WDR36 Safeguards Self-Renewal and Pluripotency of Human Extended Pluripotent Stem Cells

Abstract

Extended pluripotent stem cells (EPS cells) have unlimited self-renewal ability and the potential to differentiate into mesodermal, ectodermal, and endodermal cells. Notably, in addition to developing the embryonic (Em) lineages, it can also make an effective contribution to extraembryonic (ExEm) lineages both in vitro and in vivo. However, multiple mysteries still remain about the underlying molecular mechanism of EPS cells' maintenance and developmental potential. WDR36 (WD Repeat Domain 36), a protein of 105 kDa with 14 WD40 repeats, which may fold into two β-propellers, participates in 18sRNA synthesis and P53 stress response. Though WDR36 safeguards mouse early embryonic development, that is, homozygous knockout of WDR36 can result in embryonic lethality, what role does WDR36 plays in self-renewal and differentiation developmental potential of human EPS cells is still a subject of concern. Here, our findings suggested that the expression of WDR36 was downregulated during human hEPS cells lost self-renewal. Through constructing inducible knockdown or overexpressing WDR36-human EPS cell lines, we found that WDR36 knockdown disrupted self-renewal but promoted the mesodermal differentiation of human EPS cells; however, overexpressing of WDR36 had little effect. Additionally, P53 inhibition could reverse the effects of WDR36 knockdown, on both self-renewal maintenance and differentiation potential of human EPS cells. These data implied that WDR36 safeguards self-renewal and pluripotency of human EPS cells, which would extend our understanding of the molecular mechanisms of human EPS cells' self-renewal and differentiation.

Keywords: WDR36; differentiation; hEPS cells; p53; self-renewal.

FIGURE 1

Down-expression of WDR36 during hEPS1 cell differentiation. (A) Expression of WDR36 protein in hEPS1 cells. Bars = 20 μm. (B) Flow chart of hEPS1 cells in a spontaneous differentiation model. (C,D) Representative brightfield [(C), Bar = 200 μm] and immunofluorescent images [(D), Bar = 50 μm] of hEPS1 cells in the spontaneous differentiation assay at P0, P1, and P2, separately. (E–G) Expression of pluripotent genes (E), embryonic germ layer-related genes (F), and extraembryonic differentiation-related genes (G) in hEPS1 cells cultured in N2B27 medium at P0, P1, and P2. n = 3 experiments; mean ± S. D; two-tailed Student’s t-test. *, 0.01 < p < 0.05; **, p < 0.01; no labeling indicates no statistical significance.

FIGURE 2

siRNA-WDR36 would impair self-renewal of hEPS1 cells. (A,B) WDR36 expression level in hEPS1 cells which had been transfected with WDR36 siRNA was determined using qRT-PCR (A) and western blotting (B). (C) Expression of OCT4, NANOG and SOX2 mRNA in hEPS1 cells transfected with WDR36 siRNA. (D) Immunofluorescent images of OCT4 and NANOG in hEPS1 cells with WDR36 siRNA. Bar = 50 μm. (E,F) Expression of three embryonic germ layer related genes (E) and extraembryonic differentiation related genes (F) in siRNA-WDR36 hEPS1 cells. n = 3 experiments; mean ± S.D; two-tailed Student’s t-test. *, 0.01 < p < 0.05; **, p < 0.01; no labeling indicates no statistical significance. NC-hEPS1: hEPS1 transfected with siRNA-scramble; si1-hEPS1 and si2-hEPS1: hEPS1 transfected with small interference RNA 1 or RNA 2 of WDR36 respectively.

FIGURE 3

Dox-induced WDR36 knockdown elevated differentiation potential of hEPS1 cells in an EB formation assay. (A) Representative images of WDR36-modified hEPS1 cells forming EBs in the EB medium. On day 7, EBs were collected and observed under the microscope. Bar = 200 μm. (B) Expression of mesodermal genes in WDR36-modified EBs treated with or without Dox. (C,D) Immunofluorescent images (C) and quantification intensity for immunofluorescence (D) of WDR36-modified EBs treated with or without Dox (2 μg/ml). Bar = 50 μm; n = 3 experiments; mean ± S. D; two-tailed Student’s t-test. *, 0.01 < p < 0.05; **, p < 0.01; no labeling indicates no statistical significance.

FIGURE 4

WDR36 knockdown promoted mesodermal differentiation potential of hEPS1 cells in a mesodermal-committed differentiation assay. (A) Time scheme of the mesodermal-committed differentiation assay. (B) Representative images of Dox-induced shWDR36 hEPS1 cells in mesoderm differentiation medium with or without Dox. Bar = 200 μm. (C,D) mRNA (C) and protein expression (D) of T and vimentin in sh1-hEPS1 cells treated with or without Dox. Bar = 50 μm. (E) Quantification intensity levels for immunofluorescence of T and vimentin in sh1-hEPS1 cells treated with or without Dox. n = 3 experiments; mean ± S. D; two-tailed Student’s t-test. *, 0.01 < p < 0.05; **, p < 0.01; no labeling indicates no statistical significance.

FIGURE 5

Expression of P53 pathway-related genes during the spontaneous differentiation procedure of hEPS1 cells. (A) Expression of P53, P21, MDM2, and BAX in hEPS1 cells which were cultured in the N2B27 medium at P0, P1, and P2. (B) Expression of P53, P21, MDM2, and BAX in hEPS1 cells with WDR36 siRNA. NC: siRNA-scramble; si1: small interference RNA 1 of WDR36; si2: small interference RNA 2 of WDR36. (C) Expressions of P53, P21, MDM2, and BAX in Dox-induced WDR36 knockdown EBs treated with or without Dox. (D) Expressions of P53, P21, MDM2, and BAX in Dox-induced WDR36 overexpression EBs treated with or without Dox. OE: overexpression of WDR36. n = 3 experiments; mean ± S. D; two-tailed Student’s t-test. *, 0.01 < p < 0.05; **, p < 0.01; no labeling indicates no statistical significance.

FIGURE 6

Inhibition of P53 could restore the influences of WDR36 knockdown on hEPS1 cells. (A) qRT-PCR analysis of the Dox-induced shWDR36 hEPS1 cells cultured under the condition with or without Dox. (B) Immunofluorescent images of OCT4 in Dox-induced shWDR36 hEPS1 cells. (C) Expression of T and vimentin in Dox-induced shWDR36 hEPS1 cells. (D) Immunofluorescent images of T and vimentin in Dox-induced shWDR36 hEPS1 cells. Bars = 50 μm. NC, culture condition did not contain 10 μM PFT-α. n = 3 experiments; mean ± S. D; one-way analysis of variance (ANOVA). *, 0.01 < p < 0.05; **, p < 0.01; #, 0.01 < p < 0.05; ##, p < 0.01; no labeling indicates no statistical significance.