CONICET | Buscador de Institutos y Recursos Humanos

Pluripotent stem cells (PSC) are a model to study embryologicaldevelopment and also have the potential to provide a supplyof different cell types for tissue replacement and drug screening.Owing to this, increasing efficiency of differentiation protocols is ofvital importance. The role of ECM in cell adhesion and signaling tocells through receptors such as integrins has received much attention.Previous publications showed that specific integrins and theirligands such as fibronectin, laminins are responsible of the cellularfate. Our goal is to find out what role integrins (α3, α5 and α6) areplaying during cardiac differentiation. It will be achieved through theCRISPR-effector system where we can specifically and reversiblyinhibit gene expression allowing us to understand the contributionof integrins to cardiac development. A new specific protocol wasdeveloped in our lab and it has 80% of efficiency. We performedthe integrin expression profile on days d0, d3.5, d7.5 and d15 atboth levels: mRNA and proteins. Through qPCR, we observed thatα5 has its peak at d3.5 where increases 10 times compared to d0and its main ligand (Fibronectin) has a d7.5 peak, 50 folds higher.Integrins α3 and α6, whose ligand is laminin, show an oppositebehavior: α6 decrease an 80% at d3.5 while α3 increase 8 folds atd15. By citometry, we got a similar profile to mRNA expression andeach mesodermal or cardiac population is correlated with specificpopulation markers. All this data together proves there is a regulationalong differentiation and they change significantly on differentcell states: early mesoderm, late mesoderm or cardiomiocyte. Theseprofiles were already done in hESC and hiPSC. We are under wayto obtain CRISPR hPSC targeting these integrins, which will allowus to see how inhibition will affect the specific protocol, not onlyregarding the efficiency but also the morphology of the beatingbodies and changes in the signaling pathways.