Generation of Severe Combined Immunodeficient (SCID) Pigs: A Novel Large Animal Model for Human Stem Cell Research

Pigs are an excellent large animal model for studies of human disease because of their similarities to humans in organ size and physiology. Developing pigs with severe combined immunodeficiency (SCID) would offer a powerful tool for human biomedical research. I report the generation of a "double knockout" SCID pig containing inactivating mutations in the genes encoding interleukin-2 receptor subunit gamma (IL2RG) and recombination activating gene 1 (RAG1) using transcription activator-like effector nucleases (TALENs). SCID pigs have no functional T, B, or NK lymphocytes and have an atrophic, non-functional thymic remnant, with loss of lymph nodes and Peyer's patches. Their spleens lack germinal centers and red/white pulp differentiation. Serum IgM, IgG, and IgA are absent in SCID pigs.;Allogeneic bone marrow transplantation (BMT) with T cell depleted porcine bone marrow was performed on SCID pigs. Intravenous BMT led to transient reconstitution of only the T cell lineage, while intraosseous BMT reconstituted T cells, B cells, and low levels of NK cells. Engrafted T cells after allogeneic BMT predominantly displayed an activated, memory phenotype, likely due to the absence of a functional thymus and extrathymic differentiation of peripheral T cell progenitors. Transplanted pigs that showed strong B cell repopulation also expressed plasma IgM and IgG up to WT levels, but not IgA. While irradiation led to earlier engraftment following allogeneic BMT, it was also quite toxic to the pigs, while higher levels of late engraftment were achieved without irradiation.;To generate SCID pigs reconstituted with human lymphocytes, human CD34+ cells were transplanted into SCID pigs, leading to successful engraftment with human T cells, which repopulated the blood, spleen, thymus, and bone marrow, as well as human B cells, which repopulated the blood and spleen. In utero intrahepatic transplantation of human donor cells was significantly more effective than postnatal intraosseous transplantation. Engrafted human T cells predominantly expressed a naive phenotype, with lineage marker expression largely consistent with normal thymic T cell development. Human IgM was present in the plasma, reflecting the presence of functional B cells. These findings constitute the first successful construction of a "humanized" SCID pig reconstituted with a human immune system. The humanized pig represents a powerful new animal model for numerous biomedical disciplines, particularly for the development of novel approaches to human transplantation and stem cell therapy.