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Durczak, Paulina Maria (2023) Deciphering the role of BMP4 signalling and gene regulation in the specification of human liver progenitor cells. PhD thesis, University of Nottingham.

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Abstract

Early hepatic specification and organogenesis can be modelled in vitro using human induced pluripotent stem cells (hiPSCs). These models apply differentiation protocols to direct hiPSCs through all the key developmental stages to accurately reflect in vivo development. Bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signalling are crucial for the specification of hepatic progenitors during early liver development. While the signalling cascades of these two morphogens are well characterized, the mechanisms by which they promote hepatic cell fate choice and hepatic gene expression in anterior foregut endoderm (FE) cells is not very well understood.

In this project, we characterize hiPSCs-based model of early liver development and apply it to understand the role of BMP signalling in hepatic specification. We confirm that BMP4 signalling is also necessary for liver progenitor cells (LPCs) specification from FE during hiPSCs differentiation. Using RNA sequencing (RNA seq.) we examine transcriptome changes induced by BMP4 during the transition from FE to LPC stage. Overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA) analysis revealed early activation of hepatocyte-specific functions such as lipid and protein homeostasis, haem metabolism or coagulation, while at the same time, cell adhesion and locomotion related genes are downregulated indicating preparation for cell migration out of the forming liver bud. We also notice upregulation of all four FGF receptors upon BMP signalling indicating at possible cross talk between the two pathways. The RNA seq. also detected a number of BMP4 upregulated transcription factors (TFs), several of these TFs are known for their roles in multiple developmental processes. Among them TBX3, previously reported to have a role in hepatic specification in mice, and two other TBX family members: TBX2 and TBX20. As a preliminary screen, we used a published, optimized protocol for creating inducible knockdown hiPSC lines to assess the importance of TBX and other TFs for the process of LPC specification. Double knockdown of TBX3 and TBX20 TFs significantly disrupted the hepatic induction process as shown by decreased expression of early hepatic genes such as TTR, AFP, AAT and ALB. Further studies are necessary to confirm and further characterize the role of TBX TFs for hepatic specification.

Our study demonstrates that application of hiPSCs derived models for the study of development can aid the understanding of molecular mechanisms driving early liver specification and improve our understanding of human embryology and organogenesis. This knowledge can also be used to created more efficient differentiation platforms that can yield more mature, functional and clinically relevant populations of hiPSC-derived hepatocytes.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Hannan, Nicholas R.F. Guruprasad, Aithal
Keywords:	Hepatic specification; Organogenesis; Human induced pluripotent stem cells; Hepatic progenitors; Liver development; Transcription factors; Hepatocytes
Subjects:	W Medicine and related subjects (NLM Classification) > WI Digestive system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	75725
Depositing User:	Durczak, Paulina
Date Deposited:	13 Dec 2023 04:40
Last Modified:	13 Dec 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/75725

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