C19MC microRNA cluster regulates the transition to a gastrulation-like stage in a model of human early development using human pluripotent stem cells

ALAN MIQUEAS MÖBBS; XIMENA GARATE; MARIA AGUSTINA SCARAFIA; SHEILA LUCIA CASTAÑEDA; NATALIA LUCÍA SANTÍN VELAZQUE; DAIANA MARTIRE GRECCO; FRANCIA MARCOS; MARIA SOLEDAD CONSENTINO; ARIEL WAISMAN; GUSTAVO EMILIO SEVLEVER; ALEJANDRA GUBERMAN; CARLOS DANIEL LUZZANI; LUCIA NATALIA MORO; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Virtual

Congreso; LXV Reunion Anual de Sociedades de Biociencias; 2020

Sociedad Argentina de Investigacion Clinica (SAIC)

Human pluripotent stem cells (hPSC) have the capacity to self-renew and differentiate in vitro into all the cell types of the organism. We previously described the miRNome during the in vitro cardiac differentiation od hPCS. It revealed a yet unexplored group of 56 microRNAs transcribed during pluripotency and whose expression decayed during the early mesoderm stage. These microRNAs are clustered ina primate-specific 100 kb sequence at chromosome 19, hence known as C19MC. To ascertain a possible role of this cluster during early human embryogenesis, we generated a C19MC-defficient human induced pluripotent stem cell line using CRISPR/Cas9. When maintained under pluripotency conditions, these cells displayed no evident changes in the cell cycle, apoptosis, or differentiation markers compared to control cells. However, this line was drastically impaired to differentiate into cardiac and endothelial lineages, evaluated both by RT-qPCR, flow cytometry and a total lack of contractile cardiomyocytes. Since cardiac and endothelial lineages share a common mesodermal progenitor, we evaluated different markers, and found that early mesodermal genes MESP1/2, TBX6 and EOMES were downregulated compared to wild type cells, while the early ectoderm marker SOX2 was upregulated. Since the balance between TBX6 and SOX2 has been reported as critical for gastrulation, we next explored the genome-wide transcriptional changes by performing an RNA-seq at 0h and 24h of cardiac induction in the parental and C19MC line, a period that in vitro recapitulates gastrulation. Gene ontology analysis showed a global dysregulation of the differentiation pathways ERK/FGF, VEGF, NODAL, as well as focal adhesion related genes. We then performed an Ago-IP RNAseq experiment in pluripotency conditions with miR-520a over-expression and found an enrichment of WNT, FGF, and NOTCH signaling pathway genes. Thus, we show that the C19MC miRNA cluster has a critical role during early mesodermal specification.