The objective of this project is to engineer defines factors that are potent in transactivating downstream target genes for the generation of induced pluripotent stem cells (iPSCs). We have successfully completed all the tasks listed in our first grant year.
1. Screening of factors that potentiate iPSC induction:
The low induction efficiency of induced pluripotent stem cells (iPSCs) is a major challenge to the translation of this laboratory discovery into patient-tailored regenerative medicine. In the first grant year, we screened endogenous growth factors and hormones that are able to enhance the induction of iPSCs. We found that human thyroid hormone triiodothyronine (T3), a wildly used clinic drug, was potent in enhancing the conversion of human skin cells into iPSCs. The potentiation of stem cell induction is related to metabolic remodeling activity by the hormone. We further identify the activation of the PI3K/AKT signal pathway as an underlying mechanism for the T3-mediated potentiation of iPSC induction. This finding thus sheds light on a new research direction for screening factors and small chemicals to promote iPSC induction, particularly by the activation of the PI3K/AKT signaling pathway. These data have been published in the journal of Biomaterial (2012;33:5514-5523). The support of the CIRM grant has been acknowledged in the acknowledgement section of the paper.
2. Mechanisms underlying iPSC induction:
The extremely inefficient process of iPSC induction suggests the presence of a strong epigenetic block that must be overcome before the cells achieve pluripotency. We thus investigated the epigenetic mechanisms underlying the low efficiency of existing approaches. We found that the activation of the endogenous pluripotent factors, like Oct4, Nanog, Sox2, was the key in iPSC induction. In order to identify an epigenetic feature unique to iPSC, we collected iPSCs and un-reprogrammed cells (URCs). Comparison of local chromatin structure revealed that in each case, there is an intra-chromosomal structure that enables the activation of endogenous stemness genes, a critical step towards the successful iSPC induction. These findings highlight the importance of chromosomal structure as critical epigenetic mediators of cell reprogramming and suggest future directions for improving iPSC induction by promoting chromatin remodeling.
3. Library screening of the engineered iPSC inducer factors:
The binding of virally expressed defined factors to the downstream target genes is not a limiting factor in the iPSC induction. Instead, the remodeling of chromatin structure, thus the activation of endogenous stemness genes, represents a critical epigenetic block preventing iPSC induction. We thus used molecular approaches to engineer the OCT4 protein factor in order to enhance the remodeling of intra-chromosomal interaction. We have constructed about twenty Oct4 constructs that contain a strong transactivating domain (TA). We identified four constructs that significantly transactivate the Oct4 and Nanog promoter activities, and activate the expression of endogenous stemness genes. We are going to test how to use these constructs to generate the safest iPSCs for future preclinical study.