Human pluripotent stem cells are increasingly used for cell-based regenerative therapies worldwide, with the use of embryonic and induced pluripotent stem cells as potential treatments for a range of debilitating and chronic conditions. However, with the level of chromosomal aneuploidies the cells may generate in culture, their safety for therapeutic use could be in question. This study aimed to develop sensitive and high-throughput assays for the detection and quantification of human pluripotent stem cell aneuploidies, to assess any changes in their positioning in nuclei, as well as investigate the possible roles of lamins in the accumulation of aneuploidies. Using Droplet Digital PCR™, we optimised the detection of aneuploid cells in a predominantly diploid background. An assay was established for the sensitive detection of up to 1% of mosaicism and was used for the monitoring of low-level chromosome copy number changes across different cell lines, conditions and passages in the human pluripotent stem cells. In addition, fluorescence in-situ hybridisation was used to map genes ALB and AMELX on chromosomes 4 and X, respectively, in karyotype-stable chromosome X aneuploid lymphoblastoid cell lines. Our results demonstrated significant alternations in the gene loci positioning in the chromosome X aneuploid cell lines. Using the same established method, the positioning of ALB and AMELX was monitored, alongside the genomic instability with ddPCR™, in the different human pluripotent stem cell lines, conditions and passage. We demonstrated a highly plastic nuclear organisation in the pluripotent stem cells with many changes occurring within a single passage. Furthermore, these results were not exclusive to a single cell line or condition, regardless of the presence or absence of feeder cells and of passage number, and the flexibility of the chromatin organisation remained throughout the duration of the study. We demonstrated high levels of genomic instability with recurrent gains and losses in the AMELX copy number in the human embryonic stem cells during the course of our study, however no significant changes in their gene loci positioning from these abnormalities were observed. xvi | P a g e Additionally, we observed reduced levels of lamin B2 in the aneuploid lymphoblastoid cell lines and complete loss in some hPSC samples. Our results support recent findings that suggest a link between lamin B2 loss and the formation of chromosome aneuploidies in cell culture. In conclusion, our data demonstrates several key novel findings. Firstly, we have established a sensitive technique for the detection of up to 1% mosaicism, which to our knowledge is the most sensitive assay currently available. Secondly, we showed significant changes in the gene loci positioning between aneuploid and diploid cell lines. Thirdly, utilising our novel ddPCR™ assay, we demonstrated the karyotypical instability of hPCSs with consistent gains and/or loses of gene copy numbers in a short period of time in culture. When studying the effects of different growth conditions, we showed that the karyotypical instability was not exclusive to a single condition or a combination of conditions, and what is more, the karyotypical abnormalities detected were not observed to change the gene positioning of hPSCs significantly, with the genome organisation remaining plastic. Finally, our results support a potential association of lamin B2 loss and karyotypical instability. We conclude that more sensitive and robust techniques need to be readily used by clinicians for the screening of potential therapeutic hPSCs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:765045 |
| Date | January 2018 |
| Creators | Henry, Marianne Patricia |
| Contributors | Bridger, J. ; Hawkins, R. |
| Publisher | Brunel University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://bura.brunel.ac.uk/handle/2438/17081 |
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