Cardiomyocytes develop from anterior primitive streak cells induced by ß-catenin activation and the blockage of BMP signaling in hESCs.

Cardiomyocytes arise from cells that migrate to the mid-to-anterior region of the primitive streak (PS) during embryogenesis. We previously showed that canonical Wnt/ß-catenin pathway signaling leads to the development of nascent PS populations from human embryonic stem cells (hESCs) and that synergistic activation of the Wnt/ß-catenin pathway and inhibition of bone morphogenetic protein (BMP) signaling by Noggin induced the formation of anterior PS cells. We herein demonstrate that anterior PS cells induced by the activation of ß-catenin with Noggin differentiate into functional cardiomyocytes when cultured in suspension with BMP4 and fibroblast growth factor 2 (FGF2). All aggregates generated from the anterior PS cells developed into contracting cells demonstrating their cardiac potential. More than 30% of the cells in each aggregate were α-actinin-positive cardiomyocytes. In addition, these cardiomyocytes could be easily purified up to 80% by simple size fractionation. In contrast, the posterior PS cells induced by ß-catenin activation without Noggin showed poor cardiac potential. These results show that the commitment to a cardiac lineage in vitro occurs through similar cellular and molecular signaling pathways involved in cardiac development in vivo, thus providing a valuable culture model for studying early cardiac developmental events in hESCs.

A 3D sphere culture system containing functional polymers for large-scale human pluripotent stem cell production.

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

Dynamic link between histone H3 acetylation and an increase in the functional characteristics of human ESC/iPSC-derived cardiomyocytes.

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.

A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

Combination of functional cardiomyocytes derived from human stem cells and a highly-efficient microelectrode array system: an ideal hybrid model assay for drug development.

Human pluripotential stem cells including both embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) possess self-renewing potency and pluripotentency and can differentiate into virtually any somatic cell type. These features are a distinct advantage for the generation of specific types of human tissue cells in vitro for continuous use in drug development. Recently, an assay system for drug-induced QT interval prolongation using hESC/hiPSC-derived cardiomyocytes and microelectrode arrays (MEA) has been developed. Drug-induced QT interval prolongation (DIQTIP) can lead to sudden cardiac death and is a major safety concern for the drug industry. Regulatory authorities such as the US FDA and the European Medicines Agency require in-vitro testing of all drug candidates to identify potential risk of DIQTIP prior to clinical trials. To reduce the risk of DIQTIP, a routine assay system for in vitro electrophysiological properties using cell-based assays is effective and necessary in early phase of drug discovery. This review discusses developments over the last couple of years for a qualified drug testing method and provides some examples of how hESC/hiPSC-derived cardiomyocytes are beginning to find a practical use for drug discovery and development.

Progressive maturation in contracting cardiomyocytes derived from human embryonic stem cells: Qualitative effects on electrophysiological responses to drugs.

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.

Rapid induction of large chromosomal deletions by a Cre/inverted loxP system in mouse ES Cell hybrids.

Loss of heterozygosity by whole or partial loss of chromosomal regions is crucial to genetic disorders, cancers and diseases. It is difficult to analyze the mechanisms of pathogenesis caused by large-scale chromosomal abnormalities due to the extreme rarity of this mutagenesis. Using a Cre/inverted loxP system, we have generated a chromosome elimination cassette (CEC) that induces a selective loss of embryonic-stem-cell-derived chromosomes in undifferentiated embryonic stem cell-somatic cell hybrids. Here, due to the increased expression of Cre, rapid formation of Cre recombination products and immediate loss of CEC-tagged chromosomes were detected by fluorescence in situ hybridization. Cre also initiated intrachromosomal recombination between identical short sequences outside loxP, leading to large chromosomal deletions of CEC-tagged regions. The Cre-mediated antiparallel synapses likely act as a scaffold to bring the identical short sequences into close proximity for recombination. This CEC technology might allow better understanding of the modulator sequences responsible for the tangled structure formation and its solution mechanism, inducing mitotic recombination leading to chromosomal deletions.

Targeted chromosome elimination from ES-somatic hybrid cells.

To engineer a stem cell genome, we developed a technology for targeted elimination of chromosomes from mouse embryonic stem (ES)-somatic hybrid cells. Here we demonstrate the use of a universal chromosome elimination cassette (CEC) for elimination of a single embryonic stem cell (ESC)-derived chromosome 11 or 12, and also both copies of chromosome 6, which harbor pluripotency-associated genes including Nanog. We attribute hybrid-cell pluripotency to the expression of Nanog from the reprogrammed somatic-cell nuclei.

Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase.

Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal (lambdamax = 460 nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells.