Dr. Ulrich Martin, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Germany, discussed experiments in which murine and human induced pluripotent stem (iPS) cells were generated from somatic cells using transcription factors.

Murine somatic cells were reprogrammed to become iPS cells using the transcription factors, Oct3/4, Sox2, Klf4, C-Myc, Nanog, and Lin28. A standard ES cell (ESC) differentiation protocol was used to induce differentiation of the iPS cells into cardiomyocytes. Immunostaining confirmed expression of cardiac markers by these cells. The iPS-derived cardiomyocytes showed field potentials similar to those in mESC-derived cardiomyocytes and spontaneous synchronous Ca²+ transients.

Similar techniques are used to generate iPS cells from adult human fibroblasts. To address possible problems with using cells from aged individuals, Dr. Martin generated iPS cells from human cord blood endothelial cells (hCBEC). Oct3/4, Sox2, Nanog, and Lin28 were used with a lentiviral vector; C-Myc and Klf4 were not used, to avoid oncogenic potential. Immunostaining showed that the hCBEC-iPS cells expressed several ESC markers. The induction factors decreased during differentiation of the hCBEC-iPS cells but Oct4 remained switched on. The differentiated cells expressed differentiation markers for all three germ layers.

When stained for cardiac markers, expression of cardiac troponin T, sarcomeric actinin, and Cx43 was observed, indicating functional cardiomyocytes. The cells exhibited spontaneous contraction and field potentials comparable to mESC-derived cardiomyocytes. Coupling of the hCBiPS cardiomyocytes was demonstrated by the presence of spontaneous synchronous Ca²+ transients.

An issue in generating large numbers of iPS cells for clinical applications is monitoring and controlling differentiation status. To resolve this, a transgenic clone was generated from the iPS cells, creating a huOct4-promoter-GFP reporter iPS line. Quantitative RT-PCR showed a significant decrease of eGFP expression during differentiation. Human iPS cells in suspension cultures expressed eGFP, indicating their undifferentiated state.

The next step is up-scaling generation and enrichment of ESC-derived cardiomyocytes. A colleague of Dr. Martin’s developed a technique using a synthetic medium that produces >99% cardiomyocytes following differentiation and genetic selection. In a total yield of 5x10⁷/50 mL, there were 7x10⁶ cardiomyocytes/mL.

Murine and human iPS cells were able to generate contracting cardiomyocytes with a functional β-adrenergic pathway and characteristic Ca²+ fluctuations that express typical cardiac markers. Undifferentiated primate ESCs and human iPS cells can be expanded in suspension culture. Future work will include comparative analysis of different iPS cell clones, correlation of phenotypes to expression and methylation patterns, and application for cell transplantation and myocardial tissue engineering.

Dr. Shinsuke Yuasa, Keio University School of Medicine, presented data from experiments on embryonic stem (ES) cell and induced pluripotent cell (iPS) generation and differentiation into cardiomyocytes. ES cells have strong proliferation and differentiation capabilities. Studies in mouse embryos revealed that the BMP antagonist, noggin, is transiently expressed in the heart forming region. Further studies confirmed that noggin promotes cardiac myocyte differentiation. The developing hearts of mouse embryos also specifically expressed X receptor, suggesting this receptor is involved in cardiac development. X factor added to ES cell culture significantly increased the incidence of beating embryoid bodies. These experiments showed that noggin improves cardiomyocyte differentiation and X factor increases cardiomyocyte proliferation.

Dr. Yuasa also performed experiments using primate ES cells, obtained from marmosets and monkeys. These studies showed that primate ES cells can differentiate into functional cardiomyocytes with normal cardiomyocyte structure and the ability to produce an action potential.

A study comparing ES cells, Nanog-iPS cells, and Fbx-iPS cells showed that all three types have similar morphology. All three cell types can differentiate into cardiomyocytes that express cardiac specific markers, indicating that ES cell- and iPS cell-derived cardiomyocytes have normal cardiomyocyte structure. Cardiac differentiation efficiency was compared among ES cells and five iPS cell lines. The ES cells and two nanog-iPS lines (Ng20D17, Ng38C2) had a high incidence of beating colonies. The two Fbx-iPS cell lines (FbxWT1, Fbx4-3) showed late cardiomyocyte differentiation efficiency.

Human iPS cells can be induced to differentiate into cardiomyocytes using a standard human ES cell differentiation method. Cardiomyocytes derived from human iPS cells express cardiac specific protein, have normal cardiomyocyte structure, and produce an action potential. The field potential of human iPS cell derived cardiomyocytes was measured using a multiple electrode array (MEA), demonstrating Na+, Ca2+, and K+ currents. Exposing the cardiomyocytes to a sodium channel inhibitor (quinidine) reduced the field potential amplitude, showing that the cells express a functional sodium channel. A similar experiment using a potassium channel inhibitor (E4031) showed that the cells express a functional potassium channel. Exposure to isoproterenol increased the beating frequency of the cardiomyocytes, demonstrating that they had normal function.

In this series of experiments, Dr. Yuasa generated cardiomyocytes from mouse and primate ES cells and from mouse iPS and human iPS cells. These experiments showed that human iPS cell derived cardiomyocytes are identical to human cardiomyocytes in both structure and function. The data from these studies will lead to future cardiac regeneration therapy in human patients.