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.
- Actin Cytoskeleton
- Adherens junctions
- Embryoid Body
- Induced Pluripotent Stem Cells