All surgery was performed under anesthesia, and all efforts were made to minimize suffering. Mouse Recipients and Donors Accurately time-dated pregnant C57BL/6 mice were used as recipients at embryonic day 10 (E10; 10 days post conception). skin grafts was observed in all recipient (5 out of 5) mice 6 weeks after birth. Induction of anti-GFP antibodies was completely inhibited. Cytotoxic immune reactivity of thymic cells against cells harboring GFP was suppressed by ELISPOT assay. Conclusions In this study, we utilized early gestational placental injection targeting the murine fetus, to transfer donor cells carrying a foreign protein into the fetal circulation. This approach is sufficient to induce both 2-Deoxy-D-glucose humoral and cell-mediated immune tolerance against the foreign protein. Introduction Induction of immunologic response is usually a major problem in replacement therapies for inherited disorders such as hemoglobinopathies, immune deficiencies, or certain inborn errors of metabolism. When allogeneic transplantation is performed after birth, intensive immunosuppression and myeloablation is required to avoid rejection or graft versus host disease. Immune tolerance created by exposure to antigen may facilitate postnatal replacement therapies.[1,2] It is well known that under specific circumstances, early gestational exposure to a specific antigen can induce antigen specific tolerance. In humans, the window for tolerance induction is usually thought to be limited to the first trimester, ending after approximately 14 weeks gestation.[3,4] Chorionic villus sampling (CVS) is widely utilized for prenatal diagnosis and has been demonstrated to be feasible and safe when performed at 10 to 14 weeks of gestation. Thus, the technique used for CVS is an attractive approach to deliver cells and or foreign antigens to the fetus with appropriate timing to achieve fetal tolerance. Historically, there have been previous studies utilizing intraplacental bone marrow transplantation in the early gestational mouse model. The classical studies of Fleischman and Mintz [5, 6] exhibited hematopoietic engraftment and chimerism after intraplacental injection of hematopoietic cells, but tolerance was not investigated. However, in those studies, the placenta was blindly injected, and delivery of the cells to the fetal circulation was inconsistent. In this study, we utilized high-resolution ultrasound guidance in the murine model to inject bone marrow cells expressing a foreign protein (GFP) into the fetal side of the placental circulation, mimicking the CVS procedure. We then analyzed tolerance for the immunogenic GFP protein after birth. Methods Ethical Statement All procedures in this study were carried out in strict accordance with the guidelines for animal experimentation from the Animal Research Committee of Osaka University and that of National Cerebral and Cardiovascular Center. The protocol was approved by the Animal Research Committee, Osaka University (Pemit Number: 24-079-018), and National Cerebral and Cardiovascular Center (Permit Number: 13018). All surgery was performed under anesthesia, and all efforts were made to minimize suffering. Mouse Recipients and Donors Accurately time-dated pregnant C57BL/6 mice were used as recipients at embryonic day 10 (E10; 10 days post conception). Donor cells were from C57BL/6TgN(act-EGFP) OsbY01 mice (kindly provided by Dr. Okabe, Osaka University, Genome Information Research Centerreferred to as B6GFP in this report) that have been maintained in our breeding colonies. Injected mice were housed in the Laboratory Animal Facility at National Cardiovascular Center Research Institute. The experimental protocols were approved by the Institutional Animal Care and Use Committee at the National Cardiovascular Center Research Institute. Preparation of Donor BMCs Adult GFP+ BMCs (B6GFP-BMCs) were isolated from 8 week old B6GFP mice MSH4 by flushing the tibiae, femurs and iliac bones with Ca/Mg-free phosphate-buffered saline (PBS) using a 26-gauge needle. After filtration through a 40-m nylon mesh filter, B6GFP-BMCs were centrifuged at 440 x for 5 2-Deoxy-D-glucose minutes at 4C. After the red blood cells were lysed with lysing buffer, the B6GFP-BMCs were counted and suspended in PBS at a density of 4 x 107 cells/ml for injection. Intra-Chorionic Villi Injection (ICVI) We used an ultrasound-guided injection system (Vevo 2100, VisualSonics, Toronto, Canada) to precisely identify two layers of the murine placenta, 2-Deoxy-D-glucose which consist of the labyrinth and spongiotrophoblast layer and the maternal decidua (Fig 1). The labyrinth is the area of nutrient and gas exchange between the fetal and maternal circulations. It exists around the fetal side of the placenta and is equivalent to the chorionic villi in the human placenta. Thus, we defined cell transplantation into the labyrinth as intra-chorionic villi injection (ICVI) in this study. Pregnant mice at E10 were anesthetized with isoflorane (3.5% for induction, 2% for maintenance) and.