Numerous in vitro models endeavour to mimic the characteristics of primary human hepatocytes
for applications in regenerative medicine and pharmaceutical science. Mature hepatocyte-like cells
(HLCs) derived from human induced pluripotent stem cells (hiPSCs) are one such in vitro model. Due
to insufficiencies in transcriptome to proteome correlation, characterising the proteome of HLCs is
essential to provide a suitable framework for their continual optimization. Here we interrogated the
proteome during stepwise differentiation of hiPSCs into HLCs over 40 days. Whole cell protein lysates
were collected and analysed using stabled isotope labelled mass spectrometry based proteomics.
Quantitative proteomics identified over 6,000 proteins in duplicate multiplexed labelling experiments
across two different time course series. Inductive cues in differentiation promoted sequential
acquisition of hepatocyte specific markers. Analysis of proteins classically assigned as hepatic markers
demonstrated trends towards maximum relative abundance between differentiation day 30 and 32.
Characterisation of abundant proteins in whole cells provided evidence of the time dependent transition
towards proteins corresponding with the functional repertoire of the liver. This data highlights how
far the proteome of undifferentiated precursors have progressed to acquire a hepatic phenotype and
constructs a platform for optimisation and improved maturation of HLC differentiation.