Abstract
BackgroundCellular 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.MethodsHuman 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.ResultsWe 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.ConclusionsHigh 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0425-x) contains supplementary material, which is available to authorized users.
Highlights
Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries
Autophagy is a prominent feature of reprogramming cells and stable Induced pluripotent stem cell (iPSC) The autophagy–lysosome and ubiquitin–proteasome systems are the two major pathways that cells employ to selectively target long-lived and misfolded proteins, protein aggregates, damaged mitochondria, or unwanted cytoplasmic components and organelles [22]
Structures of endosomal/lysosomal nature with double membranes were clearly evident under high magnification, which were filled with an array of cell debris and organelles (Fig. 1j–l)
Summary
Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Induced pluripotent stem cell (iPSC) technology enables conversion of patients’ somatic cells into embryonic stem (ES)-like cells [1] which can be differentiated into the major cell types in the body, raising the expectation of personalized medicine to treat patients with their own somatic tissue-derived cells. This technology offers an opportunity to generate human cell models of diseases for therapeutic development, which has not kept pace with pharmaceutical investment in recent decades. There are challenges in phenotyping iPSCs prior to the development of screening assays, which include lengthy differentiation durations and heterogeneity of the end cell products
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