^fl/fl</i>) and after Cre-mediated excision (<i>Scl^ex/ex</i>). (<b>B</b>) Gene expression analysis by qRT-PCR of MKD1 and MkPs in SCL expressing cells (dark grey bars: MKD1 <i>Scl^fl/fl</i>, white bars: <i>Cre;Scl^+/+</i> MkPs) and upon Cre-mediated <i>Scl</i> excision; <i>Scl^ex/ex</i> (black bars: MKD1 <i>Scl^ex/ex</i>; light grey bars: <i>Cre;Scl^ex/ex</i> MkPs). The <i>y</i>-axis represents the enrichment in cDNA sequences normalised to <i>Gapdh</i> gene control sequences. For MKD1, the histograms show the mean ± SD of 3 independent experiments, p<0.05. For the MkPs, the data show one representative experiment out of 2. (<b>C</b>) (Top) Schematic representation of the mouse <i>Gp6</i> proximal promoter. The location of the E box (E), Gata (G), Sp1 and Ets motifs is indicated, in bp relative to the transcription start site (+1). P1 to P4 show the location of the primer pairs designed for real-time PCR. Not to scale. (Bottom) ChIP analysis over the <i>Gp6</i> locus using material isolated from MKD1 cells (left) and primary megakaryocytes derived from 5-FU treated mouse (right). The antibodies used are indicated. The data show the mean ± SD of 3 independent experiments; enrichment over no antibody, *p<0.05. (<b>D</b>) Trans-activation assays in 3T3 (left) and MKD1 cells (right). Top, the luciferase gene is under control of a 330 bp fragment of the <i>Gp6</i> promoter. The reporter constructs with point mutations in the E box (Emut), Gata (Gatamut) and Ets motifs (Etsmut) are shown below. (Left) The graph shows relative luciferase activity measured in 3T3 cells transiently transfected with the luciferase reporter construct (GP6-330) and vectors expressing the indicated transcription factors. The mean ± SD of four independent experiments performed in duplicate is shown. *, p<0.05. (Right) The graph shows relative luciferase activity measured in MKD1 cells transiently nucleotransfected with the different luciferase reporter constructs as indicated. The mean ± SD of three independent experiments performed in duplicate is shown. *, p<0.05.</p

Generation of MK clones showing different degrees of differentiation.

<p>Generation of MK clones showing different degrees of differentiation.</p

MKD1 cell line exhibits characteristics of primary MkPs.

<p>(<b>A</b>) Outline of the strategy used to isolate immortalized MK clones. (<b>B</b>) (Left) MGG staining reveals small cells with a high nuclear/cytoplasmic ratio (arrow heads) and large cells with multilobulated nuclei (arrows). (Right) AchE staining (red arrow). The photographs were taken using an Olympus BX60 microscope with a Qimaging camera (Surrey, BC). Openlab version 3 software (Improvision, Coventry, UK) was used for image acquisition and images were exported into Adobe Photoshop version CS2 (Adobe Systems, San Jose, CA). (<b>C–D</b>) Analysis of MKD1 cells by flow cytometry. Cells were stained for lineage-specific markers (C) and MK-specific markers (D). The hatched histograms represent the staining with the indicated antibody and the open histograms correspond to the isotype control. One representative experiment out of 3 is shown. (<b>E</b>) Comparison of the FACS-profile of MKD1 cells (top) to that of primary MkPs (bottom), defined as Lin⁻ sca1⁻IL7-R⁻ Thy1⁻, ckit⁺CD41⁺, Fcγ^low CD9⁺. (<b>F</b>) Gene expression profile in MKD1 cells. Analysis by real-time RT-PCR of levels of expression of MK and erythroid-specific markers from mRNA isolated from MKD1 (white bars), primary MkPs (black bars) and Day3 MkPs cultivated with a cocktail of cytokines (grey bars). The <i>y</i>-axis represents enrichment in cDNA sequences normalised to <i>Gapdh</i> gene control sequences. The data show the mean ± SD of 3 independent experiments. (<b>G</b>) Valproic acid (VPA)-induced differentiation of MKD1 cells. Percentages of CD41⁺ (Top left) and CD42b⁺ (Top right) MKD1 cells after 3 days of VPA treatment (25, 50 and 100 µg/ml). The histograms represent the mean ± SD of 3 independent experiments. *, p<0.05. (Middle) Facs plots showing the percentage of high CD41 expressing cells before and after VPA treatment (25 and 50 µg/ml). (Bottom) Ploidy analysis of MKD1 cells after VPA treatment for 7 days. The cells were gated on CD41 high expressing cells. The histograms represent the percentage of the different class ploidy for each condition (white bars: Epo, IL3, grey bars: Epo, IL3, VPA 25 µg/ml, black bars: Epo, IL3, VPA 50 µg/ml). The data show the mean ± SD of 3 independent experiments. *, p<0.002.</p

SCL-mediated regulation of the cell-cycle regulator p21 is critical for murine megakaryopoiesis

Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation