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Access Type
WSU Access
Date of Award
1-23-2012
Degree Type
Thesis
Degree Name
M.S.
Department
Biochemistry and Molecular Biology
First Advisor
Jianjun Wang
Abstract
The unlimited self-renewal and pluripotency characteristics of human embryonic stem (hES) cells allow them to have the unique potential to be utilized in the study and treatment of a plethora of diseases, such as neurodegenerative diseases (Parkinson's disease, Amyotrophic Lateral Sclerosis, Alzheimer's disease), diabetes, spinal cord injury, heart diseases and cancers. However, moral and political issues with the use of human embryos in research prevent utilization of these human ES cells for clinical application. In addition, the risk of immunologic rejection by the patients further impedes its applications in human disease treatments.
In 2006, Yamanaka's group demonstrated that mouse ESC-like cells could be generated from mouse somatic cells by ectopic expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc) in these somatic cells. These generated ESC-like cells are termed induced pluripotency stem cells (iPSCs). In 2007, several groups generated human iPSCs following a similar strategy by Yamanaka's group, which reprograms human somatic cells to reset their clock back to human ESC-like cells. Since these human iPSCs can be directly generated from human skin cells obtained via skin biopsy, this new technology raised great interest in the scientific and medical community for stem cell based personalized regenerative medicine.
While Yamanaka's method has been successful in generation of iPSCs, it utilizes viral delivery vehicles to deliver four reprogramming genes and these viral vehicles can integrate into chromosome and cause high rates of mutagenesis and unpredictable genetic dysfunction. This raises a major concern of the safety when these iPSCs are used for human clinical applications. To address this safety issue, a new protein based method for producing patient-specific iPS cells has been developed. In this method the transcription factors, or reprogramming proteins, are directly delivered to cells to generate protein-induced pluripotent stem cells (piPSCs). This method solved the most challenging safety hurdles, enabling iPSC generation without genetically altering the originating cells. The delivered proteins eventually degrade and would be absent by the time iPSCs would be used for experiments or therapies.
However, the current iPSC/piPSC technology faces four major challenges: inefficient, time-consuming, complicated/expensive, and low quality. The current iPSC technology, including gene delivery and protein delivery, obtain a conversion of only 0.001-1% of human somatic cells into iPSCs. Such low conversion efficiency makes iPSC generation a stochastic process, causing major difficulties in studying cell-reprogramming. The current methods take 4-8 weeks to generate human iPSCs (hiPSCs). This renders iPSC technology too expensive and useless for the disorders that demand rapid treatment. These challenges are particularly critical for protein-induced cell reprogramming, because generation of human piPSCs takes 8 weeks with a conversion efficiency of only 0.001%.
Through the use of the three novel techniques developed in my lab: high-level bacterial expression method, QQ-protein transduction method, and an in vivo protein refolding technique, we have developed a simple, straight-forward reprogramming protocol that can efficiently produce piPSCs within a short time. We have collected critical data to demonstrate that we could generate piPS cells in 10 days with ~80% conversion efficiency using the Yamanaka's four factors. We further demonstrated that piPS cells could be generated using Nanog as the only reprogramming factor and using Oct4/Nanog or Sox2/Oct4/Nanog. These data indicate that Oct4 is dispensable and Nanog may serve as a better reprogramming factor to generate high-quality piPSCs since an enhanced level of non-oncoprotein Nanog maintains pluripotency of ESCs, whereas an enhanced oncoprotein Oct4 level causes differentiation and possible tumorigenesis.
Our novel piPSC technique significantly speeds up the entire process of generating patient-specific piPSCs. It will remove the oncoproteins as reprogramming factors during cell reprogramming, making piPSCs safer for possible human applications. This highly-efficient piPSC technology will allow piPS cell generation to be a predictable process, making the mechanistic studies of protein-induced cell reprogramming more reliable and accurate. It allows us to quickly generate a panel of disease-specific piPSCs as the starting materials for generating surrogate models of human diseases, to gain valuable insight into the pathophysiology of the diseases, to discover new prognostic biomarkers, and to ensure a continuous supply of afflicted cell types for drug screens and discovery.
Recommended Citation
O'Connor, Carrie Elizabeth, "Generation and characterization of the protein-induced pluripotent stem (pips) cells" (2012). Wayne State University Theses. 136.
https://digitalcommons.wayne.edu/oa_theses/136