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

NEIMAN G.; GARATE X; SANTIN N; SEVLEVER G; MIRIUKA S; SCARAFIA A; GUBERMAN A

Congreso; Reunion Conjunta de Biociencias; 2017

Human pluripotent stem cells (hPSCs), with their abilityto differentiate into mature cell types, represent a strong system tostudy human development and disease, and efficacy of drugs beforeclinical trials. Also, these cells provide an unlimited source of ?rawmaterial? for regenerative medicine therapies. However, details ofthe molecular mechanisms interconnecting the extracellular matrixthrough integrins of hPSC remain unclear and how this influence onself-renewal and stem cell fate. Our main goal is finding out the roleof integrin a5 by disrupting alpha5-fibronectin interaction, in hPSCand during cardiac differentiation. To investigate the role of this pro-tein, the knockdown of integrin a5 was induced in the hES3-hESCline with a DOX inducible KRAB repressor through a variant of cris-pr-cas9 system. hESC line showed a strong down-regulation of inte-grin a5 by flow cytometry, reducing the protein expression in an 80%of cells (n=3) after 72h of 500ng/ml DOX treatment. mRNA level wasalso checked by qRT-PCR and it was 4 times less expression (n=3)than without treatment. As we noticed that cells had a slower pro-liferation when integrin a5 was inhibited, a XTT cell viability assaywas done after 72h of treatment and interestingly cell proliferationwas decreased in a 20% (n=4). Since self-renewal is impaired, weare now interested to see how this affects the cell-cycle by usingan EDU Proliferation Assay. As well, during a cardiac differentiationprotocol (Lian, et al.), we observed that α5 has its mRNA and proteinexpression peak at d3.5 (early mesoderm) and it is 10 times higherthan hPSC (n=3). Owing to this, we are now working on the inhibi-tion of integrin a5 in order to find out if it is also involved during theearly mesoderm differentiation.