Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes-In Vitro Studies.

Jerzy Wiater,Ryszard Słomski,Maria Skrzyszowska,Jacek Jura,Marcin Samiec,Marek Romek,Kamil Wartalski,Zdzisław Smorąg

doi:10.3390/ijms22189683

Abstract

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

Highlights

Nowadays, the use of the porcine cells, tissues, and organs seems to be the response to the contemporary scarcity of organs for transplantation
The regions of localization noticed for rhα1,2-FT and rhα-Gal A enzymes were determined by immunofluorescence staining of the in vitro cultured porcine transgenic epidermal keratinocytes (PEKs) stemming from hFUT2 or hGLA mono-transgenic (Figure 1), hFUT2×hGLA bi-transgenic (Figure 1), and nontransgenic (Figure 2) pigs served as a control group (CTR nTG)
Homogenously dispersed regions associated with fluorescently immunostaining the rhα-Gal A molecules were found to occur in whole cytoplasm of both transgenic PEK variants as follows: hGLA (Figure 1b) and hFUT2×hGLA (Figure 1d)

Summary

Introduction

The use of the porcine cells, tissues, and organs seems to be the response to the contemporary scarcity of organs for transplantation. Genetically engineering triggering the simultaneous coexpression of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) enzymes, which are encoded by hFUT2 and hGLA transgenes, appears to be a promising approach to overcome the HAR of porcine cell, tissue, and organ xenotransplants [11,12]. Both endogenous porcine α1,3-GT and the transgenically expressed rhα1,2-FT utilize N-acetyllactosamine (LacNAc) residues for the purposes of their biocatalytic activities. No research has ever been conducted to comprehensively estimate the concomitant semiquantitative profiles (RAs) of enzymatic rhα1,2-FT and rhα-Gal A immunoproteins, and enhanced abrogation of Galα1→3Gal epitopes in the extracorporeally proliferating PEK lines that were previously established from dermal explants of the hFUT2×hGLA bi-transgenic pigs

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Results

Discussion

Conclusion