Abstract 16384: Isoproteronol Facilitated Cardiomyocyte Generation From Human Pluripotent Stem Cells
Background: While various strategies for cardiomyocyte (CM) differentiation from human pluripotent stem cells (hPSCs), which include embryonic stem cells and induced pluripotent stem cells have been reported, these methods are highly variable and inefficient. Accordingly, we aimed to develop a novel two-dimensional CM differentiation protocol, which can efficiently generate CMs.
Methods and Results: To direct the differentiation of hPSCs to the cardiac lineage, we designed a stepwise protocol that is divided into four distinct phases: 1) hPSC expansion, 2) mesoderm induction, 3) cardiac lineage commitment, and 4) cardiomyogenesis. In phase 1, we used a two dimensional culture method to avoid the variability resulting from embryoid body formation. Undifferentiated hESCs were directly transferred onto Matrigel-coated plates and cultured under mTeSR® media for cell expansion. Next, to enhance mesodermal differentiation, several different combinations of growth factors known to have a role in mesodermal development were tested and compared by qRT-PCR using mesodermal markers including T (Brachyury) and MESP1. As a result, we found that a combination of BMP4 (10ng/ml), Activin A (3ng/ml) and FGF2 (5ng/ml) was the most efficient for mesodermal differentiation. To induce cardiac lineage commitment in phase 3, we tested five different previously reported methods and found that supplementation with endodermal cell line (END-2) conditioned media induced the highest expression of cardiac lineage markers such as KDR, NKX2.5, GATA4, and ISL1. Finally, in phase 4, treatment with a beta-adrenergic receptor agonist, isoproterenol for 4 days or longer most efficiently generated spontaneously beating CMs indicating the importance of beta-adrenergic receptor activation during CM differentiation and maturation. These beating CMs demonstrated significant expression of CM-specific markers such as beta MHCs and cTnT.
Conclusions: Here we developed a novel two-dimensional protocol, which can generate CMs at high yield. This newly developed system for CM differentiation will serve as a novel and efficient platform for generating functional CMs suitable for cardiac regenerative therapy.
- © 2011 by American Heart Association, Inc.