Immune tolerance induction using fetal directed placental injection in rodent models: a murine model.

OBJECTIVES:Induction of the immune response is a major problem in replacement therapies for inherited protein deficiencies. Tolerance created in utero can facilitate postnatal treatment. In this study, we aimed to induce immune tolerance towards a foreign protein with early gestational cell transplantation into the chorionic villi under ultrasound guidance in the murine model. METHODS:Pregnant C57BL/6 (B6) mice on day 10 of gestation were anesthetized and imaged by high resolution ultrasound. Murine embryos and their placenta were positioned to get a clear view in B-mode with power mode of the labyrinth, which is the equivalent of chorionic villi in the human. Bone marrow cells (BMCs) from B6-Green Fluorescence Protein (B6GFP) transgenic mice were injected into the fetal side of the placenta which includes the labyrinth with glass microcapillary pipettes. Each fetal mouse received 2 x 105 viable GFP-BMCs. After birth, we evaluated the humoral and cell-mediated immune response against GFP. RESULTS:Bone marrow transfer into fetal side of placenta efficiently distributed donor cells to the fetal mice. The survival rate of this procedure was 13.5%(5 out of 37). Successful engraftment of the B6-GFP donor 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 humoral and cell-mediated immune tolerance against the foreign protein

Successful delivery of GFP-BMCs to mouse embryos by ICVI at E10.

<p>A: GFP positive cells were detected in the fetal body and heart at 30 minutes after injection (x10). B: Naïve fetus at E10 (x10). C-D: E17 mouse liver and thymus at 1 week after ICVI (x40).</p

E10 embryo imaged by power Doppler mode.

<p>The two layers of the placenta, which consist of the labyrinth and spongiotrophoblasts layer and the maternal decidua. The white dotted line indicates the border between these two layers.</p