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

MÖBBS AM, GARATE X, SCARAFÍA MA, CASTAÑEDA S, SANTINVELAZQUE NL, MÁRTIRE GRECO D, FRANCIA MG, CONSENTINO MS, WAISMAN A, SEVLEVER GE, GUBERMAN A, LUZZANI CD,MORO LN, LA GRECA AD, MIRIUKA SG

Congreso; SAIC 2020; 2020

Human pluripotent stem cells (hPSC) have the capacity to self-renewand differentiate in vitro into all the cell types of the organism.We previously described the miRNome during the in vitro cardiacdifferentiation of hPSCs. It revealed a yet unexplored group of 56microRNAs transcribed during pluripotency and whose expressiondecayed during the early mesoderm stage. These microRNAs areclustered in a primate-specific 100 kb sequence at chromosome 19,hence known as C19MC. To ascertain a possible role of this clusterduring early human embryogenesis, we generated a C19MC-deficienthuman induced pluripotent stem cell line using CRISPR/Cas9. When maintained under pluripotency conditions, these cellsdisplayed no evident changes in the cell cycle, apoptosis, or differentiationmarkers compared to control cells. However, this line wasdrastically impaired to differentiate into cardiac and endothelial lineages,evaluated both by RT-qPCR, flow cytometry and a total lackof contractile cardiomyocytes. Since cardiac and endothelial lineagesshare a common mesodermal progenitor, we evaluated differentmarkers, and found that early mesodermal genes MESP1/2, TBX6and EOMES were downregulated compared to wild type cells, whilethe early ectoderm marker SOX2 was upregulated. Since the balancebetween TBX6 and SOX2 has been reported as critical for gastrulation,we next explored the genome-wide transcriptional changesby performing an RNA-seq at 0h and 24h of cardiac induction in theparental and C19MC line, a period that in vitro recapitulates gastrulation.Gene ontology analysis showed a global dysregulation of thedifferentiation pathways ERK/FGF, VEGF, NODAL, as well as focaladhesion related genes. We then performed an Ago-IP RNAseq experimentin pluripotency conditions with miR-520a over-expressionand found an enrichment of WNT, FGF, and NOTCH signaling pathwaygenes. Thus, we show that the C19MC miRNA cluster has acritical role during early mesodermal specification.