Role of integrins (α3, α5 and α6) along cardiac differentiation through silencing by CRISPR-effector system.

GABRIEL NEIMAN; FERNANDA MESQUITA; XIMENA GARATE; ADRIANA BASTOS CARVALHO; ANTONIO CARLOS CAMPOS DE CARVALHO; ALEJANDRA S. GUBERMAN; GUSTAVO SEVLEVER; SANTIAGO G. MIRIUKA

Mar del Plata

Congreso; SAIC SAI SAFE 2016; 2016

SAIC

Pluripotent stem cells (PSC) are a model to study embryological development and also have the potential to provide a supply of different cell types for tissue replacement and drug screening. Owing to this, increasing efficiency of differentiation protocols is of vital importance. The role of ECM in cell adhesion and signaling to cells through receptors such as integrins has received much attention. Previous publications showed that specific integrins and their ligands such as fibronectin, laminins are responsible of the cellular fate. Our goal is to find out what role integrins (α3, α5 and α6) are playing during cardiac differentiation. It will be achieved through the CRISPR-effector system where we can specifically and reversibly inhibit gene expression allowing us to understand the contribution of integrins to cardiac development. A new specific protocol was developed in our lab and it has 80% of efficiency. We performed the integrin expression profile on days d0, d3.5, d7.5 and d15 at both levels: mRNA and proteins. Through qPCR, we observed that α5 has its peak at d3.5 where increases 10 times compared to d0 and its main ligand (Fibronectin) has a d7.5 peak, 50 folds higher. Integrins α3 and α6, whose ligand is laminin, show an opposite behavior: α6 decrease an 80% at d3.5 while α3 increase 8 folds at d15. By citometry, we got a similar profile to mRNA expression and each mesodermal or cardiac population is correlated with specific population markers. All this data together proves there is a regulation along differentiation and they change significantly on different cell states: early mesoderm, late mesoderm or cardiomiocyte. These profiles were already done in hESC and hiPSC. As a general conclusion, once we have the CRISPR hPSC lines, we are going to be able to see how inhibition will affect the specific protocol, not only with regard to efficiency but also the morphology of the beating bodies and changes in the signaling pathways.