The generation of immunodeficient mice that support the engraftment of human immune systems has enabled the in vivo study of human immune system development and function(1). Early efforts to engraft human immune systems into mice utilized the C.B-17 strain bearing the Prkdc scid ( scid, severe combined immune deficiency) mutation (2), which permitted low levels of human immune cell engraftment after injection with peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) but overall immune system function was limited (1). The development of NOD -scid mice (3) improved engraftment of human immune systems but overall function and levels of take were still suboptimal for the study of human immunobiology (4, 5). The introduction of immunodeficient Prkdc scid ( scid ), or recombination activating 1 or 2 gene ( Rag1 or Rag2 ) knockout mice bearing a mutated IL-2 receptor gamma chain ( IL2r γ null ) facilitated greatly the in vivo engraftment and function of human immune cells (6-9). The IL2rγ-chain is required for high affinity signaling of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 cytokines, and IL2r γ null mice have severe defects in NK-cell activity in addition to T and B cell development (10). These new strains of immunodeficient IL2r γ null mice are now being used for studies of human hematopoiesis, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine (11).
NOD.Cg-Prkdc scid Il2rg tm1Wjl /Sz (NODscid IL2r γ null or NSG) mice support engraftment of human HSC from a variety of sources, including G-CSF mobilized peripheral blood, bone marrow aspirates, umbilical cord blood, and fetal liver (12-15). In vivo human hematopoietic repopulation through transplantation of human CD34+ HSC in NSG recipients allows high levels of human HSC engraftment, differentiation of human T cells in the murine thymus and human B cells, differentiation of human myeloid subsets and human immune function in vivo (12-14, 16-20). A critical aspect for generating HSC-engrafted immunodeficient mice is the use of standardized protocols that enable consistent and robust human immune system development. For example, age of the mouse recipient has important implications for development of human immune cell subsets. One study has shown that newborn NSG mice support more efficient human T cell development after HSC injection than adult NSG mice (8-12 weeks) (21).
HSC-injection into newborn mice is challenging, as the injection sites (intrahepatic, intracardiac and facial vein) require technical expertise and in some instances survival is problematic (6). Here we describe a standard protocol for the engraftment of human HSC into 21-day old NSG mice using an intravenous injection approach. A description of the multi-parameter flow cytometry used to monitor human immune system development is shown. The kinetics of human immune system development in 21-day old NSG mice were compared to those in HSC-engrafted newborn NSG mice.