There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues.
Freshly isolated fetal liver-derived CD34(+) hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2(-/-)gamma(c)(-/-) mice, frozen and ex vivo expanded CD34(+) cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays.
After fetal liver/thymus tissues were coimplanted into the irradiated Rag2(-/-)gamma(c)(-/-) mice, with frozen and ex vivo expanded CD34(+) hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity.
The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2(-/-)gamma(c)(-/-) mice.
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2(null)Il2rγ(null) strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34(-) cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2(null)Il2rγ(null) humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.
[Show abstract][Hide abstract] ABSTRACT: Immunodeficient mice bearing targeted mutations in the IL2rg gene and engrafted with human immune systems are effective tools for the study of human hematopoiesis, immunity, infectious disease, and transplantation biology. The most robust human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver hematopoietic stem cells (often referred to as the BLT [bone marrow, liver, thymus] model). To evaluate the NOD-scid IL2rγ(null) (NSG)-BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG-BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG-BLT mice develop a fatal graft-versus-host disease (GVHD)-like syndrome, which correlates with the activation of human T cells and increased levels of human Ig. Onset of GVHD was not delayed in NSG mice lacking murine MHC class I or II and was not associated with a loss of human regulatory T cells, or absence of intrathymic cells of mouse origin (mouse CD45+). Our findings demonstrate that NSG-BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD-like pathologic changes.
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cell (HSC) transplantation has the potential to treat a variety of human diseases including genetic deficiencies, immune disorders, and to restore immunity following cancer treatment. However, there are several obstacles that prevent effective HSC transplantation in humans. These include finding a matched donor, having sufficient numbers of cells for the transplant, and the potency of the cells in the transplant. Ethical issues prevent effective research in humans that could provide insight into ways to overcome these obstacles. Highly immunodeficient mice can be transplanted with human HSCs and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multi-lineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. As such, humanized mice serve as a model for human HSC transplantation. A variety of conditions have been analyzed for their impact on HSC transplantation to produce humanized mice, including the type and source of cells used in the transplant, the number of cells transplanted, the expansion of cells with various protocols, and the route of introduction of cells into the mouse. In this review, we summarize what has been learned about HSC transplantation using humanized mice as a recipient model and we comment on how these models may be useful to future pre-clinical research to determine more effective ways to expand HSCs and to determine their re-populating potential in vivo.
Stem cells and development 08/2013; 23(1). DOI:10.1089/scd.2013.0265 · 3.73 Impact Factor
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