Cellular Reprogramming: Dissecting the Molecular Mechanism and Enhancing Efficiency

Cellular Reprogramming: Dissecting the Molecular Mechanism and Enhancing Efficiency

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01628
Approved funds: 
$1,458,000
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
Pluripotent stem cells have a remarkable potential to develop into virtually any cell type of the body, making them a powerful tool for the study or direct treatment of human disease. Recent demonstration that induced pluripotent stem (iPS) cells may be derived from differentiated adult cells offers unprecedented opportunities for basic biology research, regenerative medicine, disease modeling, drug discovery and toxicology. For example, using patient-derived iPS cells, one can model diseases in vitro and screen for drugs in ways never before possible, enabling the identification of promising new therapeutic candidates earlier in the drug discovery process. In addition, iPS cell derivatives represent an ideal source for autologous cell replacement therapies, as they would not be rejected upon transplantation back into the patient. While it is clear that iPS cells hold great promise for finding therapies for diseases, there are significant hurdles that need to be overcome before full clinical potential is realized. The mechanism of iPS cell derivation is largely elusive, and the process used to generate them is very inefficient and needs to be improved in significant ways. Currently, iPS cells are generated by forced expression of four molecular factors using genome-integrating viruses. This may lead to mutations and altered differentiation potential of iPS cells, as well as tumorogenesis if transplanted back into the patient. The inefficient and stochastic nature of the reprogramming process indicates that additional, as yet unidentified mechanisms may exist and contribute to iPS cell generation. Finally, increasing the efficiency of current iPS cell derivation protocols will increase the ability to generate large panels of patient-specific iPS cell lines. We propose to use a human cell-based assay to identify small molecules that can enhance the efficiency of iPS cell generation. Our strategy will allow us to identify small molecules that target events essential for derivation of iPS cells, as well as those that replace one or more of the four virally-delivered factors. We will use the identified small molecules to discover regulatory pathways and molecular targets involved in induction of pluripotency, gaining valuable insight into the mechanism of cellular reprogramming. Application of these small molecules themselves, as well as novel approaches derived from mechanism of action studies, will help overcome issues associated with viral integration and has the potential to transform personalized cell-replacement therapies as well as accelerate drug discovery based on iPS cell-derived disease models.
Statement of Benefit to California: 
California’s health care system faces significant challenges as millions of children and adults suffer from a host of incurable illnesses. It is expected that health care costs will continue to rise as California’s citizenry ages and requires treatments for age-related, chronic metabolic, cardiovascular, and neurodegenerative disorders. Both the measureable economic impact on California’s health care system and the incalculable emotional suffering of affected individuals, their families and communities, make it an imperative to develop novel therapeutic treatments to address these mounting medical and economic societal challenges. Recognizing the potential utility of novel stem cell technologies to address California’s unmet medical needs, California voters approved Proposition 71 which created the California Institute for Regenerative Medicine (CIRM), an agency that administers funds to support stem cell research that has the greatest potential for development of novel regenerative medical treatments and cures. The CIRM Basic Biology Awards II program is intended to fund studies that will lay the foundation for future stem cell-based translational and clinical advances. In keeping with this mission, our proposed research program aims to discover new methods for producing human induced pluripotent stem cells (iPS cells) on an industrial scale and in an efficient manner; and to develop a better understanding of the mechanisms underlying cellular reprogramming. As such, our research program will help accelerate the realization of the full potential of iPS cells in cell-based regenerative medicine therapies and drug discovery. Our proposed research program will benefit the State of California and its citizens in several ways. Firstly, our research program will lay the foundation for future stem cell-based clinical and translational advances to treat and manage one of California’s most pressing unmet medical needs. Secondly, execution of our research program will create new jobs in the academic, biotechnology and pharmaceutical sectors throughout California. Funding from CIRM will be expanded with additional funding from the applicant to augment achievement of the aims of this project. CIRM funding will leverage other sources of investment in this project to help ensure California’s continued future as a world leader in biomedical innovation and translational medicine for the benefit of human health. Lastly, our proposed research program will stimulate California’s economy by creating new enabling tools and technologies that can be broadly adopted across the life science industry, thus promoting development across the academic institutions and biopharmaceutical companies that create biomedical discoveries and advances. These activities will continue to strengthen California’s leadership position at the forefront of the stem cell and regenerative medical revolution of the 21st century.
Progress Report: 

Year 1

iPS cell lines display high variability with respect to their growth properties, differentiation ability and disease phenotype manifestation; this is a major challenge for both in vitro disease modeling and drug screening, as well as cell replacement therapy. The cause for this variability is currently unknown and heterogeneity of the starting fibroblast population, composition of reprogramming factors, and viral integration into the genome have all been proposed to contribute to variability among different iPSC clones derived from the same starting population. In particular, the use of DNA viral vectors to deliver the reprogramming factors was suggested to be the key obstacle for eventual use of iPSCs in cell replacement therapies. Recently, several methods have been developed to derive integration-free iPS cells, but they mostly suffer from unacceptably low reprogramming efficiency or labor-intensive delivery of reprogramming factors. The goal of this work is to produce integration-free iPS cells with high efficiency. To date, we developed a high-throughput platform to screen small molecules that enhance reprogramming in the presence of 3 transcription factors required for reprogramming of fibroblast cells - KLF4, SOX2, and OCT4. These efforts included assay development, characterization of our engineered stable cell lines expressing reprogramming factors under control of an inducible promoter, and optimization for high-throughput screening. We screened over 100,000 compounds from our small molecule library, comprising compounds with broad chemical diversity and covering multiple cellular target classes. We identified 130 hit candidates in our primary screen, however, in follow-up assays none of these compounds showed an effect comparable with that of a positive control compound. Moving forward, we plan on detailed characterization of other integration-free reprogramming methods to determine the differences between integrating vs. non-integrating methods for reprogramming fibroblasts. We will assess the quality and variability of iPS cell lines, as well as their potential and variability of differentiation and disease phenotype manifestation in integration-free iPSCs, and compare them to DNA virus-derived iPSCs. Our results should contribute to the understanding of the source of variability between iPS cells and bring us closer to reaching the ultimate goal: production of integration-free human iPS cells with high efficiency.

Year 2

The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN’s derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutamate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. Importantly, these iPSC astrocytes can be directed to an anterior versus posterior (brain versus spinal cord) identity. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons, in order to identify the appropriate co-culture conditions where iPSC-derived neurons reach functional maturity as assessed by neuronal markers and electrophysiological activity. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific disease processes in neurodegeneration and neuroinflammation through the ability to combine in co-culture either disease versus healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem cell-based model systems to better replicate in vivo physiological and pathological processes.

Year 3

The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN's derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutumate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific diseases processes in neurodegeneration and neuroinflammation through the ability to combine co-culture either in disease vs. healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem-cell based model systems.

© 2013 California Institute for Regenerative Medicine