Integrin alpha-5 is critical for the early stages of human pluripotent stem cell cardiac differentiation

GABRIEL NEIMAN; MARIA AGUSTINA SCARAFIA; ALEJANDRO DAMIÁN LA GRECA; ARIEL WAISMAN; NATALIA LUCÍA SANTÍN VELAZQUE; XIMENA GARATE; ALAN MIQUEAS MÖBBS; CARLOS DANIEL LUZZANI; LUCIA NATALIA MORO; GUSTAVO EMILIO SEVLEVER; ALEJANDRA GUBERMAN; SANTIAGO GABRIEL MIRIUKA

Los Angeles

Congreso; International society for Stem Cell Research; 2019

ISSCR

Adult heart has limited regenerative capacity that is insuficient to compensate for cell death after myocardial injury. Recently, there have been significant advances in the differentiation of cardiomyocytes from human pluripotent stem cells (hPSC) for cell replacement therapies. It is well-known that stem cell niche plays a key role in growth and maintenance of pluripotency and it deeply influences stem cell differentiation potential. Therefore, our goal was to analyze how interactions between integrins and the extracellular matrix are involved in cardiac differentiation. FIrst, we performed an expression profile of integrins, whose ligands are fibronectin (FN) and laminin, in hPSC and in two stages of cardiac differentiation: mesodermal progenitor cells (MPC) and cardiomyocytes (CM). We found a significant down-regulation of integrins a3, a4 and a6 and up-regulation of a5 in MPC but a subsequent reduction at CM stage. These results were consistent in two different protocols: from a monolayer and through the derivation of 3D.embryoid bodies. We then focused on the a5 integrin regulation, thus we engineered a DOX-inducible CRISPRi-KRAB cell line to repress a5 expression in the first three days of differentiation. Our results showed asignificant down-regulation of T-brachyury and TBX6 by approximately 50% in dox-treated protocols. Furthermore, the mRNA expression of epithelial to mesenchymal transition (EMT) markers ZEB1 and ZEB2 was also decreased. Taken togeteher, these results suggest that a5 is important for the EMT process and for giving rise to MPC. FInally, at day 15, CM yield was reduced from 60% ± 6% to 22 ± 5% and cardiac markers NKX2-5 and cTnT were significantly down-regulated. We also found thet not only there was a reduction on CM differentiation but also on CM contractility. The latter was measured by using a specific contractibility software and found that the contraction and peak amplitude of CM were strongly impaired. In summary, we showed that integrin a5 has a significant impact in human cardiac differentiation from hPSC through the alteration of early stages od mesoderm commitment.