Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

Ahrens

et al. 2019

Preprint

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 and 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+ selected cord blood cells into the fetal Art-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ 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.One sentence summaryWe have generated a T- B- NK- SCID pig model through site directed mutagenesis of IL2RG in a naturally occurring Artemis null background and show successful engraftment of human T and B cells in blood and lymphoid organs after in utero injection of human hematopoietic stem cells.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Novel engraftment and T cell differentiation of human hematopoietic cells inArt^-/-IL2RG^-/SCID pigs

Ahrens

et al. 2019

Preprint

“…As of this writing, very few studies have been conducted in relation to introducing human HSCs into non‐murine models. Non‐SCID lambs and piglets can support the development of human T, B, NK, and myeloid lineages when HSC cells were injected in the fetal intraperitoneal space via in utero injection s with human HSCs . Additionally, the pig thymus can support the development of a wide range of human T cells .…”

Section: Discussionmentioning

confidence: 99%

Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs

Cunnick

Powell

et al. 2018

Xenotransplantation

Background: Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human-induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig.Methods: Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess whether porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12, non-blocking antibody 2D3, and nonspecific IgG1 antibody and exposing to human or porcine monocytes. Results:We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed, and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were preincubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity of porcine monocytes with human cells requires interaction between porcine SIRPA and human CD47. AUTH O R CO NTR I B UTI O N S ANB designed and performed experiments, analyzed data, and wrote the manuscript; JEC, CLL, EJP, and TKE designed experiments, reviewed data, and edited manuscript; EJP performed statistical analysis; SEC genotyped piglets, managed SCID pig colony, and helped with sample collection; and CKT designed experiments, reviewed data, and edited manuscript. O RCI D Adeline N. Boettcher

“…Another potential method to increase engraftment is to condition the fetuses prior to human cell injection. Plerixafor, a drug that mobilizes stem cells out of bone marrow (58), has previously been utilized for in utero injections of human cells into sheep fetuses to improve engraftment of human cells (59). Plerixafor is an agonist for CXCL12 on stromal cells, which binds to CXCR4 on hematopoietic stem cells (HSC) (58).…”

Section: Improving Human Cell Engraftment In Scid Pigsmentioning

confidence: 99%

“…Administration of plerixafor mobilizes sheep HSC out of bone marrow, providing more available niches for human HSC to engraft. Goodrich et al (59) described that administration of plerixafor along with injection of CD34 + CXCR4 + human stem cells and mesenchymal stem cells improved chimerism (in peripheral blood) 5 weeks after transplantation from 2.80 to 8.77%. Now that T − B − NK − SCID pigs and biocontainment facilities are available for extended postnatal follow-up, a similar regimen could be administered to SCID pig fetuses prior to in utero injection with human stem cells.…”

Section: Improving Human Cell Engraftment In Scid Pigsmentioning

confidence: 99%

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART−/−IL2RG−/Y SCID Pigs

Ahrens

et al. 2020

Front. Immunol.

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.