Inhibition of PTEN Tumor Suppressor Promotes the Generation of Induced Pluripotent Stem Cells

Abstract 3122

Pioneering work by Yamanaka's group has demonstrated that mammalian somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the ectopic expression of Oct3/4 (also called Pou5f1), Klf4, Sox2 with or without c-Myc (OKSM) [Takahashi, K., and Yamanaka, S. (2006). Cell 126, 663–676, Takahashi et al. (2007). Cell 131, 861–872, Nakagawa et al., (2008) Nat Biotechnol 26, 739–740.]. The iPSCs are similar to embryonic stem cells (ESCs) in their morphology, and their abilities of differentiation to three germ layers in vitro and in vivo.

Comparing with ESCs, iPSCs have less concerns regarding the ethically controversial limitation or immune rejection. And iPSCs have great potential of clinical application for personalized stem cell-based therapies. However, the efficiency of iPSC generation is quite low (0.01∼1%), which is considered to be an impediment of the clinical utility of iPSCs. Although the molecular mechanisms of reprogramming process during the generation of iPSCs have not been fully understood, senescence and apoptosis induced by the ectopic expression of OKSM have been considered to be the major road block in the process of iPSC generation. Therefore the new strategy of iPSC generation which can alleviate such hurdles during the reprogramming process should be developed.

The signaling pathway mediated by the activation of phosphoinositide 3-kinase (PI3K) has been shown to play crucial roles for various aspects of cell biology including cell survival, proliferation, migration, metabolism and vesicular trafficking in variety of cell types. In addition previous studies have shown that PI3K pathway is an important regulator of ES cell cycle by promoting G0 to G1 transition and ES cell proliferation. In this study we hypothesized that activation of PI3K pathway might facilitate the reprogramming process for iPSC generation. To test this hypothesis we first induced the expression of OKSM in mouse embryonic fibroblasts (MEFs) lacking Pten which is a negative regulator of PI3K pathway. About 7 days after the retroviral transduction of OKSM into Pten^−/− MEFs, ES like round-shaped colonies were found (Figure 1). Twenty-two days after the retroviral transduction, the efficiency of iPSC generation was examined by the number of colonies having alkaline phosphatase (AP) activity. We found that significantly higher number of AP⁺ colonies were formed from Pten^−/− MEFs (74 ± 15) compared to Pten^+/− or wild-type MEFs (17 ± 5 and 16 ± 2, respectively). Moreover the number of SSEA1⁺ colonies induced by the expression of OKSM significantly increased from Pten^−/− MEFs (103 ± 2) in comparison to Pten^+/− or wild-type MEFs (40 ± 9 and 21 ± 9, respectively). Similar results were obtained when OKS were transduced without c-Myc.

Next we tested the hematopoietic differentiation in vitro with the reprogrammed MEFs by using embryoid bodies (EBs) formation assay. After the following colony forming unit (CFU) assay, we observed the higher percentage of hematopoietic cells from reprogrammed Pten^−/− MEF group comparing with controls, indicating that reprogrammed Pten^−/− MEFs had the higher potential to differentiate into hematopoietic cells. Continuous activation of PI3K pathway may cause the transformation of the cells; therefore to efficiently and safely generate iPSCs transient activation of the pathway combined with the transduction of OKSM would be desirable. To establish transient activation of the PI3K pathway we used Pten inhibitor, Dipotassium Bisperoxo(5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpV), during the generation of iPSCs. The efficiency of AP⁺ cell-generation from MEFs by transducing Yamanaka factors with bpV was approximately 9%. This was 2 times higher than that by transducing Yamanaka factors without bpV in our hand. These AP⁺ cells showed a normal karyotype and an ability of differentiation into three germ layers in immunodeficient mice. Therefore ectopic expression of OKSM in the presence of Pten inhibitor can establish the efficient generation of iPSCs. Overall, transient activation of PI3K pathway by Pten inhibition offers a highly efficient method generating iPSCs, and might contribute to the establishment of human regenerative medicine with iPSCs.