Injection of allogeneic bone marrow cells into the portal vein of swine inutero

The ability to safely manipulate the immune system of the developing fetus carries the hope of effective treatment strategies for certain congenital d isorders that can be diagnosed during gestation. One possible intervention is the induction of specific transplantation tolerance to an adult donor wh o could provide tissue after birth without the need for immunosuppression. Although the introduction of allogeneic stem cells to a developing immune s ystem has been shown to result in hematopoietic chimerism, donor-specific t ransplantation tolerance has not been demonstrated in a large animal model. In previous reports of in utero stem-cell transplantation, the cells were injected into the fetus by an intraperitoneal route. We sought to improve u pon this technique of cell transplantation by developing a method for the s afe delivery of allogeneic stem cells directly into the hepatic circulation of fetal swine. In the second phase of our study, we determined if adult a llogeneic bone marrow cells delivered to the fetus by this intravascular ro ute could result in result in hematopoietic chimerism and donor-specific tr ansplantation tolerance. A method of successful intravascular injection was designed in which a lapa rotomy was performed on a sow at midgestation (50-55 days) to administer 1 cc of inoculum into the portal vein of each fetus using transuterine ultras ound guidance and a 25-gauge spinal needle. In one sow, 10 piglets were inj ected with saline to test safety, and 8 piglets were born. For transplantat ion of stem cells to the fetuses, donor bone marrow was harvested from a ge netically defined miniature swine. In one sow the marrow was injected witho ut T-cell depletion resulting in abortion. In the third sow, the marrow was depleted of T-cells to less than 0.01% using magnetic beads conjugated to anti-CDS monoclonal antibodies. No chimerism was detected in these offsprin g. Only in the fourth sow where the T-cell depletion was reduced to about 1 % of the cells in the inoculum did one animal demonstrate chimerism. This p iglet showed reproducible blood chimerism (0.95% donor cells) detected by h ow cytometry measurement of monoclonal antibodies to the donor MHC. In addi tion, this animal demonstrated hyporesponsiveness to donor lymphocytes in a n MLR assay while reacting strongly to third-party stimulator cells. A spli t-thickness skin graft from the donor was accepted, and a third-party graft was rapidly rejected. (C) 2000 Academic Press.