Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs

In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

Osteogenic and Fluorescent Characterization of Pig-Derived Mesenchymal Stem Cells Between eGFP Transgenesis and Non-viral Plasmid Transfection

BackgroundWidely used in recent years, mesenchymal stem cells (MSCs) expressing enhanced green fluorescent protein (eGFP) can be tracked during migration to injury sites, while also supporting tri-lineage differentiation. However, the relationship between the expression of green fluorescence and the magnitude of osteogenic differentiation is not clearly defined. Despite increasing use of eGFP-MSCs derived from the transgenic pigs and non-viral eGFP plasmid transfected MSCs in recent years, it remains unclear which cells are suitable for tracking during osteogenic differentiation, and whether the transfected plasmid alters osteogenic potential.MethodsWe compared the expression of green fluorescence and the magnitude of osteogenic differentiation between eGFP MSCs from a transgenic pig (group 1) and non-virally transfected eGFP-MSCs using transIT®-2020 (group 2). Non-transfected MSCs were used as control group (group 3). We also use a scaffold to compare the osteogenic induction environments created by 2-D monolayer cultures and 3-D cultures, respectivelyResultsIn the monolayer culture, flow cytometry from day 7 to day 28 showed that the percentage of green fluorescent cells in groups 1 and 2 were 99.6% and 59.7% of total cell counts, respectively. Quantification showed that eGFP expression peaked on day 7, decreased after day 14, and plateaued to day 28 in group 1 and group 2. Significant aggregation of eGFP over bone-like nodules was appreciated in group 1. In 3-D culture, eGFP expression increased from day 7 to day 28 in both groups, and was higher in group 1 than in group 2 at each time point. Osteogenic profiles and immunohistochemistry showed more significant osteogenic activity in group 1 and group 3 than in group 2. ConclusionsThe expression of eGFP in the test groups did not significantly change after osteogenic induction. However, quantification data was different in monolayer and 3-D cultures due to spatial limitations, differing extracellular environments, and heterogeneous cell morphology and methods of division. Osteogenic profiles and immunohistochemistry data confirmed that osteogenic potential did not change in transgenic pig-derived MSCs. However osteogenic potential decreased in pig MSCs (pig MSCs) treated with the transfection reagent, likely from related toxicity.