Fetal bone engraftment reconstitutes the immune system in pigs with severe combined immunodeficiency

lab animal Article https://doi.org/10.1038/s41684-024-01439-7 Fetal bone engraftment reconstitutes the immune system in pigs with severe combined immunodeficiency Check for updates 1,7 1,7 2 1 1 Kaylynn Monarch , Junchul Yoon    , Kyungjun Uh    , Emily Reese , Diana Canaveral Restrepo , Darling Melany de Carvalho Madrid1, Laurie Touchard1, Lee D. Spate1, Melissa S. Samuel1,3, John P. Driver1, Ji-Hey Lim4, Sarah Schlink5, Kristin M. Whitworth1,3, Kevin D. Wells1,3, Randall S. Prather    1,3, Paula R. Chen    1,6 & Kiho Lee    1,3 Genetic modification of genes such as recombination activating gene 2 (RAG2) or interleukin-2 receptor-γ (IL2RG) results in pigs exhibiting severe combined immunodeficiency (SCID). Pigs presenting a SCID phenotype are important animal models that can be used to establish xenografts and to study immune system development and various immune-related pathologies. However, due to their immunocompromised nature, SCID pigs have shortened lifespans and are notoriously difficult to maintain. The failure-to-thrive phenotype makes the establishment of a breeding population of RAG2/IL2RG double-knockout pigs virtually impossible. Here, to overcome this limitation, we investigated whether reconstituting the immune system of SCID piglets with a fetal bone allograft would extend their lifespan. Following intramuscular transplantation, allografts gave rise to lymphocytes expressing T cell (CD3, CD4 and CD8), B cell (CD79α) and natural killer cell (CD335) lineage markers, which were detected in circulation as well in the spleen, liver, bone marrow and thymic tissues. The presence of lymphocytes indicates broad engraftment of donor cells in the recipient SCID pigs. Unlike unreconstituted SCID pigs, the engrafted animals thrived and reached puberty under standard housing conditions. This study demonstrates a novel method to extend the survival of SCID pigs, which may improve the availability and use of SCID pigs as a biomedical animal model. Pigs displaying severe combined immunodeficiency (SCID) are a valuable preclinical model in biomedical research. The lack of functional lymphocytes in these animals allows the successful engraftment of cells and tissues without host rejection1–4. Pigs lacking functional recombination activating gene 2 (RAG2) and interleukin-2 receptor subunit-γ (IL-2Rγ) do not have B, T and natural killer (NK) cell populations5, which resembles the phenotype of the NOD SCID gamma (The Jackson Laboratory) mouse models that have been extensively used for xenograft and disease studies6. SCID pigs have also been used for human xenograft and disease studies1–4,7; however, a limited number of studies has been performed using SCID pigs owing to difficulties producing and maintaining these animals8. Nevertheless, SCID pig models offer advantages over murine models due 1 to their size and physiology being more comparable to humans, thus serving as a better context for the development of treatments and procedures that permit them to fill the niche for a more translational model9,10. One example is in cancer research, where SCID pigs can form tumors from human cells, which are of similar size to tumors found in humans and which are often impossible to grow in mice2. In addition, genetic variation in pigs better represents the genetic variation found in humans than highly inbred mouse strains, which is useful for the development of treatments and pharmaceuticals11. Despite these advantages, current SCID pig models are expensive and resource intensive to produce; and even under intensive management practices, SCID pigs do not live long enough to reach sexual maturity5,12. Division of Animal Science, College of Agriculture Food and Natural Resources, University of Missouri, Columbia, MO, USA. 2Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea. 3National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA. 4 Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA. 5Office of Animal Resources, University of Missouri, Columbia, MO, USA. 6United States Department of Agriculture – Agriculture Research Service, Plant Genetics Research Unit, Columbia, MO, USA. 7These authors contributed equally: Kaylynn Monarch, Junchul Yoon. e-mail: ; Lab Animal Article https://doi.org/10.1038/s41684-024-01439-7 a c b f Piglet 5 Transplanted bone d e Fig. 1 | Transplantation of GFP+ fetal bone fragments into SCID piglets. a, Day 79 GFP+ transgenic fetal donors under a 440–460 nm wavelength excitation light source. b, Humerus and scapula bones collected from fetal donors were broken into fragments (arrow) before insertion. c, The insertion of GFP+ bone fragments (arrows) into the first implantation site on the left side of the incision site on the dorsal neck adjacent to the sixth cervical vertebra. d, GFP+ bone fragments (arrow) prior to insertion into the second implantation site on the opposite side of the incision. e, GFP+ bone fragments (arrow) after insertion into the second implantation site on the right side of the incision. f, A SCID piglet 1 week posttransplant with a palpable lump where the bone fragments were inserted. Arrow denotes the location of the first implantation site on the left side of the incision. This failure-to-thrive phenotype has prevented the establishment of breeding pigs that are homozygous for SCID mutations. Consequently, only founder animals are directly utilized for study, which could confound results owing to the prevalence of mosaicism13–15 or cloning defects16–18 depending on the route used to produce these animals. To avoid these issues, SCID pigs can be generated by breeding heterozygous animals; however, the process would take years due to the prolonged gestational length (114 days) of pigs and months to reach sexual maturity across multiple generations. Moreover, breeding heterozygous carriers produces a low fraction of homozygous knockout pigs (25%). Furthermore, the breeding scheme is not effective if more than one gene needs to be disrupted. For instance, pigs lacking functional RAG2 or IL-2Rγ do not thrive; therefore, obtaining RAG2/IL2RG double-knockout pigs through breeding is nearly impossible with an expectation of 1 in 16 piglets with double-null genotype after the breeding of double-heterozygous parents. Hematopoietic stem cell transplantations (HSCTs) have been performed to extend the lifespan of homozygous SCID pigs; however, few animals with successful engraftments have been reported4,19. Conventional HSCT requires conditioning of the recipient, T cell depletion of bone marrow and donor matching20,21. Performing total body irradiation or chemotherapy on large animals, such as pigs, is prohibitive due to the high expense and specialized facilities these procedures require. In this study, we investigated the feasibility of a simplified process of reconstituting the immune sys (...truncated)