A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

Abstract

We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A, bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture, and were subsequently treated with noggin for three days. Thereafter, the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs, which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers, NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker, E-4031, an Ikr trafficking inhibitor, pentamidin, and a slowly activating delayed rectifier potassium current (Iks) channel blocker, chromanol 293B, and was increased by treatment with a beta-receptor agonist, isoproterenol. At a low concentration, verapamil, a calcium channel blocker, increased the beating rate of the beating EBs, while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium. Key words human induced pluripotent stem cell; myocardial differentiation; embryonic bodyThe cardiotoxicity of drug candidates is one of the crucial findings that limits drug development. A large number of pharmaceutical companies hope to evaluate the risk of cardiotoxicity of drug candidates using human cardiomyocytes in the early stage of development. We have been trying to examine the embryotoxicity of drug candidates in vitro using human induced pluripotent stem (iPS) cells. A simple and highly efficient protocol for inducing the myocardial differentiation of human iPS cells using biological substances has been strongly desired, especially a system using the embryonic body (EB, spheroids of iPS cells) method.Primary human myocardium is difficult to obtain commercially because the cells do not proliferate in culture. Embryonic stem (ES) cells and iPS cells from humans are able to differentiate into myocardium, but the mechanisms are different from those required for ES and iPS cells of mice, and the induction has generally been inefficient. The myocardium is differentiated from the mesoderm, one of the three germ layers generated during the differentiation of pluripotent stem cells. Bone morphogenetic protein-4 (BMP-4), activin-A and wnt-3a are mesoderm inducers, and play a crucial role in its development.

A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells

“…The mES cells are differentiated spontaneously into cardiomyocytes after the withdrawal of leukemia inhibitory factor (LIF), which helps maintain the pluripotency of the undifferentiated mES cells, from culture medium (Evans and Kaufman, 1981;Martin, 1981;Wobus et al, 1984). We previously developed a simple protocol to differentiate iPS cells into cardiomyocytes within a week by the application of biological substances (Aikawa et al, 2015), which made it possible to carry out the cardiac differentiation assay. In a previous study, the teratogenicity effect of thalidomide had already been predicted (Aikawa et al, 2014).…”

“…The experimental protocol of the iPST in the present study was a partially modified of a previously reported protocol as follows: the drug treatment period was prolonged to 6 days (from 4 days) depending on an cardiac differentiation period, the ReLeSR™ as a cell dissociation reagent was used instead of dispase to equalize the number of cells seeding, and the author's original prediction criteria, established by assay of the mouse cells, was improved to the classification of the type for developmental toxicity as the developmental toxicity prediction model (Aikawa et al, 2014(Aikawa et al, , 2015. In the present study, the iPST was validated internally using various reference drugs.…”