QUAKING KNOCK OUT INDUCES AN ALTERED PLURIPOTENT STATE WITHOUT COMPROMISING CARDIAC MESODERM COMMITMENT

COLLI, CAROLINA; GUADALUPE AMIN; FEDERICO SEVLEVER; MARIA AGUSTINA SCARAFIA; ALAN MIQUEAS MÖBBS; LUCIA NATALIA MORO; ARIEL WAISMAN; DAMIÁN LA GRECA, ALEJANDRO; MIRIUKA, SANTIAGO GABRIEL

Mar del Plata

Congreso; LXVII Reunion Anual de Sociedades de Biociencias; 2022

Sociedad Argentina de Investigacion Clinica (SAIC)

Circular RNAs (circRNA) were found to participate in the differentiation of human pluripotent stem cells (PSC). They originate from backspliced junctions during processing of pre-mRNAs, producingcovalently-closed stable molecules. Formation of circRNA depends on several factors, including the activity of the RNA binding protein Quaking (QKI). QKI5 increases during epithelial-to-mesenchymaltransition (EMT), and therefore it could be necessary for the first stages of cardiac differentiation. To test this, we aimed to generate and characterize QKI knock out (KO) lines in PSC. For generatingthe KO cells (QKI-KO) we targeted a genomic region comprising the promoter region and first exon of QKI gene using CRISPR, affecting expression of relevant isoforms (5/6/7). We corroborated the success of our strategy by PCR and Sanger sequencing on genomic DNA and by RTqPCR and Western blot to confirm the absence of QKI expression. Next, we studied the pluripotent state of QKI-KO cells by assessing their proliferation and differentiation capacities. We observed that, while wild type (WT) and KO cell lines were morphologically indistinguishable, QKI-KO cells showed an increase in cells in S phase compared to WT (51,3%±1.9 vs 41,1%±1.4), demonstrated by measuring EdU incorporation by flow cytometry. Differentiation to cardiac mesoderm was apparently not affected in QKI-KO cells as evaluated by expression analysis of EMT markers (EOMES, MIXL1, PDGFRα) 24, 48h and 72h after addition of CHIR. However, pluripotency marker genes (OCT4, SOX2 and NANOG) were not downregulated after CHIR addition, indicating that QKI-KO cells might have an altered exit from pluripotency. In summary, we successfully generated a KO cell line for QKI that evinces increased proliferation rates, sustained expression of pluripotent markers during early EMT and differentiates into cardiomyocytes. Further experiments will be directed to characterizing the different cardiac cell types obtained compared to WT.