Rapamycin Regulates Autophagy and Cell Adhesion in Induced Pluripotent Stem Cells

Tilo Kunath, Areechun Sotthibundhul, Katya McDonagh, Alex Von Kriegsheim, Amaya Garcia-Munoz, Agnieszka Klawiter, Kerry Thompson, Kapil Dev Chauhan, Janusz Krawczyk, Veronica McInerney, Peter Dockery, Michael J Devine, Frank Barry, Timothy O'Brien, Sanbing Shen

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features, produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined.
Methods: Human iPSCs were investigated by microscopy, immunofluorescence and immunoblotting to detect autophagy machinery. Cells were treated with Rapamycin to activate autophagy and Bafilomycin to block autophagy during iPSC maintenance. High concentrations of Rapamycin treatment, unexpectedly, resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with Rapamycin treatment.
Results: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5 and LC3B. Block of autophagy by Bafilomycin induces iPSC death and Rapamycin attenuates the Bafilomycin effect. Rapamycin treatment up-regulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of Rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of Rapamycin in mediating iPSC detachment and differentiation.
Conclusions: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. Therefore, this research highlights the potential of Rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.
Original languageEnglish
JournalStem Cell Research and Therapy
Volume7
Issue number166
DOIs
Publication statusPublished - 15 Nov 2016

Keywords

  • Actin Cytoskeleton
  • Adherens junctions
  • Autophagy
  • Differentiation
  • Embryoid Body
  • Induced Pluripotent Stem Cells
  • Rapamycin

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