Autophagy in Human Embryonic Stem Cells

Abstract

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

The role of protein clearance mechanisms in organismal ageing and age-related diseases

“…Notably, the degradation of damaged proteins is triggered on the first signs of mESC differentiation via induction of PA28ab 69,70 . Furthermore, both ESCs exhibit higher autophagy activity on early differentiation 71 . In addition, induced pluripotent stem cells generated from patients with PD show more autophagic vacuoles when differentiated into dopaminergic neurons 72 suggesting an active rejuvenation step to generate a pool of 'healthy' cells.…”

At the Intersection of the Pathways for Exocytosis and Autophagy

“…Exocytosis and autophagy are essential for a number of common biological processes, including; the immune response (Govind, 2008;Minty et al, 1983;Murray et al, 1998;Ostenson et al, 2006), cell growth (Brennwald & Rossi, 2007;Orlando & Guo, 2009;Wei & Zheng, 2011;Zhang et al, 2005), cell proliferation and apoptosis (Kundu, 2011;Shin et al, 2011;Zeng et al, 2012), and multicellular organism development (Gutnick et al, 2011;Hu et al, 2011;Sato & Sato, 2011;Tra et al, 2011). Autophagy and exocytosis both involve membrane trafficking and fusion events and so similar groups of molecular machinery may be required for both processes: such as GTPase proteins, that facilitate membrane tethering and SNARE proteins which are involved in membrane fusion.…”

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection

“…The usual number of autophagosomes seen by 3D animation was 100 per infected cell, but sometimes approached 200 per cell. In contrast, a typical nonstressed cell usually has fewer than 4 autophagosomes (3,4). When monolayers were inoculated with VZV-infected cells, the traditional method for VZV infection, autophagy was again easily seen after enumeration of autophagosomes and immunoblotting for the LC3-phosphatidylethanolamine conjugate (LC3-II).…”