Human hematopoiesis and blood cell formation in pigs

In a recent study, Yong-Guang Yang and colleagues developed a severely immunodeficient pig model that supports long-term engraftment and multilineage differentiation of human hematopoietic stem/progenitor cells (HSPCs).1 This breakthrough addresses key limitations of current humanized mouse models, including their small size and limited lifespan, and offers a promising platform for the large-scale production of human immune cells, potentially functioning as an in vivo bioreactor. The development of large animal models that can replicate human hematopoiesis has been a longstanding challenge in biomedical research.2 While immunodeficient mice have played a crucial role in the study of human immune cell development and function,3 their physiological differences from humans and limited lifespan constrain their utility for long-term studies and large-scale human cell production. Although various large animals have been tested as recipients for human grafts, pigs present several advantages as preclinical models due to their closer physiological and anatomical similarities to humans, longer lifespan, and larger body size.4 Moreover, pigs are considered the most suitable source animals for clinical xenotransplantation, primarily owing to these advantages and their amenability for the production of gene-edited pigs through somatic nuclear transfer and cloning, which is essential for generating severely immunodeficient pigs. However, previous efforts to humanize pigs have been hindered by poor engraftment and limited immune cell reconstitution.5 In a recent report,1 the authors first developed a severe immunodeficient pig with genetic inactivation of recombination-activating gene 1 (Rag1) and interleukin 2 receptor subunit gamma chain (IL2rg), referred to as the RG pig. Although the RG pig demonstrated the capacity to support the engraftment of human HSPCs in the bone marrow, human cells were not detected in the blood or spleen, indicating that human cells are unable to survive once they exit the bone marrow, likely due to rejection or an inadequate tissue microenvironment in RG pigs. Given that the RG pig lacks T, B, and natural killer (NK) cells, macrophages, which have been shown to reject xenogeneic cells due to the absence of cross-reaction between donor CD47 (also known as integrin-associated protein [IAP]) and the recipient signal regulatory protein α (SIRPα),6 are likely responsible for immune rejection. CD47 acts as a critical "don't eat me" signal that inhibits phagocytosis through interaction with SIRPα, a highly immunosuppressive receptor on macrophages.7 Building upon earlier study demonstrating that macrophages developing in CD47-deficient mice do not recognize CD47 as a "don't eat me" signal,8,9 the authors further deleted Cd47 in the RG pig (referred to as the RGD pig) and demonstrated significantly higher levels of human cell reconstitution throughout the tissues in human HSPC-transplanted RGD pigs. Following human HSPC transplantation, RGD pigs exhibited sustained (>200 days) and elevated levels of human hematopoietic cells throughout the tissues, including bone marrow, spleen, liver, and blood, with peak blood concentrations ranging from 23.8% to 99.3%. Flow cytometric and single-cell RNA sequencing analyses confirmed the multilineage differentiation of human hematopoietic cells, comprising CD3+ T cells, CD19+ B cells, CD14+ monocytes (including CD14+CD16− classical, CD14+CD16+ intermediate, and CD14+CD16++ non-classical monocytes), dendritic cells (DCs; inclusive of CD11c+CD123− myeloid DCs and CD11c−CD123+ plasmacytoid DCs), as well as cytokine-producing CD56+CD16− and cytotoxic CD56+CD16+ NK cells. Histological examination revealed the formation of white pulp structures comprised of human immune cells within the spleen. Furthermore, the thymi of these RGD pigs were structurally normal, featuring a well-organized cortex and medulla populated by phenotypically normal human thymocytes. RGD pigs demonstrated superior capacity compared to immunodeficient mice in supporting durable and robust human thymopoiesis and T cell development following human HSPC transplantation; in the latter, human T cell production is transient due to the rapid involution of the recipient mouse thymus.3 This finding aligns with previous observations in pig thymus-grafted immunodeficient mice.10,11 Lymphocyte receptor gene rearrangements and repertoire formation are essential for their function.12 The authors conducted single-cell profiling of T cell receptor (TCR) variable–diversity–joining (V(D)J) rearrangements and identified a diverse array of V(D)J chain elements at the TCRα and TCRβ loci in human thymocytes and splenic T cells developing in RGD pigs, akin to that observed in humans.13 Furthermore, most human thymocytes within the pig thymus were found to be monoclonal, indicating that these thymocytes were generated de novo through thymopoiesis rather than being contaminated by circulating T cells, which are typically multiclonal. Additionally, human B cells developing in RGD pigs exhibited a wide variety of immunoglobulin (Ig) V(D)J elements. Notably, bone marrow and splenic human B cells displayed significant differences in the frequencies of V(D)J segment usage, reflecting that negative selection occurs during B cell maturation in pigs. The production of human IgM and IgG antibodies further substantiates the functionality of human B cells developing in these RGD pigs. Finally, the authors conducted functional analyses to confirm the functionality of human T cells developed in RGD pigs. Following incubation with anti-CD3/CD28 antibodies, human T cells derived from RGD pigs exhibited significant activation, demonstrated by the upregulation of CD25 and CD69 expression, proliferation, and production of inflammatory cytokines, including IL-2, interferon-γ, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF) α, and soluble Fas ligand (sFasL). RGD pig-derived human CD4 T cells were capable of differentiating into various T cell subtypes, including Th1 (induced by IL-12), Th2 (induced by IL-4), Th17 (induced by transforming growth factor-β [TGF-β] and IL-6), and the regulatory T cells (Tregs) (induced by TGF-β). Furthermore, transduction with an anti-human CD19 chimeric antigen receptor (CAR) enabled human CD8 T cells derived from RGD pigs to specifically target and eliminate human CD19+ B cell lymphoma cells. These results confirm that both human CD4 and CD8 T cells developed in RGD pigs are functional. In summary, the RGD pig developed by Yong-Guang Yang and colleagues demonstrates promise in supporting human hematopoiesis and blood cell differentiation. In addition to serving as a large animal model with a human-like immune system, the RGD pig may also function as a potential live bioreactor for producing human hematopoietic and immune cells for clinical therapies. However, in vivo tests, which are crucial for assessing the functions and therapeutic potential of T cells, have not been conducted in the current study. Furthermore, human thymopoiesis and T cell functions could be enhanced through the transgenic expression of human leukocyte antigens (HLAs) in the pig, as the authors suggested in their report. Given its sufficient body size and the absence of immune rejection against human grafts, the RGD pig is anticipated to have significantly broader applications, including but not limited to stem cell-based regeneration studies, the production of human organs or larger organoids, and advancements in regenerative medicine. ACKNOWLEDGMENTS Y.T. is supported the Natural Science Foundation of Jilin Province (YDZJ202201ZYTS101).