Pten-mediated Gsk3β modulates the naïve pluripotency maintenance in embryonic stem cells

Abstract

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency-naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten −/− mouse ESCs with the CRISPR-Cas9 system and verified that Pten −/− ESCs maintained naïve pluripotency by blocking Gsk3β activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3β modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.

Pten Regulates Cardiomyocyte Differentiation by Modulating Non‐CG Methylation via Dnmt3

“…Intriguingly, Pten −/− EBs had a lower spontaneous beating rate than WT EBs ( Figure 2 a ; Videos S2 , S3 , Supporting Information). The cells that differentiated from Pten −/− ESCs tended to be neural stem cell‐like cells, which is consistent with our previous findings that Pten loss promoted neural ectoderm differentiation, [ 29 ] and WT ESCs could differentiate into spontaneously beating cardiomyocytes (Figure 2b ; Videos S4 , S5 , Supporting Information).…”

“…[ 10 ] To explore the function of Pten in cardiomyocyte differentiation, we generated Pten ‐deficient mouse ESCs using the CRISPR‐Cas9 system as described previously. [ 29 ] Cardiomyocytes were generated from wild‐type (WT) and Pten −/− ESCs using the differentiation protocol described above. Intriguingly, Pten −/− EBs had a lower spontaneous beating rate than WT EBs ( Figure 2 a ; Videos S2 , S3 , Supporting Information).…”

“…Phosphorylation of the C‐terminal of PTEN at S380, T382, and T383 is shown to inhibit its phosphatase activity. [ 31 ] We utilized the PTEN‐A3 mutant (S380A, T382A, and T383A) ESC line described previously to augment the phosphatase activity, [ 29 ] and the cardiomyocytes derived from PTEN‐A3 mutant ESCs displayed a higher expression level of cardiomyocyte genes (Figure S3d , Supporting Information). In addition, we measured the level of PTEN expression at different time points during cardiomyocyte differentiation and found that PTEN expression was potently induced on day 5 and decreased in the following days (Figure S3e , Supporting Information).…”

“…Mouse ESCs were cultured as previously described. [ 29 ] Briefly, mouse ESCs were cultured in DMEM/F12 containing GlutaMAX and sodium pyruvate (Life Technologies, 10565) with 15% fetal bovine serum (Hyclone, SH30071), non‐essential amino acids solution (Life Technologies, 11140), HEPES (Life Technologies, 15630080), β ‐mercaptoethanol (Life Technologies, 31350) with 1 × 10 3 units/ml LIF (Millipore, ESG1106), 1 × 10 −6 m PD0325901 (Sigma, PZ0162), and 2.5 × 10 −6 m CHIR99021 (Sigma, SML1046). The mouse ESCs were grown on gelatin‐coated plates and the medium was replaced every other day.…”

Temporal regulation of Pten is essential for retina regeneration in zebrafish

“…Overexpression of ptena and ptenb mRNA resulted in a drastic decline in the number of MGPCs in 4dpi retina (Figures EV2G and EV2H). Similar results were observed when Pten activity was blocked using a pharmacological inhibitor SF1670 (Spinelli et al, 2015;Wang et al, 2020) in regenerating retina at 4dpi (Figures 2D and 2E). The 1016tuba1a:GFP transgenic zebrafish retina also had an elevated GFP expression because of SF1670 treatment (Figure EV2I), which supports the enhanced the proliferation of MGPCs seen in the wild-type retina.…”