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Updated: Dec 9, 2025
In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells
Published on: August 9, 2017
3'UTR enhances hCD47 cell surface expression, self-signal function, and reduces ER stress in porcine fibroblasts
Nora Hosny1,2, Anders W Matson1, Ramesh Kumbha1
1Department of Surgery, Schulze Diabetes Institute, University of Minnesota School of Medicine, Minneapolis, MN, USA.
Introduction:
Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated region (3'UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. The aim of this study was to investigate the impact of a modified form of the 3'UTR on the expression, localization, and function of hCD47 in transfected porcine cells.
Methods:
hCD47 constructs with and without the modified 3'UTR were knocked into the GGTA1 locus in porcine fetal fibroblasts using CRISPR. Flow cytometry of the transfected cells was used to analyze hCD47 localization. Endoplasmic reticulum (ER), mitochondrial, and oxidative stress were examined by gene expression analysis and confocal microscopy. Phagocytosis of transfected cells by human macrophages was measured by flow cytometry, and stimulation of human/non-human (NHP) primate lymphocytes by the cells was examined using a PBMCs proliferation assay.
Results:
Cells transfected with the construct lacking the 3'UTR (hCD47(3'UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3'UTR (hCD47(3'UTR+)) decreased intracellular expression of hCD47 by 36% and increased cell surface expression by 53%. This was associated with a significant reduction in cellular stress markers and a higher level of protection from phagocytosis by human macrophages. Furthermore, hCD47(3'UTR+) porcine cells stimulated significantly less proliferation of human/NHP T cells than hCD47(3'UTR-) cells.
Conclusion:
Our results suggest the potential benefits of using hCD47 constructs containing the 3'UTR to generate genetically engineered hCD47-expressing donor pigs.
