SummaryIncreasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets.

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.

Expression of peroxisome proliferator-activated receptor (PPAR) α was investigated in adiponectin knockout mice to elucidate the relationship between PPARα and adiponectin deficiency-induced diabetes. Adiponectin knockout (Adp−/−) mice were generated by gene targeting. Glucose tolerance test (GTT), insulin tolerance test (ITT), and organ sampling were performed in Adp−/− mice at the age of 10 weeks. PPARα, insulin, triglyceride, free fatty acid (FFA), and tumor necrosis factor α (TNFα) were analyzed from the sampled organs. Adp−/− mice showed impaired glucose tolerance and insulin resistance. Additionally, PPARα levels were decreased and plasma concentration of triglyceride, FFA and TNFα were increased. These data may indicate that insulin resistance in Adp−/− mice is likely caused by an increase in concentrations of TNFα and FFA via downregulation of PPARα.

We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the β cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129+Ter/SvJcl×C57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1-/-) mice.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer in the clinic. Further discovery of novel drugs or therapeutic protocols that enhance efficacy requires reliable animal models that recapitulate human immune responses to ICI treatment in vivo. In this study, we utilized an immunodeficient NOG mouse substrain deficient for mouse FcγR genes, NOG-FcγR−/− mice, to evaluate the anti-cancer effects of nivolumab, an anti-programmed cell death-1 (PD-1) antibody. After reconstitution of human immune systems by human hematopoietic stem cell transplantation (huNOG-FcγR−/− mice), four different programmed death-ligand 1 (PD-L1)-positive human cancer cell lines were tested. Among them, the growth of three cell lines was strongly suppressed by nivolumab in huNOG-FcγR−/− mice, but not in conventional huNOG mice. Accordingly, immunohistochemistry demonstrated the enhanced infiltration of human T cells into tumor parenchyma in only nivolumab-treated huNOG-FcγR−/− mice. Consistently, the number of human T cells was increased in the spleen in huNOG-FcγR−/− mice by nivolumab but not in huNOG mice. Furthermore, human PD-L1 expression was strongly induced in the spleen of huNOG-FcγR−/− mice. Collectively, our results suggest that the anti-cancer effects of anti-PD-1 antibodies can be detected more clearly in NOG-FcγR−/− mice than in NOG mice.