Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude

• Main Author: Tan, Wen Ting
• Other Authors: Irene Gallego Romero
• Format: Final Year Project
• Language: English
• Published: 2017
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/70538
• Institution: Nanyang Technological University

Due to the nature of human evolutionary and adaptation studies, access to cells and tissues have been largely limited. The use of induced pluripotent stem cells (iPSCs) as a renewable source of cells allows us to cross this hurdle and explore the possibilities of more sustainable research. Individuals adapted to high altitudes shared a region of the Denisovan haplotype in their genome and EPAS1 downregulation was proposed as the molecular basis for their lower-than-expected haemoglobin level. To facilitate the validation of this hypothesis, in this project, we attempted to reprogram seven lymphoblastoid cell lines (LCLs) into iPSCs by introducing integration-free episomal vectors encoding reprogramming factors through electroporation. Four reprogramming protocols with varying media changes schedules were tested for optimisation and we observed that the length of recovery period after electroporation, electroporation density, plating density and electroporation conditions were all determining factors of reprogramming efficiency. We derived optimal reprogramming conditions: a post-electroporation recovery period in LCL growth media of at least 72 hours; electroporation using one pulse of current at 1350V for 30 milliseconds; using 300,000 to 500,000 cells for electroporation and successfully established four iPSC lines using our optimised version of the Stem Cell Technologies Protocol.