In our studies of life-supporting GalT-KO pig-to-baboon kidneys, we have found that some recipients developed increased serum creatinine with growth of the grafts, without histologic or immunologic evidence of rejection. We hypothesized that the rapid growth of orthotopic pig grafts in smaller baboon recipients may have led to deterioration of organ function. To test this hypothesis for both kidneys and lungs, we have assessed whether the growth of outbred (Yorkshire) organ transplants in miniature swine is regulated by intrinsic (graft) or extrinsic (host environment) factors. Yorkshire kidneys exhibited persistent growth in miniature swine, reaching 3.7x their initial volume over 3 months vs. 1.2x for miniature swine kidneys over the same time period. Similar rapid early growth of lung allografts was observed and, in this case, led to organ dysfunction. For xenograft kidneys, a review of our results suggests that there is a threshold of 25cm3/kg for kidney graft volume/recipient body weight that induces cortical ischemia in transplanted GalT-KO kidneys in baboons. These results suggest that intrinsic factors are responsible, at least in part, for growth of donor organs and that this property should be taken into consideration for growth-curve mismatched transplants, especially for life-supporting organs transplanted into a limited recipient space.

Bioengineered lungs consisting of a decellularized lung scaffold that is repopulated with a patient’s own cells could provide desperately needed donor organs in the future. This approach has been tested in rats, and has been partially explored in porcine and human lungs. However, existing bioengineered lungs are fragile, in part because of their immature vascular structure. Herein, we report the application of adipose-derived stem/stromal cells (ASCs) for engineering the pulmonary vasculature in a decellularized rat lung scaffold. We found that pre-seeded ASCs differentiated into pericytes and stabilized the endothelial cell (EC) monolayer in nascent pulmonary vessels, thereby contributing to EC survival in the regenerated lungs. The ASC-mediated stabilization of the ECs clearly reduced vascular permeability and suppressed alveolar hemorrhage in an orthotopic transplant model for up to 3 h after extubation. Fibroblast growth factor 9, a mesenchyme-targeting growth factor, enhanced ASC differentiation into pericytes but overstimulated their proliferation, causing a partial obstruction of the vasculature in the regenerated lung. ASCs may therefore provide a promising cell source for vascular regeneration in bioengineered lungs, though additional work is needed to optimize the growth factor or hormone milieu for organ culture.

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

Background We have recently demonstrated that human‐CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra‐bone bone marrow transplantation (IBBMTx) in a pig‐to‐baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. Methods Baboons received GalTKO‐hCD47/hCD55Tg (n = 5) or ‐hCD55Tg (n = 1) or ‐hCD46/HLA‐E Tg (n = 1) pig IBBMTx. Macrochimerism, anti‐pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47‐ porcine lungs 1‐3 months later. Results All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti‐pig antibody levels decreased over time and anti‐pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1‐4 days). Conclusion This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.