Gene Expression Analysis of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Restricted; Files Only

Jeong, Woo (Fall 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/tt44pn88z?locale=es
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Abstract

Blood vessels play an essential role in transporting oxygen and nutrients to tissues, leading to tissue homeostasis. Cardiovascular ischemic diseases, such as peripheral artery disease, are highly linked with damaged and dysfunctional blood vessels. The damaged blood vessels restrict efficient blood supply to tissues, leading to shortages of oxygen and nutrients and ultimately to dysfunctional tissues. Human induced pluripotent stem cells (hiPSCs), which have an unlimited proliferation capacity to differentiate into any type of somatic cells without ethical issues, were cultured under a fully defined and clinically compatible system to differentiate hiPSCs into endothelial cells (ECs). The resultant hiPSC-derived ECs (hiPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic properties. However, the gene expression profile that facilitates endothelial and proangiogenic characteristics has been only partially explored. To expand understanding, we used RNA sequencing to identify differentially expressed (DE) genes and enriched pathways that significantly contribute to genuine EC features and proangiogenic attributes. Total RNA of hiPSC-ECs were marked at 6 EC development timepoints: Day0, Day2, Day4, Day8, Day14 before sorting, and Day14 after sorting; we compared all the timepoints to Day0. We discovered that gene ontology (GO) terms for biological processes were enriched in hiPSC-ECs in EC differentiation, EC proliferation, EC migration, and positive regulation of angiogenesis. Furthermore, we identified 28 DE genes among 1252 DE genes that significantly contribute to the endothelial and proangiogenic characteristics by comparing hiPSC-ECs (Day14 after sorting) to hiPSC (Day0). The results provide new insight into a transcriptomic understanding of hiPSC-ECs, and the identified DE genes may serve as therapeutic markers of hiPSC-ECs. 

Table of Contents

Introduction…………………………………………………......…………………………….…........................……..1

Methods………………………………………………………………….....……………….…….................................3

Results……………………………………………………………………………......…………..........................……..10

            Understanding of general workflow of RNA-seq data………………..…..........…………….............10

            Quality control of RNA-seq data………………………………..........…............................................12

            Detection of potential sample outliers and the sources of variation………………………............13

            Application of DESeq2 generalized linear model…...................................................................15

            Summarization of DE analysis…………..........……………............................................................17

            Detection of relative expression levels of DE genes..………………………….............….………......18

            Distribution of DE genes during EC differentiation and enrichment........................................20

            Identification of enriched GO terms……………………………...........................................……..…21 

            Identification of DE genes with respect to biological and statistical significance….............….23

            Process to distinguish notable genes......................................……………………………….........…25

            Determination of meaningful genes……………………….............................................................35

            Confirmation of enriched endothelial and proangiogenic properties over time……..............…36

Discussion…………………………………………………………………………………...................................…....37

Conclusion.................……………………………………………………………………...............................……...40

References……………………………………………………………………………………..................................…..41

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