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  • Rejection might also have occurred as iPSC CMs might fail

    2018-10-24

    Rejection might also have occurred as iPSC-CMs might fail to suppress recipient NK kisspeptin in MHC-matched transplantation. MHC class I molecules such as MHC-E, which are expressed on almost all cells in the body, bind to receptors on NK cells to inhibit cytotoxic activity and, following the immune reaction of NK cells (Janeway et al., 2001), are recognized as self. Since the expression levels of MHC class I genes in iPSC-CMs were low in this study, the grafts might have been recognized as non-self and rejected by recipient NK cells. Consistent with this, MHC-matched transplantation using TAC only or without immune suppressants induced elevated expression of the NK cell markers FcγR III and NKG2D in the grafts (Figure S1E), which might suggest that NK cell-related immune rejection might occur during MHC-matched iPSC-CM transplantation if the recipient did not receive appropriate immunosuppressants. There were a few limitations to this study. The group sizes in this study were very small, which limits statistical robustness. In addition, we mainly performed subcutaneous transplantation of iPSC-CMs using silicone sheets to evaluate engraftment and rejection by direct observation of the transplanted iPSC-CMs and histological analysis of the grafts. This might not be the optimal location or the appropriate method as clinical applications would be expected to include transplantation to the heart without foreign material, which could provoke the host inflammatory response (Tang and Eaton, 1995) and might have worsened rejection. Notably, the iPSC-CMs transplanted into the heart in this study exhibited poorer survival compared with a previous report (Chong et al., 2014) in which human embryonic stem cell-derived cardiomyocytes were shown to survive for 3 months in the immune-suppressed macaque. This discrepancy might be due to the difference between the healthy heart in this study and the myocardial infarction model in the study of Chong et al., or might result from the different immunosuppressive regimens, both of which are expected to have a significant influence on the survival of transplanted cardiomyocytes. Further study would be needed for the detailed evaluation of immunological rejection and survival of the transplanted iPSC-CMs in disease model hearts according to the clinical applications.
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank Professor Shinya Yamanaka of the Center for iPS Cell Research and Application, Kyoto University, who kindly provided the cynomolgus macaque iPSCs. We also thank Seiko Eiraku, Akima Harada, and Hiromi Nishinaka for their technical support. This study was supported by the Japan Science and Technology Agency as a part of the project, Center for the Development of Myocardial Regenerative Treatments Using iPS Cells.
    Introduction The intestinal epithelium is a dynamic tissue that relies on integration of cell division, differentiation, migration, and apoptosis. Intestinal tissue homeostasis and regeneration are facilitated by multipotent tissue stem cells that have the ability to differentiate into multiple mature cell types. Two types of stem cells are currently proposed to reside in small intestinal crypts: cycling crypt base columnar (CBC) cells and +4 reserve cells (Barker, 2014; Clevers, 2013). CBC stem cells maintain daily homeostasis, while their reserve equivalents have been postulated to play a role in tissue regeneration upon injury (Barker, 2014; Clevers, 2013). The functional study of ISCs has been made possible by the recent characterization of ISC markers such as Lgr5, Olfm4, or Sox9 for CBC cells, and Bmi1, Hopx, Lrig1, or Sox9 for their presumed quiescent counterparts (Barker et al., 2007; Gracz and Magness, 2014; Gracz et al., 2010; Powell et al., 2012; Sangiorgi and Capecchi, 2008; Takeda et al., 2011). Currently, the isolation of pure ISCs is primarily restricted to the use of targeted murine reporter alleles of ISC markers. However, the fidelity and specificity of these genes to mark ISCs is still controversial (Munoz et al., 2012; Tan and Barker, 2014). The most widely used reporter for CBC cell isolation is the Lgr5-Gfp knockin mouse model (Barker et al., 2007), which has facilitated the isolation and characterization of CBC stem cells in many studies (van der Flier et al., 2009). However, this transgenic mouse model has several limitations: (1) the reporter cassette is prone to being silenced in over two-thirds of all crypts resulting in mosaic expression of the Gfp allele (Barker et al., 2007; Munoz et al., 2012); (2) LGR5 constitutes the receptor for R-SPONDINS (Carmon et al., 2011; de Lau et al., 2011; Glinka et al., 2011), potent WNT signal enhancers and stem cell growth factors, and the potential haploinsufficiency induced by the loss of one Lgr5 allele (replaced by the Gfp reporter cassette) cannot be excluded; and (3) the extensive breeding required to cross genetically modified mouse models with the Lgr5-Gfp reporter strain.