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PTEN Regulates the Pluripotent State and Lineage Fate Choice in Human Embryonic Stem Cells

From Stem Cells
By Stuart P. Atkinson

In mouse, Pten plays a negative role in the self-renewal of embryonic and adult cell types (Stiles et al, Korkaya et al and Groszer et al) through negative regulation of PI3K/Akt signalling and while studies have shown the importance of PI3K/Akt signalling in embryonic stem cells (ESCs) (Ding et al and Storm et al), the role of PTEN in human ESCs (hESCs) is relatively unknown.

To analyse the role of PTEN in hESCs, Alva et al from the group of April D. Pyle at the University of California, USA, generated stable PTEN knockdown ESC lines (H9 and HSF1) were generated using lentiviral mediated short hairpin RNA, leading to a 60-80% knockdown of PTEN RNA and protein, while the cells karyotype remained stable.   However, spontaneous differentiation of PTEN-KD-hESCs was reduced as compared to control hESCs.   Low density assays, which assay colonies arising from single cells, showed that PTEN-KD-hESCs gave rise to an increased number of colonies and had higher levels of TRA-1-81. Passaging colonies from these experiments found that PTEN-KD-hESCs had between a 3 and 4-fold increase in the number of colonies after each passage. These cells also showed a 2-fold decrease in apoptosis suggesting that PTEN-KD-hESCs had an increased survival capability, and BrdU pulsing experiments demonstrated that PTEN-KD-hESCs have a higher proliferation rate. Additionally, PTEN-KD-hESCs have a decreased percentage of cells in Go/G1 and increased S/G2/M cell cycle populations.

Analysis into the mechanisms behind PTEN function found that PTEN-KD-hESCs have increased levels of phospho-AKT and phospho-S6, suggesting activation of the AKT/mTOR pathway. In mouse stem cells, Pten activity modulates Wnt signaling via indirect effects of Akt activity (Korkaya et al), although in PTEN-KD-hESCs changes in the level of phospho-GSK3ß were not observed, while β-catenin was plasma membrane bound in control and KD cells. Recent studies have shown that stabilization of β-catenin in hESCs leads to disruption of self-renewal (Lam et al and Sumi et al), and while this was observed in differentiated cells from control hESC colonies, concomitant with accumulation of β-catenin in the nucleus, this was not observed in the PTEN-KD-hESCs.

Differentiation studies of H9 PTEN-KD-hESCs found that cells grown as embryoid bodies (EB)s had decreased differentiation of all three germ lineages, while HSF1 PTEN-KD-hESCs had decreased endoderm and mesoderm, but an increase in ectoderm differentiation compared to control. H9 PTEN-KD-hESCs EBs also demonstrated the decreased PTEN levels and increased phospho-AKT levels observed previously, but did not show reduced levels of OCT4 or NANOG. However HSF1 PTEN-KD-hESCs EBs showed a minimal increase in phospho-AKT, no increase in OCT4 but an increase in differentiation, suggesting that levels of phospho-AKT are critical to regulating the differentiated state.

In directed differentiation studies all PTEN-KD-hESCs differentiated less efficiently. In depth studies of neural differentiation showed that PTEN-KD-hESCs could differentiate to PAX6+ progenitor cells but had reduced differentiation to TUJ1+ mature neural cells. PCR also showed lower mature neural markers (NEUROD1, TUJ1) and an increased level of OCT4, NANOG and PAX6 compared to controls, suggesting a block in terminal differentiation due to the lack of inhibition of the pluripotency network. Endodermal differentiation studies found a reduction on FOXA2+ cells, and a lack of reduction in OCT4 and NANOG, while mesodermal differentiation found that less CD34+PECAM+ cells were produced and OCT4 and NANOG expression was maintained. In vivo teratoma assays found that teratoma size was increased in the PTEN-KD-hESCs associated with a higher level of phospho-Histone H3 indicating increased proliferation and while they could differentiate into all three germ layers, they had an increased potential for ectoderm differentiation (NESTIN and MAP2 expression) and reduced endoderm differentiation potential.

The pluripotency network was then studied with relation to PTEN loss, by studying the heterogeneity of marker expression. In PTEN-KD-hESCs TRA-1-81 was more uniform with a higher percentage of positive cells, while OCT4+ and NANOG+ cells were increased, although not SOX2+ cells as compared to controls. Other pluripotency factors (TDGF1, UTF1, DPPA2, DPPA5 and DNMT3B) were also increased as measured by QPCR.   Studies have previously shown that the cells with the highest levels of pluripotency associated marker expression have lower early progenitor markers (e.g. PAX6 and GATA4) and vice versa (Laslett et al), and in PTEN-KD-hESCs PAX6 and GATA4 were minimally expressed suggesting a boost in the pluripotent nature of these cells.

Taken together, these results demonstrate that PTEN-KD-hESCs have increased self-renewal, survival, and proliferation, increased phosphorylation of AKT and S6, but not activation of WNT signaling and generate more proliferative teratomas. Overall, pluripotency is increased and differentiation retarded with loss of PTEN in hESCs.

 

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