Abstract 18124: An Irx4+ Ventricular Progenitor Isolated by Spherical Nucleic Acid Nanoparticles

• DOI: 10.1161/circ.132.suppl_3.18124
• Publication Date: 2022-03-19T21:52:40Z
• Authors: Mitch Biermann; Annie Shao; Tianxiao Han; Gary Lyons; Timothy Kamp
• Keywords: 03 medical and health sciences; 0302 clinical medicine

While human pluripotent stem cell-derived cardiomyocytes have shown promise in engrafting and maturing in animals following myocardial infarction, transplanted cells largely exist as histologically self-contained blocks of cells with moderate or undetectable functional benefit. We therefore sought to develop methods to isolate a proliferative progenitor capable of differentiating both to functional ventricular cardiomyocytes and supportive cardiac mesenchymal cells, as these cells may hold promise for regenerative medicine. To isolate a ventricular progenitor, fluorescent spherical DNA nanoparticles (nanoflares) were synthesized to target RNA of the ventricular transcription factor Irx4 at an intermediate stage in cardiac differentiation of human pluripotent stem cells using small molecule, defined conditions without genetic modification or transfection. The synthesized nanoflare was nontoxic by Annexin V/7AAD flow cytometry and did not affect differentiation or expression of cardiac genes in RT-qPCR, including Irx4. Upon differentiation, nanoflare-FACS-separated Irx4+ cells were found to give rise to 50% cTnT+ cardiomyocytes and 46% Thy1+ mesenchymal cells. In sorted cells, we used sensitive droplet digital RT-PCR to detect early upregulation of lineage-specific genes. Relative to Irx4 -/Low cells, Irx4+ cells had significant upregulation of ventricular-specific mlc2v but not atrial genes (Hey1, CoupTF2, SLN). In characterizing their mesenchymal potency, Irx4+ cells had significant enrichment in smooth muscle actin and N-cadherin. Intriguingly, during monolayer differentiation of Irx4+ cells, a marked aggregation occurs in which Thy1+ mesenchymal cells surround and embed large assemblies of beating cTnT+ cardiomyocytes. Consistent with a matrix remodeling phenotype, both protease inbitior expression (TIMP1) and protease activity by zymography were increased in sorted Irx4+ compared to negative cells.

Conclusions: We have here isolated early ventricular progenitor cells using novel, non-invasive methods. Their remodeling activity may allow more seamless myocardial integration than differentiated cardiomyocytes and provide insights into the role of cell-cell interactions during human cardiac development.