阿部 朋行

Luminescent technology based on the luciferin-luciferase reaction has been extensively employed across various disciplines as a quantitative imaging modality. Owing to its non-invasive imaging capacity, it has evolved as a valuable in vivo bioimaging tool, particularly in small animal models in fields such as gene and cell therapies. We have previously successfully generated rats with a systemic expression of the luciferase gene at the Rosa26 locus. In this study, we transplanted bone marrow from these rats into micro-mini pigs and used in vivo imaging to non-invasively analyze the dynamics of the transplanted cells. In addition, we established that the rat-to-pig transplantation system is a discordant system, similar to the pig-to-human transplantation system. Thus, rat-to-pig transplantation may provide a clinically appropriate large animal model for pig-to-human xenotransplantation.

Sheep are typically considered as industrial animals that provide wool and meals. However, they play a significant role in medical research in addition to their conventional use. Notably, sheep fetuses are resistant to surgical invasions and can endure numerous manipulations, such as needle puncture and cell transplantation, and surgical operations requiring exposure beyond the uterus. Based on these distinguishing characteristics, we established a chimeric sheep model capable of producing human/monkey pluripotent cell-derived blood cells via the fetal liver. Furthermore, sheep have become crucial as human fetal models, acting as platforms for developing and improving techniques for intrauterine surgery to address congenital disorders and clarifying the complex pharmacokinetic interactions between mothers and their fetuses. This study emphasizes the significant contributions of fetal sheep to advancing human disease understanding and treatment strategies, highlighting their unique characteristics that are not present in other animals.

This preliminary study investigated the efficacy and safety of bone marrow-derived mesenchymal stem cell transplantation in a piglet Legg-Calve-Perthes disease (LCPD) model. The LCPD model was induced in two Landrace piglets (6- and 7-week-old, weighing 12 and 17 kg, respectively) by ligaturing the femoral neck. In the first piglet, the natural LCPD course was observed. In the second piglet, 4 weeks after ligaturing the femoral neck, simple medium and medium containing 2.44 × 107 bone marrow-derived mesenchymal stem cells were transplanted into the right and left femoral heads after core decompression, respectively. Plain radiographs were obtained every 4 weeks, and the epiphyseal quotient was calculated by dividing the maximum epiphysis height by the maximum epiphysis diameter. The piglets were sacrificed at 14 weeks postoperatively. The femoral heads were extracted and evaluated grossly, pathologically, and by using computed tomography. The transplanted cell characteristics were evaluated using flow cytometry. Flattening of the epiphysis was observed in both femoral heads of the first piglet and only in the right hip of the second piglet. The epiphyseal quotients immediately and at 14 weeks postoperatively in the right femoral head of the second piglet were 0.40 and 0.14, respectively, while those of the left femoral head were 0.30 and 0.42, respectively. Hematoxylin and eosin staining did not reveal physeal bar or tumor cell formation. The transplanted cells were 99.2%, 65.9%, 18.2%, and 0.16% positive for CD44, CD105, CD29, and CD31, respectively. Core decompression combined with bone marrow-derived mesenchymal stem cell transplantation prevented epiphyseal collapse.

Abstract Background Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. Methods We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. Results Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG–mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. Conclusion A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.