INHIBITION OF INTEGRIN A5 THROUGH A CONDITIONAL KNOCKDOWN IMPAIRS HUMAN PLURIPOTENT STEM CELL SELF-RENEWAL

GABRIEL NEIMAN; XIMENA GARATE; MARIA AGUSTINA SCARAFIA; NATALIA LUCÍA SANTÍN VELAZQUE; GUSTAVO EMILIO SEVLEVER; ALEJANDRA GUBERMAN; SANTIAGO GABRIEL MIRIUKA

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

SAIC - Sociedad Argentina de Investigacion Clinica

Abstract: Human pluripotent stem cells (hPSCs), with their ability to differentiate into mature cell types, represent a strong system to study human development and disease, and efficacy of drugs before clinical trials. Also, these cells provide an unlimited source of "raw material" for regenerative medicine therapies. However, details of the molecular mechanisms interconnecting the extracellular matrix through integrins of hPSC remain unclear and how this influence on self-renewal and stem cell fate. Our main goal is finding out the role of integrin a5 by disrupting alpha5-fibronectin interaction, in hPSC and during cardiac differentiation. To investigate the role of this protein, the knockdown of integrin a5 was induced in the hES3-hESC line with a DOX inducible KRAB repressor through a variant of crispr-cas9 system. hESC line showed a strong down-regulation of integrin a5 by flow cytometry, reducing the protein expression in an 80% of cells (n=3) after 72h of 500ng/ml DOX treatment. mRNA level was also checked by qRT-PCR and it was 4 times less expression (n=3) than without treatment. As we noticed that cells had a slower proliferation when integrin a5 was inhibited, a XTT cell viability assay was done after 72h of treatment and interestingly cell proliferation was decreased in a 20% (n=4). Since self-renewal is impaired, we are now interested to see how this affects the cell-cycle by using an EDU Proliferation Assay. As well, during a cardiac differentiation protocol (Lian, et al.), we observed that α5 has its mRNA and protein expression peak at d3.5 (early mesoderm) and it is 10 times higher than hPSC (n=3). Owing to this, we are now working on the inhibition of integrin a5 in order to find out if it is also involved during the early mesoderm differentiation