Abstract:

Objective To verify the existence of potential new receptor of ELABELA (ELA) in human embryonic stem cells (hESC) by Crispr/Cas9-mediated gene editing system. Methods The dynamic expression levels of ELA and its receptor APJ were examined by collecting cells at different days (0?13 d) during the directed differentiation of stem cells into cardiomyocytes. The mRNA expression levels of cardiomyocyte markers MYH6, TnnT2 and NKX2.5 were observed by adding the APJ inhibitor ML221 during the directed differentiation of stem cells to cardiomyocytes. Human embryonic kidney suspension cells (HEK-293F) were used to express recombinant ELA green fluorescent protein (GFP). To verify whether ELA binds through its C-terminus or N-terminus, two recombinant plasmids with GFP sequences which were inserted into the C-terminus or the N-terminus of ELA-32 sequence were constructed. The only known receptor for ELA, APJ-knockout hESC, was constructed and validated by using Crispr/Cas 9 gene editing technology. Two GFP-ELA recombinant proteins were added exogenously to the successfully constructed knockout cell lines and normal cell lines, and the ability of ELA to enter the cells was observed by fluorescence microscopy. Results During the stem cell period, ELA was highly expressed while APJ was basically not expressed; the peak of ELA expression was at the 6th day of differentiation while the peak of APJ expression appeared at the 3rd day of differentiation. The efficiency of stem cell directed differentiation to cardiomyocytes was not affected by exogenous addition of the APJ inhibitor ML221 (P>0.05). After DNA sequencing and Western blotting validation, the APJ-knockdown stem cell line was successfully constructed. When APJ was knocked down, exogenous addition of ELA N-terminal fluorescent protein was still able to enter the cells, while the absence of fluorescence of ELA C-terminal fluorescent protein indicated that ELA C-terminal fluorescent protein could not enter the cells. Conclusion There are other ELA receptors in hESCs that are not APJ. Otherwise, the binding sites of ELA and its receptor are mainly located at the C-terminal of ELA.

Key words: human embryonic stem cell (hESC), ELABELA, APJ, gene editing