Efficient creation of induced pluripotent stem cells for myocardial regeneration therapy

Takahiro Sogo¹, Tomohide Takaya², Asako Shigeno¹, Yoji Kojima², Koji Hasegawa³, Teruhisa Kawamura¹.

Career-Path Promotion Unit for Young Life Scientists, Kyogo University, Kyoto, Japan.
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.

World Congress of Cardiology Scientific Sessions 2010 (Beijing, China), 2010/06/19 (Poster).

Abstract

Although myocardial regeneration therapy emerged as an important approach for end-staged heart failure, there remain several clinical problems regarding the application of embryonic stem (ES) because of the ethical issue surrounding the manipulation of oocytes and immune rejection. Recent studies have shown that mouse and human somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells which display properties of self-renewal and pluripotency similar to ES cells. Thus, this strategy provides an opportunity to perform regeneration therapy using patient-specific somatic cells without problems accompanied with usage of ES cells. Although iPS cells can be generated by forced expression of defined factors including pluripotency factors (Oct4 and Sox2) and oncogenes (Klf4 and c-Myc), the low frequency and tendency to induce malignant transformation compromise the utility of this powerful approach for patient use. To address both issues, we investigated the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that the p53 pathway is activated in response to signaling associated with reprogramming. Reducing signaling to p53 through expression of mutant forms of one of its negative regulators or deleting or silencing p53 or its target gene, p21, or antagonizing apoptosis enhanced three factor (Oct4/Sox2/Klf4)-mediated reprogramming of mouse fibroblasts. Since Klf4 has also been reported to have oncogenic properties when overexpressed, we investigated whether cells with reduced p53 expression could be converted into iPS cells using only two factors, Oct4, and Sox2. Notably, decreasing p53 protein levels by p53 shRNA expression enabled fibroblasts to give rise to iPS cells (p53KD-2F-iPS cells) capable of generating adult chimeric mice using two factors. Furthermore, these iPS cells can efficiently differentiate into beating cardiac myocytes. While p53 pathway inactivation will be key, this cannot be done on a permanent basis as this would increase the probability of malignant transformation and the generation of unstable genomes that would mitigate use for understanding many diseases. Rather, transient inhibition using chemical antagonists or reversible approaches that avoid genetic disruption will be required. Our results above may provide insights into reprogramming mechanisms and suggest new routes to more efficient creation of patient-specific iPS cells minimizing the use of oncogenes towards myocardial regeneration therapy.