Single-cell analyses to decipher cell fate reprogramming

• Main Author: Xing, Qiaorui
• Other Authors: Koh Cheng Gee
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/138344
• Institution: Nanyang Technological University

Cellular reprogramming suffers from low efficiencies, resulting in heterogeneous populations. Therefore, ensemble measurements of the mixed population often mask the rare cells which are on the route to successful reprogramming. To de-convolute the heterogeneity, I have presented the single-cell roadmap of the human cellular reprogramming process, through time-course analysis of scRNA-Seq and scATAC-Seq libraries. From the scRNA-Seq analysis, sub-populations with varied stemness potentials were identified for the reprogramming cells of various stages. Molecular signatures specific to each sub-population were characterized. Moreover, a panel of surface markers was developed to enrich for the intermediate cells with diverse cell-fate trajectories and reprogramming propensities. On the other hand, scATAC-Seq analysis revealed the stage-specific TF regulatory networks of reprogramming. The activity of some TFs, such as FOS-JUN-AP-1 family, diminished, whereas TFs belonging to the family of TEAD, OCT, KLF, SP, FOX, YY2, and SRY were responsible for the opening of chromatin during the reprogramming. Interestingly, lineage related TFs, including GATA-TAL, SOX, NRL, PAX6, HNF4G, and RXR family, were enriched transiently in the intermediary stages of reprogramming. Importantly, FOS-JUN and TEAD4 were found to be the most variable TFs contributing to the heterogeneity of D8 cells. Furthermore, the crucial switch from a FOSL1- to a TEAD4-centric expression was described, which collectively regulates genomic accessibility, the cell-lineage transcription program, and the network of functional downstream modulators favoring the acquisition of the pluripotent state. Altogether, this study illuminates the multitude of diverse routes transversed by the individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic parts-list of the reprogramming machinery.