Determining the Signalling Pathways that Govern Human Naive Pluripotency

Conventional or “primed” human embryonic stem cells (hESCs) rely on FGF and TGFβ signalling for self-renewal, and occupy a developmentally advanced state of pluripotency comparable to mouse EpiSCs. Recent reports demonstrate that a naïve state of human pluripotency can be consistently derived either through transient histone deacetylase inhibition mediated resetting of conventional hESCs or via isolation of the inner cell mass. Long-term propagation of this state can be achieved using a cocktail of MEK, GSK3 and PKC inhibition in conjunction with leukaemia inhibitory factor (LIF) supplementation (t2iLGö) and a feeder layer of inactivated mouse embryonic fibroblasts. However, the way in which this signalling environment is interpreted in order to maintain naïve pluripotency remains unclear. I demonstrate a substrate consisting of a high concentration of tissue-derived laminin in combination with t2iLGö is sufficient to replace the feeder layer. Cultures maintained under these conditions are karyotypically normal, maintain a naive pluripotent transcriptional profile and exhibit reduced aberrant expression of mesodermal and endodermal lineage markers. I utilise the increased stringency of this culture system in combination with small molecule inhibitors to examine the roles of FGF, Activin/Nodal and JAK/STAT signalling in human naïve pluripotency. Naïve hESCs proliferate and maintain pluripotency marker expression in the presence of FGF receptor inhibition. In contrast, TGFβ signalling inhibition leads to rapid downregulation of human specific naïve pluripotency marker, KLF17, followed by the eventual collapse of the naïve transcription factor circuitry. Naïve hESCs self-renew in both the absence of LIF and presence of JAK/STAT inhibitors. However, further investigation of JAK/STAT signalling identified the increased potency of Interleukin 6 (IL-6) over LIF to activate the JAK/STAT pathway. Supplemental IL-6 improves colony-forming capacity under self-renewing conditions and attenuates differentiation following inhibitor withdrawal. Furthermore, prolonged activation of IL-6 signalling suppresses expression of GATA2 and GATA3 and upregulates KLF4 transcripts. Finally, I investigate whether ablation of PKCι is sufficient to replace the activity of the PKC inhibitor, Gö6983. Established naïve cultures that are PKCι null continue to express naïve markers and suppress upregulation of lineage makers following withdrawal of Gö6983. Furthermore, ablation of PKCι in conventional ESCs enables the maintenance of NANOG expression and the emergence of KLF17 expression in the absence of Gö6983 during histone deactylase mediated resetting.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2018

Author dc:creator

• Myers, Samuel Philip

Advisor dc:contributor.advisor

• Smith, Austin

• Human
• Naive
• Pluripotency
• Signalling

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2ef789d8-249e-4053-a5b2-b0ef0d9397a9/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

Author Identifier

0000-0003-4019-9184

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/277414