Date of Completion

7-28-2015

Embargo Period

7-26-2016

Keywords

Stem Cell, Reprogramming, Genetics, Transcriptional Model

Major Advisor

Craig E. Nelson

Associate Advisor

David J. Goldhamer

Associate Advisor

Xiuchun (Cindy) Tian

Associate Advisor

Charles Giardina

Associate Advisor

Barbara Mellone

Field of Study

Genetics and Genomics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Forced ectopic expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC (OSKM) can directly reprogram various somatic cells into induced pluripotent stem cells (iPSCs). These reprogrammed cells offer great potential as a source for patient-matched regenerative therapies thanks to their striking molecular and phenotypic similarity to embryonic stem cells. However, despite years of research, this process remains highly inefficient and produces considerable cellular heterogeneity. Moreover, long latency has stalled the effort to understand the mechanisms and molecular changes underlying the reprogramming process. To improve and facilitate the development of efficient and rapid reprogramming strategies, a clear understanding of fundamental reprogramming mechanisms is essential.

In this work, we use single-cell transcript profiling, fluorescence-activated cell sorting (FACS), and mathematical modeling to provide a precise mathematical framework describing the dynamics of pluripotency gene expression during reprogramming by OSKM. Additionally, we generated a reprogramming progression axis that precisely measures the progression of individual cells towards pluripotency. We found that the stochastic phase of reprogramming is an ordered probabilistic process with independent gene-specific dynamics. Furthermore, we demonstrated that polycistronic viral (OSKM) delivery produces significantly higher reprogramming efficiencies as compared to monocistronic delivery, due to premature inactivation of the individual O, S, K, or M vectors in the monocistronic method. Finally, we show that the order of gene activation is similar in two fibroblast cell types, MRC-5 and BJ, and that these two cell types take divergent paths upon reprogramming factor induction, followed by convergence later in the reprogramming process.

The results of our work emphasize the important value of precise mathematical modeling and of the reprogramming progression axis in understanding fundamental reprogramming mechanisms. This work lays the foundation for the measurement and mechanistic dissection of treatments that enhance the rate or efficiency of reprogramming to pluripotency.

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