title: Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells.
abstract: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined.
  
  
  
  Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with 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.
  
  
  
  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 upregulates 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.
  
  
  
  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 a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.|Chulabhorn International College of Medicine, Thammasat University, Patumthani, 12120, Thailand.
authors.forename: Areechun
authors.initials: A
authors.lastname: Sotthibundhu
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Katya
authors.initials: K
authors.lastname: McDonagh
authors.affiliation: Systems Biology Ireland, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Alexander
authors.initials: A
authors.lastname: von Kriegsheim
authors.affiliation: Systems Biology Ireland, Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Amaya
authors.initials: A
authors.lastname: Garcia-Munoz
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Klawiter
authors.affiliation: Centre for Microscopy and Imaging, Anatomy, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Kerry
authors.initials: K
authors.lastname: Thompson
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Kapil Dev
authors.initials: KD
authors.lastname: Chauhan
authors.affiliation: Department of Haematology, Galway University Hospital, Galway, Ireland.
authors.forename: Janusz
authors.initials: J
authors.lastname: Krawczyk
authors.affiliation: HRB Clinical Research Facility, National University of Ireland Galway, University Road, Galway, Ireland.
authors.forename: Veronica
authors.initials: V
authors.lastname: McInerney
authors.affiliation: Centre for Microscopy and Imaging, Anatomy, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Peter
authors.initials: P
authors.lastname: Dockery
authors.affiliation: MRC Center for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK.|Department of Molecular Neuroscience, Institute of Neurology, University College London, London, WC1N 3BG, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Devine
authors.affiliation: MRC Center for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK.
authors.forename: Tilo
authors.initials: T
authors.lastname: Kunath
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Frank
authors.initials: F
authors.lastname: Barry
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Brien
authors.affiliation: Regenerative Medicine Institute, School of Medicine, National University of Ireland Galway, Galway, Ireland. sanbing.shen@nuigalway.ie.
authors.forename: Sanbing
authors.initials: S
authors.lastname: Shen
mesh_terms: D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000187:drug effects / Q000502:physiology; D058732:Embryoid Bodies / Q000187:drug effects / Q000502:physiology; D005855:Germ Layers / Q000187:drug effects / Q000502:physiology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D020123:Sirolimus
keywords: Actin cytoskeleton; Adherens junctions; Autophagy; Differentiation; Embryoid body; Induced pluripotent stem cells; Rapamycin