DEPTOR is a stemness factor that regulates pluripotency of embryonic stem cells

Abstract

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.

Keywords: Cell Differentiation; Cell Signaling; Embryonic Stem Cell; Mammalian Target of Rapamycin (mTOR); Neurogenesis.

FIGURE 1.

DEPTOR is down-regulated during mESCs differentiation. A, representative image of DEPTOR, OCT4, and NANOG protein levels in undifferentiated and differentiated R1 mESCs with quantitation of at least three biological replicates. B, Deptor, Oct4, Nanog, and Zfp42 mRNA expression in undifferentiated and differentiated mESCs analyzed using qPCR. C, morphological changes of differentiated R1 mESCs analyzed under phase contrast. Scale bar, 20 μm. D, representative immunofluorescence image showing the loss of DEPTOR and Oct4 and the presence of differentiation markers Pax6 and Doublecortin with differentiation. Scale bar, 50 μm. E, Deptor, Oct4, Nanog, and Zfp42 mRNA expression upon Oct4 knockdown in mESCs. ***, p < 0.0005. Error bars, S.E.

FIGURE 2.

Kinetics of DEPTOR and Oct4 expression during mESCs differentiation. R1 mESCs were differentiated for 10 days on Matrigel-coated plates in the presence of IGF and inhibitors of TGF-β and BMP. Cells were collected before the start of differentiation (D0) each day for 10 days after the initiation of differentiation (D1–D10). A, Deptor and Oct4 mRNA expression were measured by qPCR. B, protein expression was measured by Western blot analysis. In both cases, actin was used as a control. C, differentiation of R1 mESCs was further analyzed at day 10 by immunofluorescence for the presence of differentiation markers β-tubulin, and NeuN. Scale bar, 50 μm.

FIGURE 3.

mTORC1 kinase activity and levels of mTOR interacting proteins change upon mESCs differentiation. A, a time course of mTORC1 activity during differentiation was analyzed by measuring S6 phosphorylation. B, the ratios of phospho-S6 (pS6) to total S6 (tS6) protein levels at these time points is shown. C, representative Western blots of mTOR and specific interacting protein levels in undifferentiated and differentiated R1 mESCs. Protein samples collected for undifferentiated and differentiated R1 mESCs were also analyzed for phospho-mTOR (pmTOR) Ser-2448 and Ser-2481 and total mTOR (tmTOR) levels. Actin was used as a loading control for all of the gels. The phospho-mTOR level was further normalized to total mTOR. D, quantitation of at least three biological replicates of experiments shown in C (*, p < 0.05; **, p < 0.005; ***, p < 0.0005). Error bars, S.E.

FIGURE 4.

DEPTOR is down-regulated during hESCs differentiation. H1 hESCs were differentiated for 2 weeks, and DEPTOR, OCT4, and PAX6 expression was analyzed using qPCR. Data for three biological replicates are shown. Error bars, S.E.

FIGURE 5.

DEPTOR knockdown promotes mESCs differentiation. A, Deptor, Oct4, Nanog, and Zfp42 mRNA expression upon DEPTOR knockdown in R1 mESCs. B, representative blot of DEPTOR levels upon DEPTOR knockdown. C, representative phase-contrast image of R1 mESC morphological changes observed upon DEPTOR knockdown using shRNA2. The insets show higher magnification images of colony edges. Error bars, S.E.

FIGURE 6.

Validation of DEPTOR knockdown-mediated mESC differentiation. A, R1 mESCs were infected with control or DEPTOR shRNA4, and cells were selected using puromycin. Cells were then grown in the absence of LIF, mRNA was extracted, and qPCR was performed for Deptor, Oct4, Nanog, and Zfp42 as in Fig. 1. Actin was used as an internal control. B, morphological change of R1 mESCs upon DEPTOR knockdown in A was analyzed under phase contrast, and representative images are shown. C, Hnf4A and Pax6 mRNA expression mESCs after knockdown of DEPTOR using shRNA4. Error bars, S.E.