Abstract 13139: Single Gene Cardiopoiesis Induced in Human Pluripotent Source

Abstract

Introduction: The process of engaging stem cells towards a cardiopoietic (CP) state was established using a protein-based cocktail consisting of multiple growth factors. Although this regenerative enhancement protocol has been successfully tested in advanced clinical trials, it remains resource-intensive. Recent efforts have focused on streamlining cardiopoiesis through a protein-independent alternative that leverages a transfection-based mRNA approach. Microencapsulated modified messenger RNA (M3RNA) was utilized to deliver brachyury (T), an early mesodermal transcription factor to provoke early lineage specification in pluripotent cells. Hypothesis: Transfection of a human pluripotent stem cell source with T induces early expression of CP transcription factors to accelerate cardiac differentiation. Methods: Human pluripotent stem cells were transfected with T and maintained for 14 days. Expression of the cardiopoietic markers MEF2C, GATA4, and Nkx2.5 was analyzed using RT-qPCR, in tandem with MYH6 at days 10 and 20. Differentiation proficiency was independently corroborated by assessing Mef2c and Nkx2.5 expression by IF. Results: Following transfection with T, Nkx2.5, MEF2C and GATA4 were significantly induced at 72h (60-, 20- and 40-fold, respectively, p<0.01 for all 3 markers vs baseline), reflecting timing of differentiation initiation. At 5 days post differentiation, expression of the cardiac-specific Nkx2.5 increased by 5 fold vs control and the cell cycle regulator CHIR (p<0.01), suggesting successful persistent induction of cardiopoeisis. At 10 and 20 days of differentiation, RNA expression of MYH6 was 10-15 times induced in human pluripotent cells transduced with T (p < 0.0001, and p < 0.001 vs control, respectively), compared to 2 times (p<0.05, p<0.01 vs control, respectively) in CHIR treated cohorts. Accordingly, at 42 days post-induction of differentiation, a greater number of cells expressed MLC2V and cTnT (p<1e-05, <1e-07, respectively vs control), driving a robust beating pattern. Conclusion: This study implements a single gene T M3RNA-based approach to readily induce cardiac differentiation from pluripotent sources, exceeding available commercial platforms.