Flatt, Luke
(2017)
Developing an automation-compatible quantitative and functional assay for the assessment of pluripotency.
PhD thesis, University of Nottingham.
Abstract
Many of the protocols utilized in human pluripotent cell (hPSC) culture remain far from standardized from laboratory to laboratory. Lack of standardization and the knock-on effects of variable culture systems can have dramatic effects on the genetic and epigenetic status of in vitro cultured pluripotent stem cells (Miura et al, 2009; Kruta et al, 2014). Moves to create more defined culture systems have addressed matrices, culture medium (Chen et al, 2011) and dissociation agents (Terstegge et al, 2007), all with the aim of refining the protocols utilized whilst maintaining pluripotency. Similar refinement in differentiation approaches through the removal of serum, or other xenogeneic products, and utilization of chemically synthesized small molecule compounds also became an area of focus in recent years.
Teratoma formation remains to be considered as the gold standard for the assessment of pluripotency (Wesselschmidt, 2011), with transcriptomic protocols becoming more frequently utilized for assessment and characterization of the pluripotent state (Bock et al, 2011). This project aimed to investigate the potential to produce a culture system, with reduced variability, and a directed differentiation protocol to each of the three embryonic germ layers, as a means of assessing the Functional Pluripotency of cultured hPSCs.
An initial culture system of mouse embryonic fibroblast-conditioned medium (MEF-CM) on Matrigel (Denning et al, 2007) was investigated to move towards chemically defined Essential8 (E8) medium and dissociation process. Initial manual experimentation demonstrated the maintenance of a stable karyotype, expression of pluripotency transcription (OCT4, SOX2 and NANOG) and cell surface antigens (SSEA4, TRA-1-60) using the defined system. To further reduce variability associated with culture, the TECAN Freedom Evo 200 automated culture platform was investigated for the potential to translate the E8 and TrypLE method to be automation compatible. Cells were stably maintained over a 13 passage period with no gain or loss of chromosomal abnormalities induced, and maintenance of pluripotency marker expression. Furthermore, 96 well plate seeding at a set density, for later use in differentiation, was demonstrated as being made more reproducible using the automated platform over manual approaches. Finally, it was demonstrated that a Matrigel substrate could be replaced with the fully defined, truncated Vitronectin N-terminal peptide, in both manual and automated processes.
The remainder of the project aimed to utilize the automated culture system developed, to optimize differentiation protocols towards each of the three embryonic germ layers; ectoderm, endoderm and mesoderm, in 96 well plate format. Replacement of growth factors for differentiation ensured that batch-variability, and cost, reduction could be attempted. Efficiency of differentiation targets of, 60 % dual-positive for lineage-specific transcription factors, were set as a minimal criterion for use in a subsequent tri-lineage differentiation assay.
To further reduce cost it was advantageous to identify early time-points during differentiation when a specific lineage could be resolved. Optimization using statistically powerful, design of experiments, approaches were applied for the ectoderm and endoderm lineages to improve lower than desired differentiation efficiencies. Overall, following optimization of density/dosage/start-point variables, efficiencies in hPSC lines cultured in E8 on the automated platform of greater than 60 % were produced for each of the three germ layers.
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