Abstract Body: Background: Human induced pluripotent stem cells (hiPSCs) and their derived cardiomyocytes (hiPSC-CMs) have been used for cell-based therapies for myocardial regeneration. Underlying paracrine signals derived from these hiPSC-CMs have been shown to play a critical role in improving the function of the ischemic myocardium. However, characterization of the transplanted cell proteome in vivo has been challenging due to the lack of tools to distinguish cell-specific proteome in a complex multicellular microenvironment.
Methods: To establish cell-specific proteome labelling, we adopted the biorthogonal non-canonical amino acid tagging (BONCAT) system in hiPSCs. We expressed the mutant mouse tRNA synthetase gene, “L274GmMetRS” in hiPSCs (L274G-hiPSCs) using lentiviral transduction to enable incorporation of the non-canonical amino acid azidonorleucine (Anl) in these cells. We assessed the pluripotency and trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo (teratoma formation assay). Furthermore, we validated the cell-specific Anl incorporation in the L274G-hiPSC-CMs both in vitro (co-culture) and in vivo (post-transplantation).
Results: Our studies showed that the L274G-hiPSCs could efficiently differentiate into all the three germ lineages and can be used to track the cell-specific proteome in their differentiated progenies, including L274G-hiPSC-CMs. Furthermore, immunostaining and western blots showed cell-specific BONCAT in L274G-hiPSC-CMs both in co-cultures (in vitro) and post-transplantation (in vivo).
Conclusion: The newly-established L274G-hiPSC line can be used to track cell-specific proteome of hiPSC-CMs in vitro and in vivo, to characterize cell-specific proteomic responses and delineate mechanisms underlying cell therapies.