To achieve a therapeutic effect with genetically corrected

To achieve a therapeutic effect with genetically corrected iPSCs for an autologous ex vivo gene therapy approach, we must still overcome several hurdles, such as the successful transplantation of iPSC-derived myogenic cells. Since MYOD1-induced muscle MM-102 from iPSCs have the ability to fuse (Goudenege et al., 2012; Tanaka et al., 2013), a corrected copy of the dystrophin gene may be able to contribute to an entire myofiber. Moreover, for long-term repopulation, the differentiation of iPSCs toward muscle progenitor cells (i.e., satellite cells) could be ideal for restoring damaged muscle in DMD patients (Darabi et al., 2012). In addition, an immunogenic response to the newly corrected gene product is possible (Mendell et al., 2010), although the response may be hindered by transient immunosuppression.
Experimental Procedures
Author Contributions
Acknowledgments We thank the anonymous DMD patient and his family for kindly providing the biopsy. We also thank Dr. Megumu K. Saito and Takayuki Tanaka for preparing the cell samples; Dr. Kazutoshi Takahashi, Dr. Yusuke Echigoya, and Emi Shoji for their technical advice; Dr. Peter Karagiannis for a critical reading of the manuscript; Dr. Knut Woltjen and Dr. Keisuke Okita for providing vectors; Yumie Tokunaga for the SNP array analysis; and Osamu Ohta for computational advice. This research was supported in part by JSPS KAKENHI, JST PRESTO, JST Yamanaka iPS Cell Special Project, and the JST Research Center Network for Realization of Regenerative Medicine. H.L.L. is a recipient of a JSPS DC1 fellowship. S.Y. is a scientific advisor of iPS Academia Japan without salary.
Introduction The cancer stem cell model proposes that the parenchymal cells of tumors are hierarchically organized (Clevers, 2011; Magee et al., 2012). At the apex of the hierarchy are cells that are uniquely capable of initiating and sustaining tumorigenesis, a property that is tightly linked to their ability to self-renew. These are the cancer stem cells (CSCs), which give rise to the phenotypically diverse and more differentiated, but nontumorigenic, offspring that make up the bulk of the tumor. Thus, cancer can be viewed as a caricature of normal development (Pierce and Speers, 1988). With some notable exceptions, such as melanoma, there is evidence supporting this model for many tumor types (Magee et al., 2012), and a hierarchical structure is even maintained to some extent in established tumor cell lines cultured in vitro (Locke et al., 2005). CSCs are thought to play a major role in driving disease recurrence, due to the intrinsically enhanced therapeutic resistance that results from high expression of multidrug transporters, enhanced DNA damage checkpoint activation and repair mechanisms, and altered cell-cycle kinetics in CSCs (Alison et al., 2012). Thus, understanding CSC biology will be critical to the development of more effective cancer therapies. CSCs are most commonly identified by fluorescence-activated cell sorting (FACS) analysis, through combinations of cell-surface markers that enrich for cell populations with enhanced tumor-initiating activity in vivo (Magee et al., 2012). However, the optimal marker combinations are very dependent on the tissue and specific cell of origin of the tumor, and even well-established markers such as CD44+CD24−/lo for breast cancer and CD133+ for brain tumors do not robustly distinguish tumorigenic from nontumorigenic cells in all patient samples (Magee et al., 2012; Visvader and Lindeman, 2012). Importantly, identification of CSCs by cell-surface marker phenotype cannot readily be used to monitor CSCs in situ in the tumor, with all the extrinsic microenvironmental cues intact. Furthermore, this approach cannot be used for real-time assessment of CSC behavior at a single-cell rather than a population level. These limitations have impeded characterization of CSCs in preclinical models, where the ability to observe the CSC directly, and monitor the behavior of individual cells in time and space, would give new insights into CSCs properties and their response to therapy.