On the road to iPS cell cardiovascular applications.

The ability to generate induced pluripotent stem (iPS) cells from somatic cells by the overexpression of a limited number of stem cell–related genes has generated great excitement and interest in the biomedical research community including cardiovascular researchers. The pioneering study by Yamanaka and colleagues showing that overexpression of Oct3/4, Sox2, Klf4, and c-Myc could reprogram mouse fibroblasts to a pluripotent state similar to that of embryonic stem (ES) cells opened major new avenues of research.1 This epigenetic reprogramming was rapidly extrapolated to the human system using either the same combination of reprogramming factors or a slightly different combination of transgenes (OCT4, NANOG, SOX2, LIN28).2–4 Like embryonic stem (ES) cells, iPS cells can be used for basic developmental biology research and also as a cell source to generate theoretically unlimited quantities of desired cell types such as cardiomyocytes. Such differentiated cells types can be used in a wide range of basic research studies and potentially in clinical applications, which not only include cellular therapies but also drug discovery and safety testing. One appealing aspect of human iPS cells compared to human ES cells is that they can be more readily generated without specialized expertise and access to human embryos, which also avoids the ethical challenges associated with human embryo research. Potentially the most powerful advantage of iPS cells over ES cells is that they can be generated from any patient to produce genetically identical pluripotent cells that can create human disease models or generate patient-specific cells for therapy. Already a number of iPS cell human disease models have been generated,5,6 and proof-of-principle iPS cellular therapies have been pioneered in mouse models.7–9 Despite the speed at which the iPS cell field is racing forward, we are just at the beginning of a long road. Many major questions remain regarding iPS cells. Will they prove to be equivalent to ES cells in their properties? In other words, will iPS cells be equally as pluripotent as ES cells and readily generate all cell lineages represented in the 3 embryonic germ layers? Will different iPS cell lines show distinct differentiation profiles which may be an advantage or disadvantage for a given application? For example, will certain iPS cell lines be less able to differentiate into cardiovascular relevant cell types compared to other lines? Another critical question for therapeutic applications is whether reprogrammed iPS cell lines are prone to tumorigenesis. Does the starting cell source matter? How does the technique of reprogramming impact iPS cell behavior? In this issue of Circulation Research, Martinez-Fernandez et al address some of these questions by carefully examining the cardiogenic potential of mouse iPS cells generated from murine fibroblasts using 3 reprogramming factors (OCT4, KLF4, and SOX2).10 Most of the detailed understanding of the developmental potential of iPS cells comes from studies using mouse iPS cell lines generated with integrating retroviral vectors encoding 4 reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc), because this technology has been available the longest, for 3 full years. Some of these 4-factor iPS cells have been demonstrated to be competent to integrate into blastocyst stage embryos and generate chimeric mice which can exhibit germline transmission.11–13 This is strong evidence of pluripotency, but initial efforts at the most rigorous developmental test of pluripotency, what is called tetraploid complementation, failed.12 In tetraploid complementation, an embryo is made tetraploid by fusing cells at the 2-cell stage. The resulting tetraploid blastocyst can develop the cells that become placenta but cannot develop the embryo proper. Stem cells transplanted at the early blastocyst stage of the tetraploid embryo will generate a viable mouse strictly from the donor cells, if the cells are truly pluripotent. Very recently, 2 studies have demonstrated using tetraploid complementation that 4-factor iPS cell lines can support development of full term viable mice.14,15 Thus, some 4-factor iPS cell lines seem to be fully capable of developing all cell types present in the adult mouse, but the hint of caution is that in the limited data available, not all tested iPS cell lines proved successful in tetraploid complementation.15 This could be because of experimental limitations and inadequate testing or fundamental differences in the iPS cell lines. Likewise, in vitro studies of cardiac differentiation of 4-factor mouse iPS cell lines have produced mixed results, with some lines showing comparable cardiogenesis to ES cells and others showing delayed cardiogenesis.16,17 Thus with the oldest of iPS cell technologies, some iPS cell lines seem to meet even the most stringent criteria for pluripotency, but it is unlikely that all 4-factor iPS cell lines will meet this standard. It was quickly recognized that chimeric animals generated from 4-factor iPS cells were at increased risk for tumorigenesis attributable to reactivation of the c-Myc transgene,11 which clearly represents a safety concern for translation of this technology to humans. However, c-Myc was soon found not to be essential for generation of iPS cells, which could be generated using 3 virally encoded factors: Oct3/4, Sox2, and Klf4 (3F iPS cells).18,19 These 3F iPS cells expressed the The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Departments of Medicine (T.J.K.), Physiology (T.J.K.), and Anatomy (G.E.L.); and Stem Cell and Regenerative Medicine Center (G.E.L.,T.J.K.), University of Wisconsin, Madison. Correspondence to Timothy J. Kamp, University of Wisconsin School of Medicine and Public Health, H6/370 Clinical Science Center–MC 3248, 600 Highland Ave, Madison, WI 53792. E-mail [email protected] (Circ Res. 2009;105:617-619.) © 2009 American Heart Association, Inc.