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

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 alpha 1,2-fucosyltransferase (hFUT2) and alpha-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 alpha 1,2-fucosyltransferase (rh alpha 1,2-FT) and alpha-galactosidase A (rh alpha-Gal A) proteins, but also the relative abundance (RA) of Gal alpha 1 -> 3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2xhGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rh alpha 1,2-FT and rh alpha-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Gal alpha 1 -> 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 Gal alpha 1 -> 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 Gal alpha -> 3Gal epitopes in hFUT2xhGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human alpha-1,2-FT and alpha-Gal A proteins followed by identification of the extent of abrogating the abundance of Gal alpha 1 -> 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.