8 research outputs found
ZFX Controls the Self-Renewal of Human Embryonic Stem Cells
<div><p>Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.</p> </div
ZFX BAC transgenic human embryonic stem cells.
<p>A. Normalized <i>ZFX</i> expression levels from G418-resistant hESC clones with the original clone names shown on the x-axis. Three clones were renamed ZFX<sup>Over1</sup>, ZFX<sup>Over2</sup> and ZFX<sup>Normal</sup> were selected for further analysis. ZFX<sup>Normal</sup> showed normal levels of ZFX but had undergone the same clonal and selection steps as ZFX<sup>Over</sup> clones. B. ZFX and actin protein levels determined by Western blot analysis of ZFX<sup>Over</sup> clones and controls. C. Oct4 (purple), SSEA-4 (green), Tra1-81 (red) and DAPI (blue) on ZFX<sup>Over</sup> clones and controls. Scale bar  = 500 µm (low) or 100 µm on the high magnification images.</p
Directed differentiation of ZFX<sup>Over</sup> clones to endoderm and neural tissue.
<p>ZFX<sup>Over</sup> clones, two control clones that express normal ZFX levels (ID1::YFPc2, Dll1::GFPc277) and H9 were directed to endoderm or neural cells, and the level of Nanog, Pax6 (neural) and CXCR4 (endoderm) mRNA at each time point was measured by quantitative PCR.</p
Gene expression analysis of ZFX<sup>Over</sup> clones compared to controls.
<p>Two independent samples of Tra1-81<sup>HI</sup>/SSEA-3<sup>HI</sup> hESCs were isolated from ZFX<sup>Over</sup> clones, ZFX<sup>Normal</sup> and H9 hESC array analysis. A. Dendrograms of each cell line after clustering analysis. B. Up- and down-regulated genes in ZFX<sup>Over</sup> compared to H9 using an adjusted p-value of 0.05 as a cutoff. C. Quantitative PCR validation of selected genes on the array.</p
ZFX knockdown impairs hESC colony size.
<p>Human ESCs were transduced with ZFX knockdown lentiviral constructs and a scrambled control before clonal replating. (A) Live cell images and (B) and the entire well stained with crystal violet seven days after replating. (C) ZFX immunofluorescence after knockdown in hESCs, and (D) ZFX quantitative immunofluorescence analysis. Each dot is the average pixel intensity of nuclear ZFX protein averaged from all cells in one microscopic field. The average pixel intensity from 50 microscopic fields derived from 3 independent experiments is shown. The crosshairs and whiskers represent the mean and SEM. All knockdowns were significantly different from the scrambled control (p<0.0001 for t-tests of each knockdown versus scrambled). 11,803 (Scr), 10,296 (Z2), 4,765 (Z3) and 6,905 (Z4) nuclei were measured in these experiments. (E) ZFX RNA levels as measured by quantitative PCR after knockdown.</p
ZFX<sup>Over</sup> cultures have a higher clonogenic capacity.
<p>A. ZFX<sup>Over</sup> clones, ZFX<sup>Normal</sup> and H9 hESCs (together grouped as controls) were dissociated into single cells before clonal replating. Cells were expanded for 10 days before fixation and staining with crystal violet. B. Colony counts between ZFX<sup>Over</sup> and control hESCs in 3 independent experiments with error bars representing the S.E.M.</p
ZFX<sup>Over</sup> clones resist spontaneous differentiation.
<p>A. ZFX<sup>Over</sup> clones and controls were expanded in conditions promoting self-renewal before SSEA-3 and SSEA-1 FACS analysis. B. ZFX<sup>Over</sup> clones and controls were expanded in suboptimal conditions before SSEA-3 and SSEA-1 FACS analysis. The quantitation compares ZFX<sup>Over</sup> clones to controls in three independent experiments and error bars represent the S.E.M. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042302#pone.0042302.s005" target="_blank">Figure S5</a> for all 3 independent experiments.</p