Fetal Immune Responses Post In Utero Transplantation of Embryonic Stem Cells, Bone Marrow and Fetal Liver Hematopoietic Stem Cells.

Introduction: We have previously shown that in utero transplanted ES cells survive and integrate into the fetus development and have established a murine model for the study of the in vivo differentiation of ES cells. The goal of this study was to monitor the fetal immune responses post in utero transplantation of ES cells, bone marrow (BM) and fetal liver (FL) hematopoietic stem cells.

Materials and Methods: Murine (MHC)-mismatched ES cells genetically engineered to express yellow fluorescent protein (YFP-ES cells) were cultured on mitomycin-treated feeder layers for four days prior to in utero transplantation (IUT) in medium containing leukemia inhibitory factor. A cell dose of 5x10⁴ YFP-ES cells ((H-2k^b) was injected intraperitoneally in the fetuses of Balb/c (H-2K^d) pregnant mice at E12- E14. BM and FL hematopoietic stem cells (H-2k^b) were transplanted in utero at a dosage of 1×10⁹ cells/kg fetal body weight into E12-E14 BALB/c fetuses (H- 2K^d). Fetal immune responses were monitored by in vitro mixed lymphocyte reaction and cytotoxicity assays performed with self (Balb/c), allogeneic ES cells, BM or FL hematopoietic cells, and 3^rd party (C3H cells). Cytokine levels (IL-2, IFN-gamma, IL-4 and IL-10) were determined in the cell culture supernatants from cytotoxicity assays. Liver tissue sections were prepared from in utero transplanted fetuses and examined for the presence of lymphocytic infiltration.

Results: In utero transplantation of YFP-ES-cells did not induce tolerance in the fetuses and was associated with increased cytokine production compared with BM and FL groups. Microscopic examination of liver sections of ES cell transplanted group revealed the presence of marked inflammatory infiltrate.

Conclusions: Embryonic stem cells transplanted in utero induce fetal immune responses and increased cytokine production associated with (MHC) upregulation.