Background: Induced pluripotent stem cells (iPSCs), cells reprogrammed with defined factors to a pluripotent embryonic stem cell (ESC)-like state from somatic cells, hold great therapeutic promise for patients. As a potential renewable source of autologous cells for individual patients, scientists have assumed these cells would not be rejected upon transplantation to their syngeneic hosts. Recent evidence suggests that even syngeneic mouse iPSCs may elicit immune responses and may be susceptible to rejection. Consequently, human iPSC-derived pancreatic tissue grafts are a suitable test graft to address the question of whether iPSC-derived grafts will be truly autologous or would be rejected by a syngeneic human immune system.The goal of this study is to elucidate the immune response evoked upon engraftment of autologous hiPSC-derived islet-like clusters (PILCs) into humanized NOD.Prkdc^scidIl2Rg^null (NSG) mice. We hypothesize that mice harboring a human immune system will accept the autologous hiPSC-derived cell grafts, while the allogeneic, non-self-derived cells will be rejected.
Methods: NSG mice were pre-conditioned with sub-lethal levels of radiation followed by introduction of the human immune system (HuNSG) via transplantation of human fetal thymus into the kidney capsule, followed by intravenous injection of autologous fetal liver hematopoietic stem cells (1.5x10⁵ CD34+ cells). HiPSCs were generated via nucleofection with pluripotency-inducing episomal vectors to deliver transcription factors to human fetal cells (CD34+, thymocytes) for transplant into HuNSG harboring either autologous or allogeneic immune systems. Once derived and characterized (teratoma, IHC, FACS) the hiPSCs were differentiated into pancreatic progenitor cells and PILCs per our differentiation protocol, and transplanted under the contralateral kidney capsule of humanized mice. Human fetal pancreata (HFP) from autologous and allogeneic donor tissues were transplanted into the humanized mice as controls. Grafts were harvested at experimental endpoints and processed for histology.
Results: Animals are evaluated for T and B cell engraftment by FACS for prior to transplant at 12 weeks and were used as recipients if there was > 25% human CD45+ cells in the peripheral blood. The engraftment levels by iterative evaluations of the mice were performed and were found to have stable engraftment levels. HiPSCs demonstrated loss of the transgene expression as confirmed by PCR after 8 passages. Characterization of the iPSCs via IHC and FACS confirmed expression of pluripotency markers (i.e. SSEA4, Oct4), demonstration of normal karyotyping and the presence of all germ layers were found in teratoma assays. We found significant T cell infiltration into allogeneic HFP and PILC transplants which correlated with failure of maturation in HFP grafts as evidenced by the absence of mature islet tissue and loss of insulin staining. In contrast, syngeneic grafts did not show hCD3+ T cell infiltration in the graft and HFP contained numerous islets with insulin staining at 4, 8 and 18 weeks post-transplant.
Conclusion: This study clarifies the adaptive immune responses to human iPSC-derived cells using pancreatic beta cell grafts in an established humanized mouse model, providing vital information before clinical translation. We have demonstrated that mice with a functional human immune system are able to mount an allogeneic immune response to hPSC-derived allografts and allogeneic tissues.