Elucidating the role of integrin alpha-6 in cardiomyocytes differentiated from human embryonic stem cells

Integrins are heterodimer transmembrane protein receptors that are composed of an α and a β subunit, which are connected by covalent interactions forming an extra-cellular head, two multi-domain legs, two single pass transmembrane helices and two short cytoplasmic tails. The two primary functions of integrins are attachment to the extracellular matrix and signal transduction from the extracellular matrix to the cell. Signaling also operates in the opposite direction: signals generated inside the cell can either enhance or inhibit the ability of integrins to bind to their ligand outside the cell (Alberts, et al., 2002). Integrin alpha-6 (ITGA6) is a particular isoform of the integrin family, which is expressed across stem cell populations and has been shown to play an integral role in human embryonic stem cell (hESC) self-renewal (Villa-Diaz 2016). During the differentiation process of hESCs to cardiomyocytes, there is a population of cells found to be Isl-1 positive and ITGA6 positive, which have been shown to produce all three cardiac cell types: endothelial, smooth muscle and cardiomyocytes. This suggests that this sub-population might be human cardiac stem cells (hCaSCs). Determining and creating a cardiac stem cell line would be beneficial in regenerative medicine, to create heart models and treatments for damaged hearts in the future. We develop the hypothesis that ITGA6 is a key protein in the formation of hCaSCs, which in turn lead to cardiomyocytes.

To test our hypothesis, we knocked out ITGA6 in hESCs using three constructs of doxycycline (DOX) inducible CRISPR-Cas9 single-guided (sg) RNAs that target specific sequences of nucleic acids in ITGA6, and we utilized a CRISPR-Cas9 as a control. We found through flow cytometry analysis that the construct denominated as CRISPR-Cas9 sg10 had the greatest effect on ITGA6 expression. Three days into our cardiomyocyte differentiation protocol, we activated the knockout with DOX. This allowed the knockout of ITGA6 by the time when it was detected as its highest expression in Isl-1+/ITGA6+ cells. This experiment was done in triplicate. Evaluation of cultures was done at day six (144 hours) and day eight (192 hours) after initiation of differentiation, by determining accumulation/sphere growth of Isl-1+/ITGA6+ cells and beating of cardiomyocytes, and compared to control groups, as an indicator of successful differentiation. Our data proved that the hypothesis was correct, as knockout of ITGA6 resulted in no large accumulations or spheres formed, and cardiomyocytes did not mature, or beat. The cultures fixed at day 6 and day 8 to perform immunocytochemistry (ICC) to test for the expression of ITGA6, Isl-1 and Oct4. The CRISPR cas9 sg10 was shown to have no expression of any of these proteins. This supports that ITGA6 is necessary for the formation of cardiomyocytes and also cardiac stem cells.