The field of drug testing currently needs a new integrated assay system, as accurate as systems using native tissues, that will allow us to predict arrhythmia risks of candidate drugs and the relationship between genetic mutations and acquired electrophysiological phenotypes. This could be accomplished by combining the microelectrode array (MEA) system with cardiomyocytes (CMs) derived from human embryonic stem cells (hESC) and induced pluripotential stem cells. CMs have been successfully induced from both types, but their maturation process is not systematically controlled; this results in loss of beating potency and insufficient ion channel function. We generated a transgenic hESC line that facilitates maintenance of hESC-CM clusters every 2 weeks by expressing GFP driven by a cardiac-specific alphaMHC promoter, thereby producing a compact pacemaker lineage within a ventricular population over a year. Further analyses, including quantitative RT-PCR, patch-clamp, and MEA-mediated QT tests, demonstrated that replating culturing continuously enhanced gene expression, ionic current amplitudes, and resistance to K(+) channel blockades in hESC-CMs. Moreover, temporal three-dimensional (3D) culturing accelerated maturation by restoring the global gene repressive status established in the adhesive status. Replating/3D culturing thus produces hESC-CMs that act as functional syncytia suitable for use in regenerative medicine and accurate drug tests.

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.

In this study, we induced differentiation of CPT II-deficient hiPSCs into mature myocytes in a highly efficient and reproducible manner and recapitulated some aspects of the disease phenotypes of CPT II deficiency in the myocyte disease models. This approach addresses the challenges of modeling the abnormality of FAO in CPT II deficiency using iPSC technology and has the potential to revolutionize translational research in this field.

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes in vitro, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA) recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.

Chromosomal deletions are widely involved in serious genetic diseases and in the pathogenesis of cancers. These deletions often generate loss of heterozygosity (LOH) of one of the alleles of a tumor suppressor gene. Because of the technical difficulty inherent in genetic manipulation studies of a chromosome-wide deficiency, it has not been experimentally determined whether chromosome deletions could be a trigger for cancer development. Using the Cre/inverted loxP system, we have developed a chromosome elimination cassette (CEC) that Cre-dependently induces whole or partial deletions of the CEC-tagged chromosomes. Most deletions are usually fatal, but diploid cells carrying small deletions have been obtained from mouse embryonic stem cells carrying a CEC transgene (CEC-ESC). Here, we further isolated various CEC-ESC clones and analyzed CEC integration sites using the fluorescence in-situ hybridization method. In 17 CEC-ESC clones possessing normal chromosome sets, 13 types of chromosomes out of 20 pairs of mouse chromosomes were tagged by CEC. Each CEC-tagged chromosome could become a future target for the creation of a Cre-inducible LOH by a combination of in vitro and in vivo genetic mutation.

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