Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo. METHODS: In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts. RESULTS: We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs. CONCLUSION: The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

Apurinic/apyrimidinic endonuclease 1 (APE1) is dispensable for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation in the immunoglobulin gene.

Activation-induced cytidine deaminase (AID) initiates DNA breakage in the variable (V) and switch (S) regions of the immunoglobulin gene, which results in somatic hypermutation (SHM) and class switch recombination (CSR), respectively. Apurinic/apyrimidinic endonuclease 1 (APE1) has been shown to be important for CSR, and is supposed to cleave at abasic sites when AID-dependently deaminated cytidine is removed by uracil DNA glycosylase. However, APE1 is unexpectedly dispensable for SHM in the S region and translocation between immunoglobulin heavy chain (IgH) and c-myc genes in the mouse B lymphoma cell line, CH12F3-2A. This suggested that APE1 is not involved in AID-dependent DNA breakage, but rather, in DNA repair. In order to investigate detailed molecular mechanisms underlying APE1's involvement in CSR and SHM, we measured apurinic/apyrimidinic (AP) sites via aldehyde reactive probe labeling. Results indicated that the frequencies of AP sites in the S regions were not different between APE1-/-/-CH12F3-2A and wild-type CH12F3-2A cells. To carry out similar experiments in SHM of the V region, we generated an APE1 knockout (APE1-/-) human Burkitt's lymphoma cell line, and compared SHM between APE1-proficient and -deficient BL2 lymphoma cells. SHM frequencies in the V regions of APE1-/-BL2 and APE1-proficient cells were also similar. Taken together, we showed that AID does not induce AP sites in the S region of the IgH gene, and that APE1 is not necessary for SHM in the V and S regions; however, it is required for DNA repair following DNA breakage in CSR.