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Fetal injection of human MSC’s as a method for xeno-tolerization

James Fisher¹, Joseph Lillegard¹, Doug Padley², Peter Wettstein¹, Scott Nyberg¹

Departments of ¹Surgery and ²Transfusion Medicine, Mayo Clinic - Rochester, Rochester, MN, United States

Introduction: Genetically-engineered murine models exist for the expansion of human hepatocytes. These models succeed for two reasons: 1- native hepatocytes are metabolically defective allowing selective growth of transplanted human hepatocytes; 2 - the murine immune system is genetically altered preventing rejection of transplanted human hepatocytes. Scale-up of metabolic defects to porcine models is under development. However, these models lack the genetic alterations necessary for tolerance. Therefore, we hypothesize that in utero injection may tolerize pigs to human hepatocytes avoiding the need for altering the pig’s immune system.

Methods: We injected 10x10^6 human mesenchymal stem cells (hMSCs) into the liver of fetal pigs at gestational day 40. Injected and non-injected piglets were delivered by cesarean-section at gestational day 115. Non-injected piglets were split into Groups 1 and 2. One in utero injected piglet survived the perinatal period and served as Group 3. Group 1 had postnatal blood draws followed by IP injection of normal saline at 2, 4, and 6 weeks. Groups 2 and 3 had postnatal blood draws followed by IP injection of 50x10^6 hMSCs at 2, 4, and 6 weeks. At 8 weeks, all groups had blood drawn and were euthanized. Cytotoxicity of piglet serum against human lymphocytes was determined at the 4 time points.

Results: Cytotoxicities are summarized in Figure 1. No cytotoxicity was detected in the serum from group 1. In contrast, Group 2 developed a robust cytotoxic response to human lymphocytes starting with the second exposure to hMSCs. Interestingly, the cytotoxic response of Group 3 was reduced significantly, indicating a hyporesponsive immune response to human cells after fetal exposure.

Conclusion: Exposure of fetal pigs to hMSCs early in gestation appears to create a state of postnatal hyporesponsiveness to other human antigens/cells. This method of tolerization may aid in the expansion of human cells in genetically-engineered pigs.