Introduction: In recent years remarkable progress has been achieved for heart/kidney xenotransplantation (XTx) in primates and humans. But there are still difficulties, especially for lung xenografts. In pig-to-primate xeno lung transplantation (XLTx), porcine lungs often fail immediately with maximum survival of one month so far [1,2]. To overcome this hurdle, it is assumed that lung-specific incompatibilities need to be addressed. This project aims to identify genetic modifications in donor pigs in vitro that could have a positive impact on the tolerance in porcine-to-human XLTx. Endothelial cells (EC) were chosen as model for the first contact between xenograft and recipients’ blood: I) Lung-specific and systemic ECs with different genetic modifications were isolated. II) EC subtypes and genotypes were characterized and functionally analyzed. III) ECs will be compared and exposed to human blood with an in vitro flow chamber model to investigate the lung-specific effects of genetic modifications.
Methods: To obtain primary lung-derived ECs, different isolation protocols were tested and compared, allowing the isolation of ECs from systemic and pulmonary arteries (PA), veins or microvasculature (MV) of porcine lungs. After confirmation of their EC characteristics (CD31, cobble stone morphology), the cells were tested for their EC subtype expression profile of DARC, CA4 and GJA5 via flow cytometry or immunofluorescence. They were also microscopically analyzed for endothelial-specific functionality by standard methods (scratch assay). Some of the ECs were selected for immortalization to obtain lung-specific cell lines with distinct genotypes for further analyses.
Results: By perfusing larger vessels and MV, ECs from different tissue origins and with variants of genetic modifications (GGTA1-KO/+CMAH-KO, ß4GALNT2-KO (TKO)/+SLAI-KO (4KO) ± hCD46.hCD55.hCD59.hHO-1.hA20) were obtained. High proportions (>90%) of the isolated cells exhibited a cobble stone morphology and stained positive for CD31. The isolated systemic cells from aorta and vena cava served as controls for the arterial (DARC⁺, CA4⁺⁺, GJA5^-) and venous (DARC⁺⁺, CA4⁺, GJA5⁺⁺) subtype. Both PA and MV ECs tend to be more like aortic than venous cells but showed slight differences in their expression levels: PA (DARC^-, CA4⁺⁺, GJA5^-) and MV (DARC⁺, CA4⁺⁺, GJA5^-).
Conclusion: Various subtypes of lung specific ECs have been obtained by isolating them from different vessels and genetically modified pigs. These subtypes can be distinguished by their DARC, GJA5, and CA4 expressions. The isolation and use of the lung-specific ECs (cell lines) under flow conditions could help to gain insides in how different genetic modifications of the donor cells can have organ-specific impacts on the outcome in XLTx.

[1] Lu, T., et al. (2019). FIMMU, 10, 3060. https://doi.org/10.3389/FIMMU.2019.03060.
[2] Burdorf, L., et al. (2022). AJT, 22(1), 28–45. https://doi.org/10.1111/AJT.16809.