Investigating the molecular mechanisms underlying the role of mTORC2 in human embryonic stem cells
File(s)
Author(s)
Tong, Li
Type
Thesis
Abstract
Mechanistic target of rapamycin complex 2 (mTORC2), one of the two mTOR complexes, regulates cell proliferation, metabolism and cytoskeleton remodelling and has a crucial role in embryonic development. Loss-of-function studies show that mTORC2-deficient (Rictor- or Sin1-null) mouse embryos fail to complete their development and die around E11 due to defective vasculature. However, much less is known about the underlying mechanisms. In addition, it is unclear about the potential roles of mTORC2 in pluripotent stem cells (PSCs). To explore the role of mTORC2 in embryonic development and human embryonic stem cells (hESCs), RICTOR-knockout (RIC-KO) hESCs have been generated in our lab. RIC-KO hESCs retain their self-renewal, maintain expression of the core pluripotent markers, and express markers of three germ layers after embryoid body (EB) differentiation. However, their differentiation to mesoderm and endoderm lineages is reduced compared to wildtype (WT) controls.
In this study, I have further characterised RIC-KO hESCs and found that their colonies are less compact with reduced cell-cell interactions and altered cytoskeleton. RNA-seq analysis reveals that downregulated genes in RIC-KO hESCs are associated with mesoderm and endoderm differentiation. To investigate the role of mTORC2 in mesoderm and endoderm specification, I employ bone morphogenic protein-4 (BMP4)-induced differentiation and show that the mesendoderm (ME) lineage is largely impeded in RIC-KO cells compared to WT controls. Interestingly, forced activation of the WNT/b-catenin pathway by GSK3 inhibitor completely rescues their defect in ME differentiation, indicating that RIC-KO possibly affects BMP4- induced activation of the WNT signalling. Furthermore, I demonstrate that impaired ME differentiation in RIC-KO hESCs results from inadequate upregulation of the canonical WNT genes, which is associated with reduced cell-cell interactions. These findings suggest that RICTOR/mTORC2 is crucial in ME differentiation in hESCs through regulating cellular interaction-mediated activation of the WNT genes and demonstrate the importance of cell-cell interactions in BMP4-induced activation of the WNT genes.
In this study, I have further characterised RIC-KO hESCs and found that their colonies are less compact with reduced cell-cell interactions and altered cytoskeleton. RNA-seq analysis reveals that downregulated genes in RIC-KO hESCs are associated with mesoderm and endoderm differentiation. To investigate the role of mTORC2 in mesoderm and endoderm specification, I employ bone morphogenic protein-4 (BMP4)-induced differentiation and show that the mesendoderm (ME) lineage is largely impeded in RIC-KO cells compared to WT controls. Interestingly, forced activation of the WNT/b-catenin pathway by GSK3 inhibitor completely rescues their defect in ME differentiation, indicating that RIC-KO possibly affects BMP4- induced activation of the WNT signalling. Furthermore, I demonstrate that impaired ME differentiation in RIC-KO hESCs results from inadequate upregulation of the canonical WNT genes, which is associated with reduced cell-cell interactions. These findings suggest that RICTOR/mTORC2 is crucial in ME differentiation in hESCs through regulating cellular interaction-mediated activation of the WNT genes and demonstrate the importance of cell-cell interactions in BMP4-induced activation of the WNT genes.
Version
Open Access
Date Issued
2024-01-10
Date Awarded
01/06/2024
License URL
Advisor
Cui, Wei
Atanur, Santosh
Publisher Department
Department of Metabolism, Digestion and Reproduction
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)