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

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. Individua...

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Main Author: Tan, Wen Ting
Other Authors: Irene Gallego Romero
Format: Final Year Project
Language:English
Published: 2017
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Online Access:http://hdl.handle.net/10356/70538
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-705382023-02-28T18:03:23Z Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude Tan, Wen Ting Irene Gallego Romero School of Biological Sciences DRNTU::Science 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. Bachelor of Science in Biological Sciences 2017-04-27T04:44:49Z 2017-04-27T04:44:49Z 2017 Final Year Project (FYP) http://hdl.handle.net/10356/70538 en Nanyang Technological University 47 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science
spellingShingle DRNTU::Science
Tan, Wen Ting
Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
description 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.
author2 Irene Gallego Romero
author_facet Irene Gallego Romero
Tan, Wen Ting
format Final Year Project
author Tan, Wen Ting
author_sort Tan, Wen Ting
title Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
title_short Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
title_full Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
title_fullStr Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
title_full_unstemmed Establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
title_sort establishing a panel of human pluripotent stem cell lines from individuals adapted to high altitude
publishDate 2017
url http://hdl.handle.net/10356/70538
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