Fellows’ research: Insights into traffic regulation in embryonic stem cells and implications for medicine
15 Jan 2019
Dr Deepa Subramanyam, Intermediate Fellow
Our study looks into mechanisms that help embryonic stem cells, which are unspecialized cells derived from embryos, maintain their ability to become several different cell types i.e. pluripotency, through the process of endocytosis. Previously published studies have hinted at the involvement of endocytosis—an energy-dependent process by which molecules gain entry into the cell—in regulating the stemness of embryonic stem cells; however, a systematic study highlighting the roles of specific molecules has been lacking. Our study demonstrates that endocytosis mediated by a vesicle-forming protein called clathrin plays a critical role in this context.
Cell replacement therapy, which involves replacing diseased, dysfunctional cells with healthy, functioning ones, is hugely dependent on stem cells. Stem cells can be differentiated into specific cell types, which can then be transferred or transplanted into patients. To achieve proper and complete differentiation, it is critical to understand how certain factors can regulate the fate of stem cells. Understanding how cellular processes such as endocytosis play a role in regulating the pluripotency of embryonic stem cells is important towards safer and more efficient cell-based therapies.
In our recently published study, we hoped to identify molecules involved in the process of endocytosis that impact the pluripotency or stemness of embryonic stem cells. Previous work has shown that the expression of genes involved in endocytosis changes when differentiated adult cells are reprogrammed into embryonic stem-cell-like induced pluripotent stem cells. In our study, we set out to identify molecules that either facilitated the process of endocytosis in stem cells and/or molecules that were being actively endocytosed and played a critical role in maintaining the stemness and pluripotency of embryonic stem cells.
To identify these molecules, we undertook a small-scale siRNA-based screen, targeting molecules that are known to be involved in membrane trafficking. These siRNAs, which reduce the expression of genes they are specific for, were introduced into mouse embryonic stem cells, and the cells were screened using a marker for stem cells, alkaline phosphatase (AP). A decrease in the expression of AP upon introduction of a siRNA specific for a particular gene, suggests that the gene may be necessary to maintain the stem cell state. Conversely, an increase in AP expression upon introduction of a gene-specific siRNA indicates that this particular gene may play a role in reducing the stemness of these cells, an essential requirement during stem cell differentiation. Based on our observations, we selected the clathrin heavy chain gene (CLTC), and demonstrated through a series of follow-up experiments that this gene was essential to maintain the stemness of mouse embryonic stem cells (mESCs).
Our results demonstrate that clathrin-mediated endocytosis (CME) is essential to maintain the pluripotency of embryonic stem cells. Our results also demonstrated that CME is required for the internalization of E-cadherin, a cell-cell adhesion molecule that primarily connects epithelial cells together. Once E-cadherin is internalized, it is recycled back to the membrane. In the same cells, CME is responsible for the internalization and subsequent degradation of transforming growth factor beta receptor 1 (TGFBR1), a receptor that transmits signals to control a downstream signalling pathway that drives the differentiation of mESCs. Thus, CME is responsible for managing the internalization and subsequent trafficking of these two molecules, E-cadherin and TGFBR1. Any disruption in CME results in an imbalance between these two molecules, pushing the stem cell towards a more differentiated fate.
The cartoon shows molecules of E-CADHERIN (E-CAD) and TGFBR1 internalized in clathrin-coated vesicles, which are transported to the endosome. Here they undergo differential sorting, with E-CAD being recycled back to the membrane, while TGFBR1 is targeted for degradation. (Image credit: Vinay Subramanyam Rajan)
In summary, our study provides important molecular insights into stem cell differentiation, which has important applications in clinical settings involving stem cell-based therapies.
Clathrin-Mediated Endocytosis Regulates a Balance between Opposing Signals to Maintain the Pluripotent State of Embryonic Stem Cells. Yadavalli V. Narayana, Chetan Gadgil, Ridim D. Mote, Raghav Rajan, Deepa Subramanyam. Stem Cell Reports. January 2019.
Banner image credit: Human stem cell embedded in a 3D matrix, Cryo SEM. Credit: Sílvia A Ferreira, Cristina Lopo and Eileen Gentleman, KCL. CC BY (wellcomecollection.org)