Robust generation of induced pluripotent stem cells by a potent set of engineered factors
Robust generation of induced pluripotent stem cells by a potent set of engineered factors
Tools and Technologies II
Stem Cell Use:
Cell Line Generation:
The recent discovery of iPSC (induced Pluripotent Stem Cell) technology marks a promising breakthrough in regenerative medicine. The beauty of the technology is its ability to convert adult mature cells into embryonic stem cells through the expression of a cocktail of essential factor genes. Thus, iPSCs bypass the ethical dilemma of using embryonic materials and eggs. In addition, the creation of iPSCs for individual patients using their own cells can avoid immune rejection and achieve successful therapeutic effects. Since its initial discovery, the method has been used to generate patient-specific stem cells for regenerative therapy and drug screening, including Parkinson disease, sickle cell anemia, Huntington disease and many other genetic diseases. It is predicted that patients may someday be treated with their own healthy versions of stem cells. The technology of iPSC induction, however, is in its infancy. Generation of iPS cells depends on the synthesis of factor proteins that regulate the developmental clock of adult cells in order to return them to the embryonic state. Viruses are a common approach to deliver factor genes into the cell but they incur the risks of gene mutation and instability. Most critically, the efficiency of iPSC induction is extremely low with all existing approaches. Without clearing these road blocks, it would be impossible to translate this technology to the clinic in the near future. In this project, we propose to fundamentally improve this technology by re-engineering the iPS-inducing factors. We will modify part of these factor proteins, which function inefficiently, to become highly potent in activating target genes related to stemness. By functional screening, we will identify the most potent set of factors for iPSC induction. In addition, we have identified a novel iPS-inducing factor in our lab. Together with those engineered factors, we will work out an ideal cocktail of factors that robustly induce iPSCs. To make virus-free iPS cells, we have developed an enzyme that specifically recognizes and removes the virus-delivered factor genes in the host cell. The removal of the viral transgene will avoid tumor formation and increase clinical safety. Finally, we will generate the safest, genetic material-free stem cells directly by using proteins produced by the genes of engineered factors. With these approaches, the generation of iPSCs will be much more robust, enabling us to create patient-specific stem cells efficiently and safely within a short period of time. We will be the first to break the current technical bottleneck by modifying their protein structure. Taken together, this project may ultimately revolutionize the existing ways to create genetically tailored stem cell lines for research and disease treatment in regenerative medicine.
Statement of Benefit to California:
Induced pluripotent stem cells (iPSCs) offer hope and promise for the therapeutic usage of personalized stem cell in regenerative medicine. Among ten leading death causes in California, five of them can directly benefit from cell-based tissue regeneration, including heart disease, stroke, Alzheimer’s disease, diabetes, and liver diseases. Currently, the economic burdens derived from these diseases are enormous. It is estimated from State of California, Department of Public Health that California taxpayers pay 48 billion annually for cardiovascular diseases, 73 billion excluding non-paid family care for Alzheimer’s disease, and 116 billion for diabetes-related diseases. By far, personated iPSC lines have been successfully made from patients with a variety of diseases, including Alzheimer’s disease, Parkinson disease, sickle cell anemia, Huntington disease, muscular dystrophy and many other genetic diseases. It is hoped that these patients may someday be treated with their own healthy versions of stem cells. However, the iPSC technology still has several shortcomings inhibiting its clinical application. Our proposed research aims at improving this technology by revolutionizing existing methods in producing iPSCs and freeing them from cancer-causing side-effects. This research will finally lead to a new way of developing personalized stem cells for therapy and possibly a cure to above mentioned diseases. The goal of this study is to robustly produce iPSCs that are safe for therapeutic applications. It is thus in line with the mission of the California Institute for Regenerative Medicine – to promote rapid progress in stem cell research leading to treatments and cures for diseases and to the growth of a stem cell and regenerative medicine industry critical to future clinical applications. Our proposed improvements in the production of iPSCs, when translated into therapeutic interventions, will directly benefit the health of California citizens and reduce the economic burden presently borne by California taxpayers. Indirectly, we believe this research will increase California’s visibility in stem cells research, attract more federal funding to sponsor future research and fulfill the wish of California citizens who voted to support stem cell research. It may also stimulate California’s economy growth by stimulating the iPSC regenerative medicine industry for the treatment or cure of diseases.
Year 1The 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.
Year 2The induced pluripotent stem cell (iPSC) technology has been profoundly advanced the area of stem cell regenerative medicine. iPSCs can be generated by overexpression of a cocktail of transcription factors. However, iPSC induction is very inefficient with all existing approaches, forming barriers to translate this technology into clinical studies. In the second grant year, we have made significant progresses by engineering iPSC-inducing factors. We optimized factor proteins based on the local chromatin loop structure, a barrier that needs to be overcome for activation of stem cell-related genes. We found that this mechanism-based engineering significantly enhanced iPSC formation. Most importantly, we demonstrated that iPSCs were able to be generated by a single engineered factor, instead of using conventional four factors. The resulting iPSCs exhibited the same potency as embryonic stem cells to differentiate into various tissue types. We believe this system will ultimately become a valuable tool for the stem cell research community in developing of patient-based stem cell regenerative therapies.
- Cell Stem Cell (2013) Intrachromosomal Looping Is Required for Activation of Endogenous Pluripotency Genes during Reprogramming. (PubMed: 23747202)
- Biomaterials (2012) Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway. (PubMed: 22575839)