Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions

Koki Hasegawa; Kazuaki Nakano; Masaki Nagaya; Masahito Watanabe; Ayuko Uchikura; Hitomi Matsunari; Kazuhiro Umeyama; Eiji Kobayashi; Hiroshi Nagashima

doi:10.1016/j.reth.2022.05.010

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions

Regen Ther. 2022 Jun 12:21:62-72. doi: 10.1016/j.reth.2022.05.010. eCollection 2022 Dec.

Authors

Koki Hasegawa¹, Kazuaki Nakano^{2

3}, Masaki Nagaya³, Masahito Watanabe^{2

3}, Ayuko Uchikura^{1

3}, Hitomi Matsunari^{1

3}, Kazuhiro Umeyama^{2

3}, Eiji Kobayashi⁴, Hiroshi Nagashima^{1

3}

Affiliations

¹ Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan.
² PorMedTec Co. Ltd., 2-3227 MIta, Tama-ku, Kawasaki, Kanagawa, 214-0034, Japan.
³ Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki 214-8571, Japan.
⁴ Department of Kidney Regenerative Medicine, The Jikei University School of Medicine - Tokyo, Japan.

Abstract

Introduction: Previously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model.

Methods: The IL2RG X^KOY pigs used in this study were obtained by mating IL2RG X^KOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity.

Results: Tumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed.

Conclusions: IL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Keywords: DPF, designated pathogen-free; IL, Interleukin; IL2RG, interleukin-2 receptor gamma; Interleukin-2 receptor gamma; KO, knock out pigs; NK cells, natural killer cells; OIDP, operational immunodeficient pig; PCR, polymerase chain reaction; Pig; SCID; SCID, Severe combined immunodeficiency; SCNT, somatic cell nuclear transfer; SD, standard deviation; U-iR, uterectomy-isolated rearing; WT, wild-type pigs; XLGD, X-linked genetic diseases; Xenotransplantation; ZFN, Zinc finger nuclease; tdTomato, tandem dimer Tomato.