Novel engraftment and T cell differentiation of human hematopoietic cells in Art-/- IL2RG-/SCID pigs

Authors: BOETTCHER, ADELINE - Iowa State University; LI, YUNSHENG - Iowa State University; AHRENS, AMANDA - Iowa State University; KIUPEL, MATTI - Michigan State University; Byrne, Kristen; Loving, Crystal; CINO-OZUNA, A. GISELLE - Kansas State University; WIARDA, JAYNE - Orise Fellow; ADUR, MALAVIKA - Iowa State University; SCHULTZ, BLYTHE - Iowa State University; SWANSON, JACK - McFarland Clinic Pc; SNELLA, ELIZABETH - Iowa State University; HO, CHAK-SUM (SAM) - Gift Of Hope Organ And Tissue Network; CHARLEY, SARA - Iowa State University; KIEFER, ZOE - Iowa State University; CUNNICK, JOAN - Iowa State University; POWELL, ELLIS - Iowa State University; GIUSEPPE DELL, ANNA - Iowa State University; JENS, JACKIE - Iowa State University; SATHE, SWANAND - Iowa State University; GOLDMAN, FREDERICK - University Of Alabama; WESTIN, ERIK - University Of Alabama; DEKKERS, JACK - Iowa State University; ROSS, JASON - Iowa State University; TUGGLE, CHRISTOPHER - Iowa State University

Submitted to: Frontiers in Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2020
Publication Date: 2/6/2020
Citation: Boettcher, A.N., Li, Y., Ahrens, A.P., Kiupel, M., Byrne, K.A., Loving, C.L., Cino-Ozuna, A., Wiarda, J.E., Adur, M., Schultz, B., Swanson, J.J., Snella, E.M., Ho, C., Charley, S.E., Kiefer, Z.E., Cunnick, J.E., Putz, E.J., Giuseppe, Dell A., Jens, J., Sathe, S., Goldman, F., Westin, E.R., Dekkers, J.C.M., Ross, J.W., Tuggle, C.K. 2020. Novel engraftment and T cell differentiation of human hematopoietic cells in Art-I- IL2RG-IY SCID pigs. Frontiers in Immunology. 11:100. https://doi.org/10.3389/fimmu.2020.00100.
DOI: https://doi.org/10.3389/fimmu.2020.00100

Interpretive Summary: Pigs are often used as an animal model for human research due to the similarites between human and pig organs and function. Generation of pigs without specific immune system components provide valuable information on pig immune cell function, and general immunology. Pigs genetically modified to eliminate the pig immune system allow for a model to put in human immune cells, and have a pig with a human immune system. This is referred to as humanization, and provides a model for cancer and regenerative medicine studies. Pigs with a natural mutation in a gene required for development of some immune cells were previously discovered. To futher remove the pig immne system, genetic manipulation tools were used to introduce a mutation in another gene, and pigs devoid of pig immune cells were developed and immune deficiency confirmed. Additionally, human stem cells were put into the immune deficient pig during development, and pigs with a human immune system derived. The humanized pigs can serve as a valuable model for biomedical research, and provide insight on the function of pig cells that support immune cell development.

Technical Abstract: Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34 plus selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45 plus CD3 epsilon plus cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.