Generation of functional cells and organs from iPSCs

Adoptive immunotherapy with functional T cells is a potentially effective therapeutic strategy against various types of cancers and viral infections. A major challenge however lies with the “exhaustion” (loss of cytotoxic and proliferative capacities) of antigen-specific T cells during expansion in culture. For an effective adoptive immunotherapy, what we need is not the "exhausted" T cells, but large number of "young and active" CD8+ T cells that can kill tumors or virus infected cells efficiently. To address this issue, we generated induced pluripotent stem cells (iPSCs) from EBV-specific CD8+ T cells from an EBV-infected patient. We then redifferentiated these iPSCs into CD8+ T cells or we like to call them “rejuvenated” T cells since they are newly generated and highly proleferative. These rejT cells possessed antigen-specific killing activity and exhibited TCR gene rearrangement patterns identical to those of the original T cell clone from the patient. In order to confirm in vivo efficacy of these rejT cell, we innoculated EBV-induced tumors into immunodeficient mice and after confimation of tumor growth, we injected these rejT cells. Results indicated that these rejT cells eliminated tumors more efficiently than the original EBV-specific CD8+ T cells, thus confirming in vivo efficacy of these T cells. Another aspect we worked on is generation of a functional organ in livestock animals. In the past, we have demonstrated generation of rat pancreas in mouse utilising a method called "blastocyst complementation". In ancillary work, we successfully generated exogenous-pig pancreata using the same principle. Whilst these studies prepared us to examine the feasibility of generating human PSC-derived pancreata in pancreatogenesis-disabled pigs, some ethical issues on making such “admix chimeras” have yet to be solved. A part of the concern comes from the possibility that human iPSC-derived cells contribute to neural or germ cells in chimeric animals. To overcome this issue, we attempted to restrict differentiation of PSC-derived cells into endodermal organs by introducing a gene that is important for the development of internal organs. When the expression of this gene was induced after transfer of embryo to foster mother, differentiation of ES-derived cells were directed toward interenal organs avoiding contribution of those cells in germ cells, skin and nervous systems. We termed this type of organ generation as "Targeted organ generation" and this should, in principle, reduce the ethical concern when making human-livestock chimeras. In addition, we found that the inhibition of nuclear translocation of a molecule called b-CATENIN enhances conversion of mouse EpiSCs (non-chimera forming) to naive-like PSCs (chimera forming). Since most human ES/iPSCs are considered EpiSCs and non-chimera forming, the finding is of importance for the generation of human organs in ivestock animals.

In this project, utilizing stem cells, we are trying to generate not only cells for cell therapy but transplantable organs for those with end stage organ failure. The following is a summary of our progress made in the generation of cells and organs from stem cells. Adoptive T-cell immunotherapy is a potential therapeutic strategy for combating various types of cancer. However, highly expanded T cells have not proven particularly effective. This is in part explained by exhaustion and losses of function that occur during the ex vivo expansion of T cells. For an effective adoptive immunotherapy, what we need is not the "exhausted" T cells, but large numbers of "young and active" T cells that can kill tumors. To address this issue, we have recently developed a novel system in which antigen-specific killer T cells (CTLs) can be rejuvenated by reprogramming them to induced pluripotent stem cells (iPSCs) and redifferentiating them while expanding their numbers, yielding abundant rejuvenated T cells (rejT cells). We confirmed the in vivo efficacy of these rejT cell against EBV-induced tumors inoculated in immunedeficient mice. To increase the safety of this novel rejT cell therapy, we inserted a drug inducible suicide system to T cell-derived iPSCs. Both the iPSCs and rejT cells derived from them induced cell death after administration of the inducing drug in vivo thereby demonstarting efficacy of this safety system. The results facilitate clinical application of the rejT cell therapy and other branches of iPSC-derived regenerative medicine. To help patients with end-stage organ failure waiting for organ transplantation, we are trying to generate transplantable organs in livestock animals. Previously we have demonstrated the generation of rat pancreas in mouse using interspecific blastocyst complementation technique. This time, we succeeded in the generation of mouse pancreas in rats. The generated pancreata were composed of mouse PSC-derived cells but were of rat size. Islets were prepared from these pancreata and transplanted into diabetic mice. Transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days. Because the transplanted islets contained small number of rat cells, immunosuppressive drug was given to the recipient mice for the first 5 days, but the rest of time, it was totally immunosuppression free. These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated in a xenogeneic host using the blastocyst complementation technique, our ultimate goal. In order to apply this principle to generation of human organs, human PSCs that can contribute to chimera formation are necessary. However, recent studies demonstrated that currently available human PSCs are epiblast-stage stem cells (EpiSCs) and not able to contribute to chimera formation. Using a rodent model, we discovered that if cell survival is transiently promoted by over expressing the anti-apoptotic gene (BCL2), EpiSCs and even lineage-committed progenitors can contribute to chimera formation upon injection into preimplantation embryos. We similarly showed that over expressing BCL2 in human PSCs could increase their survival following injection into sheep embryos in vitro. Having successfully engineered a platform to delete the pancreas in sheep using CRISPRs, we are now in a position to evaluate how well these modified human PSCs can contribute to pancreas formation in vivo. We believe application of our newly designed strategies may generate human PSC-derived organs whilst avoiding unwanted contributions to host gametes or neural tissues.