Tomomi Otsuji

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.

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.

A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

Summary 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.

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.

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.