Understanding and exploiting naïve human pluripotency

Thesis

Naïve human pluripotent stem cells (hPSC) represent an earlier stage in human development compared to conventional “primed” hPSC. They resemble the inner cell mass of an embryonic day (E) 5 human blastocyst and are considered developmentally equivalent to mouse embryonic stem cells (mESC). Access to this new cell type facilitates interrogation of early human development. In this thesis, I have shown that the Theunissen et al. protocol for generating naïve hPSC yields cells that most closely approximate the human E5 inner cell mass and is robustly reproducible across several induced pluripotent stem cell lines. miRNAs are essential in mouse embryogenesis for differentiation from the naïve to the primed state, but the miRNAome of the human E5 inner cell mass has yet to be determined. I have established the naïve to primed differentiation and identified the miRNAome of naïve, differentiating and primed human pluripotent stem cells by miRNAseq. This novel dataset has revealed that the largest difference in the miRNAome between mouse and the human is due to cell state rather than species. The mouse miR-290 family are the most highly expressed miRNAs in mESC and the absence of the human equivalent homologs miR- 371-373 in conventional hPSC was thought to be a difference in species. Using the miRNAseq dataset, I have shown that miR-371-373 are highly upregulated in naïve hPSC, confirming that naïve hPSC more closely resemble mESC. I also observed high expression of miR-182-5p, which downregulates the homology directed repair (HDR) pathway. This surprising finding contrasts with previous publications which suggest that naïve hPSC genomes are more efficiently manipulated by HDR. In support of this finding, I show that naïve hPSC repair Cas9 induced double strand breaks less often by HDR than primed hPSC and demonstrate that a higher proportion of naïve hPSC are in G1 phase of the cell cycle which is likely a contributor to the lower rates of HDR.

Authors: Dodsworth, B

• DOI: 10.5287/ora-pv9k8p0yk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English