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 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+ selected cord blood cells into the fetal ART−/− IL2RG−/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and 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.
Highlights
Animals with severe combined immunodeficiency (SCID) are invaluable to biomedical researchers because they are permissive to engraftment of human cells, allowing one to study developmental processes within an in vivo environment
We have demonstrated that porcine SIRPA binds to human CD47 to inhibit phagocytosis of human cells [31], indicating pigs may be permissive to human xenografts, similar to non-obese diabetic (NOD) mice
Once we confirmed the cellular phenotype of ART−/− IL2RG−/Y pigs, we investigated whether these pigs were capable of engrafting human CD34+ hematopoietic stem cells
Summary
Animals with severe combined immunodeficiency (SCID) are invaluable to biomedical researchers because they are permissive to engraftment of human cells, allowing one to study developmental processes within an in vivo environment. Within the past few years, such SCID pigs are being utilized by cancer [13], disease model [12], and stem cell therapy [7] researchers. An important step in further developing the SCID pig model is to immunologically humanize these animals through the introduction of human CD34+ hematopoietic stem cells. Similarities between human and porcine immune genes [16] suggest that human immune development would be supported in vivo within the pig [17]. Development of such a model could provide researchers with a larger humanized animal for use in cancer [13, 17], HIV, and vaccine development research
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