UCLA UCLA Electronic Theses and Dissertations Title Investigating the Mechanisms of Reprogramming and Optimizing the Generation of Potentially Therapeutically Useful Induced Pluripotent Stem Cell Derivatives Permalink https://escholarship.org/uc/item/25v79301 Author Awe, Jason Patrick Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California University of California Los Angeles Investigating the Mechanisms of Reprogramming and Optimizing the Generation of Potentially Therapeutically Useful Induced Pluripotent Stem Cell Derivatives A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular and Medical Pharmacology By Jason Patrick Awe 2014 ABSTRACT OF THE DISSERTATION Investigating the Mechanisms of Reprogramming and Optimizing the Generation of Potentially Therapeutically Useful Induced Pluripotent Stem Cell Derivatives By Jason Patrick Awe Doctor of Philosophy in Molecular and Medical Pharmacology University of California, Los Angeles, 2014 Professor James A. Byrne, Chair Human induced pluripotent stem cells (hiPSCs), derived from easily obtainable skin cells, possess enormous opportunity for autologous cellular treatment therapies, gene correction, and disease modeling without worries of ethical constraints associated with human embryonic stem cells (hESCs). Although lentiviral based reprogramming remains as one of the most popular methods for reprogramming, potentially oncogenic viral integrations in random locations throughout the genome along with non-human antigens associated with the reprogramming process thwart the clinical applications of these hiPSCs. To address these concerns we derived a hiPSC line void of any exogenous reprogramming factors and differentiated these hiPSCs into clinically relevant cell derivatives. In addition, to maintain clinical relevance, we implemented a methodology to clean our hiPSCs from non-human antigens to allow for current good manufacturing practice conditions that could help set the standard for human clinical trials with our factor-free hiPSCs. The field of stem cell reprogramming has rapidly advanced, and a new ii technique involving mRNA based reprogramming was introduced that we found to be difficult to reproduce due to an innate immune response based degradation of mRNA when introduced into the cell. To solve this problem, a small chemical compound was utilized that blocked important aspects of the innate immune response to single stranded mRNA that yielded robust and uniform expression of a key reprogramming factor. This stabilization could be important in increasing mRNA based reprogramming efficiency of hiPSC derivation. Another challenge in the hiPSC field is investigating nuanced potential differences manifested in transcriptional, epigenetic, immunological, and differentiation potentials between hESCs and hiPSCs. To help and potentially solve this problem and allow for more complete and faithful reprogramming to a hESC state, global microarray transcriptional analysis of oocyte cytoplasm was utilized to find eight putative novel shared reprogramming factors across multiple species. These factors have identifiable roles in opening up chromatin that can allow reprogramming factors to better access reprogramming loci that could confer the known reprogramming advantage that somatic cell nuclear transfer based reprogramming maintains over current direct reprogramming approaches. To address the recently observed immunogenicity issues of iPSCs, we studied the expression of two normally fetally associated genes implicated in an iPSC-specific immune response. We found high line-to-line variation between both hESC and hiPSC lines across different levels of differentiation and confirmed that current differentiation protocols derive cell types with a fetal phenotype as opposed to the adult phenotype needed for clinical applications as indicated by aberrant expression of specific fetal genes. Taken altogether, we hope these studies allow for more robust, reproducible, and clinically relevant hiPSCs that more closely resemble hESCs and maintain full ability to differentiate into clinically relevant cell types that can be used for potential human clinical trials for disease and cell replacement therapy. iii COMMITTEE PAGE The dissertation of Jason Patrick Awe is approved. Kathrin Plath Lily Wu Patricia Phelps Heather Christofk James A. Byrne, Committee Chair University of California, Los Angeles 2014 iv DEDICATION PAGE I dedicate this thesis to my amazing wife who has been incredibly supportive of my research, the long hours in the laboratory, and most importantly, of me and all my endeavors. I also dedicate my thesis to my grandmother, Nana, who has supported me unconditionally throughout my whole life in every aspect. v Table of Contents ABSTRACT OF THE DISSERTATION ................................................................................................... ii COMMITTEE PAGE ................................................................................................................................. iv DEDICATION PAGE .................................................................................................................................. v ACKNOWLEDGEMENT ........................................................................................................................... ix VITA ............................................................................................................................................................. xi CHAPTER 1: INTRODUCTION ............................................................................................................... 1 REFERENCES ..................................................................................................................................... 14 CHAPTER 2: GENERATION AND CHARACTERIZATION OF TRANSGENE-FREE HUMAN INDUCED PLURIPOTENT STEM CELLS AND CONVERSION TO PUTATIVE CLINICAL- GRADE STATUS ..................................................................................................................................... 22 Abstract .................................................................................................................................................. 23 Introduction ............................................................................................................................................ 24 Materials and methods ........................................................................................................................ 26 Results ................................................................................................................................................... 39 Discussion ............................................................................................................................................. 46 Conclusions ........................................................................................................................................... 48 Abbreviations ........................................................................................................................................ 48 Table 2.1: Human iPSC Reprogramming Efficiencies from Human Dermal Fibroblasts ...... 50 Figure 2.1. Representative colonies from the three hSTEMCCA-derived induced pluripotent stem cell lines. .................................................................................................................................. 50 Figure 2.2. Characterization of pre-excised and post-excised induced pluripotent stem cell lines. ................................................................................................................................................... 51 Figure 2.3. Expression and splicing analysis of the pre-excised and post-excised lines. .... 52 Figure 2.4. Differentiation into therapeutically relevant cell lineages. ...................................... 53 Figure 2.5. Morphology of transgene-free induced pluripotent stem cells following conversion to clinical-grade conditions. ........................................................................................ 54 References ............................................................................................................................................ 55 CHAPTER 3: BAY11 ENHANCES OCT4 SYNTHETIC MRNA EXPRESSION IN ADULT HUMAN SKIN CELLS .............................................................................................................................. 60 Abstract .................................................................................................................................................. 61 Introduction ............................................................................................................................................ 62 Materials and methods ........................................................................................................................ 64 vi Results ................................................................................................................................................... 71 Discussion ............................................................................................................................................. 75 Conclusions ..........................................................................................................................................