Chimerism and tolerance post-in utero transplantation with embryonic stem cells

Background. Clinical application of in utero transplantation (IUT) in human fetuses with intact immune systems resulted in a very low level of donor chimerism. In this study, we examined whether the fetal immune system early in the second trimester of pregnancy (13.5 dpc) can initiate immune tolerance for major histocompatibility complex (MHC)-mismatched embryonic stem (ES) cells. We also examined whether immune tolerance mechanisms respond differently to ontogenetically different stem cells. Methods. MHC-mismatched ES, fetal liver (FL), and bone-marrow (BM) cells (H-2k^b) at 1 × 10⁹ cells/kg fetal body weight were injected intraperitoneally into 13.5 dpc BALB/c fetuses (H-2K^d). Peripheral chimerism was determined in blood by flow cytometry (sensitivity≤0.1%) at monthly intervals. Donor-specific immune responses were determined by cytotoxic lymphocyte (CTL) assay, mixed lymphocyte reaction, and T helper (Th)1 and Th2 cytokine assays. Chimeric mice at the age of 9 months received postnatal boosts (PB) with minimal conditioning of 200 cGy by intravenous injection of 1 × 10⁹ of the corresponding cells/kg body weight. Results. After IUT with ES, FL, or BM cells, the level of peripheral chimerism within the first 9 months of life was 0% to 0.4%. PB with 1 × 10⁹/kg of corresponding cells resulted in a decrease in the peripheral chimerism to 0% within 2 weeks of PB. CTL and cytokine assays before and after PB demonstrated a shift toward immunity. Conclusions. Immunologic tolerance was not achieved after IUT of murine fetuses at 13.5 dpc with MHC-mismatched ES cells, and only a low level chimerism was achieved.