**4.2 Allogeneic transplantation models for cardiovascular regenerative medicine**

In pluripotent stem cell-based cardiovascular regenerative medicine, the advantage of induced pluripotent stem cells (iPSCs)over embryonic stem cells (ESCs) is the availability of autologous cells for the treatment. When iPSC products are generated autologously, better engraftment free from the risk of immune rejection after the transplantation of cell products is theoretically anticipated [69]. Human iPSCs are also expected to mitigate immune rejection after cell/tissue transplantation in human leucocyte antigen (HLA)-controlled allogeneic use, which is investigated in various animal allogeneic transplantation models [70, 71]. The allogeneic use of iPSCs is expected to avoid disadvantages of autologous iPSCs transplantation, such as the cost and time required for quality control of each individual cell line [72]. Furthermore, autologous cell products from patients with genetic disorders such as genetic cardiomyopathy may take over its diseased phenotype that would hamper the therapeutic effects of the products.

Medicetty et al. introduced allogeneic bone marrow-derived cell transplantation to a pig myocardial infarction model in which cells are delivered by catheter directly to the coronary artery. They showed a significant positive modulation of left ventricular function and remodeling [73]. Regarding iPSC-based cardiovascular regenerative medicine, Shiba et al. reported an allogeneic transplantation experiment using cynomolgus monkeys (Macaca fascicularis). iPSC-derived cardiomyocytes from major histocompatibility complex (MHC)-homozygous animals were transplanted into MHC-matched monkeys by direct intra-myocardial injection. Transplanted cardiomyocytes showed electrical coupling to the recipient's heart tissue and survived without immune rejection in monkeys treated with clinically relevant doses of immunosuppressants, whereas the transplantation of cardiomyocytes to MHC-mismatched monkeys even treated with immunosuppressants exhibited immune rejection of grafted cardiomyocytes with severe infiltration of T lymphocytes [74]. Kawamura et al. reported a cynomolgus monkey-based allogeneic transplantation experiment using cell sheets prepared from iPSC-derived cardiomyocytes. In the experiments, the monkeys with immunosuppressants could show fair engraftment of iPSC-derived cardiomyocyte sheets regardless of MHC matching, whereas even MHC-matched iPSC-derived cardiomyocyte sheets could not be sufficiently engrafted without immunosuppressants, indicating the requirement of immunosuppressants even in MHC-matched transplantation, which may prevent minor antigen-triggered immune rejection [75]. These results may indicate that the significance of MHC matching would be attenuated in iPSC-based cardiac regenerative therapy [76]. The establishment of a swine leukocyte antigen (SLA)-identified allogeneic transplantation pig model [77] and investigations of histological and molecular mechanisms of immune rejection attributed by cell transplantation would contribute to develop the strategy to avoid immune rejection associated with allogeneic human iPSC therapies as well as therapeutic mechanisms of the allogeneic transplantation.

Additionally, researchers have been struggling to establish pig somatic stem cell lines such as bone marrow-derived stem cells or live progenitor cells using somatic cell cloning technology, which may realize a syngeneic donor-recipient system in pigs [53]. Furthermore, attempts are being made to establish pluripotent stem cells from pigs such as pig iPSCs or ESCs. Xu et al. reported pig iPSCs generated by infecting pig pericytes and embryonic fibroblasts with a retroviral vector encoding Oct4, Sox2, Klf4, and c-Myc. The pig iPSCs could be differentiated into cell derivatives of all three primary germ layers *in vitro* [78]. Chakritbudsabong et al. also reported the

generation of pig embryonic fibroblast-derived iPSCs and their differentiation ability into cardiomyocytes [79]. Choi et al. reported the generation of pluripotent pig ESCs derived from *in vitro*-fertilized and parthenogenetic embryos [80]. Although iPSC generation from various animal species has been attempted and criticized [81], it will largely contribute to the autologous/allogeneic transplantation experiments in the future, which may further promote stem cell-based cardiovascular regenerative medicine.
