Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers

Abstract

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4 days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2 days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23 days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60 days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Keywords: Human CD47 transgenic; Intra-bone bone marrow transplantation; Pig-to-baboon model; Tolerance; Xenotransplantation.

Fig. 1

(a) A picture of the line system used for baboon recipients of xenografting procedures. The mesh jacket is rigidly attached to an aluminum tether through which 1–3 catheters are placed. The tethers are 4–6 ft. in length. (b) A close-up view of the swivel shown in (a), which has been unattached from the aluminum tether. As many as three catheters are securely affixed to each of three ports on the swivel. This allows the animal to move within the cage without twisting the lines. (c) A close-up view of the connection between the jacket and the tether. In this image, the jacket has been detached from the tether, showing the tape-wrapped catheters traveling through the jacket and into the tethering system

Fig. 2

A picture of the proper positioning for IBBM injection. Rt. tibial tuberosity was used for IBBM injection