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“Humanized” mice are a promising translational model for studying human hematopoiesis and immunity. Their utility has been enhanced by the development of new stocks of immunodeficient hosts, most notably mouse strains such as NOD-scid IL2rγ null mice that lack the IL-2 receptor common gamma chain. These stocks of mice lack adaptive immune function, display multiple defects in innate immunity, and support heightened levels of human hematolymphoid engraftment. Humanized mice can support studies in many areas of immunology, including autoimmunity, transplantation, infectious diseases, and cancer. These models are particularly valuable in experimentation where there is no appropriate small animal model of the human disease, as in the case of certain viral infections. This unit details the creation of humanized mice by engraftment of immunodeficient mice with hematopoietic stem cells or peripheral blood mononuclear cells, provides methods for evaluating engraftment, and discusses considerations for choosing the appropriate model system to meet specific goals.
“Humanized” mouse models of immunity refer to normal, immunocompetent mice expressing human genes via transgenesis (e.g., HLA or human immunoglobulin transgenic mice) or to immunodeficient mice engrafted with human hematopoietic and lymphoid cells or tissues. The protocols presented in this unit describe approaches for generating the latter type of humanized mice. Two basic protocols describe generating humanized mice: Basic Protocol 1 deals with hematopoietic stem cell (HSC) engraftment (human SCID repopulating cell; hu-SRC) and Basic Protocol 2 addresses engraftment with human peripheral blood mononuclear cells (PBMC) (human peripheral blood leukocyte; hu-PBL). Additionally, Alternate Protocols 1 and 2 respectively describe procedures for creating hu-SRC and hu-PBL severe combined immunodeficient (SCID) mice that differ from Basic Protocols 1 and 2 in timing and/or route of engraftment with human cells. Finally the Support Protocol outlines the steps needed to verify engraftment levels in humanized mice. These methods are complimentary, each with its individual strengths and limitations, and can be used to address different experimental questions. Creation of another type of humanized mouse, the SCID-hu mouse, a model where human fetal thymus and liver tissues are transplanted into the renal subcapsular space of immunodeficient mice, is detailed in UNIT 4.8.
The main advantage of the HSC engraftment model (hu-SRC-SCID) is that the human T and B cells develop from human stem cells engrafted in the mouse, undergo negative selection during differentiation into T and B cells, and are therefore tolerant of the mouse host. This model allows for investigation of hematopoietic lineage development and mechanisms of immune system development and the generation of primary immune responses by a naïve immune system.
The PBMC model (hu-PBL-SCID) utilizes leukocytes isolated from peripheral whole blood or spleen and allows for rapid analysis of human immune function because the transferred lymphocytes are functionally mature. This model is best suited for studies of immune function from patients with immunologic disorders, analyses of antigen recall responses, investigations of allograft rejection, and other short-term (~4-week) experiments. The sources and availability of HSC and PBMC, methods of engraftment, and evaluation of reconstitution are considered in this unit.
NOTE: Human tissues, mice engrafted with human tissues, as well as the mouse bedding and caging from humanized mice, should be considered potential biological hazards and handled with proper personal protective equipment at animal biosafety level 2 (ABSL2), in accordance with governmental and institutional biosafety guidelines.
NOTE: Immunodeficient mice should be housed in a specific pathogen free (SPF) environment, using sterile techniques and microisolator caging. See UNIT 1.2 for details on care of immunodeficient mice.
There are several factors to address before commencing with the generation of humanized mice. Outlined below are several key areas to consider. Additional details regarding vendors for immunodeficient mice and mechanisms for obtaining human tissues are provided in the Internet Resources section at the end of this chapter.
Several reports have now been published using BALB/c and NOD strain immunodeficient mice that lack the IL-2r common γ chain. While there is no data in the literature directly comparing these strains (NOD.Cg-PrkdcscidIl2rgtm1Wjll/SzJ, NOD.Cg-PrkdcscidIl2rgtm1Sug, C.129(Cg)-Rag2tm1FwaIl2rgtm1Sug, and (H2d)-Rag2tm1Fwa Il2rgtm1Krf), published reports appear to indicate similar levels of engraftment and immune function after reconstitution with HSC (Gimeno et al., 2004; Traggiai et al., 2004; Ishikawa et al., 2005; Shultz et al., 2005). In addition, (C57BL/6J × C57BL/10SgSnAi)-[KO]γc-[KO]Rag2 are also commercially available, but there have been no published reports of their successful use as recipients of human HSC or PBMC.
As of this writing, for immunodeficient IL2rγ null mice that have been used as recipients of human cells and tissues, only NOD/Lt-scid IL2rγ null mice are widely available from a mouse repository. For investigators desiring to use BALB/c-Rag2 null IL2rγ null mice, there are three approaches for obtaining this strain: (1) Breeding stock can be requested from Dr. M. Manz (see Table 15.21.1). (2) Investigators can generate this strain by crossing the commercially available C.129S6(B6)-Rag2tm1Fwe with C.129S4-Il2rgtm1Wjl/J (BALB/c-IL2rγ null cryopreserved embryos. (3) Another approach would be using the C.129S7(B6)-Rag1tm1Mom/J strain in place of the BALB/c-Rag2 null strain (see Internet Resources). Many previous strains of immunodeficient hosts, particularly those based on the NOD/Lt-scid or NOD/Lt-Rag1 null strains, are available from The Jackson Laboratory, and these mice can also be readily obtained. See Table 15.21.1 for more information.
Hematopoietic stem cells for use in generating humanized mice can be obtained from several different tissues, including umbilical cord blood (UCB; Ito et al., 2002; Traggiai et al., 2004; Ishikawa et al., 2005), bone marrow (Holyoake et al., 1999) G-CSF-mobilized peripheral blood (Shultz et al., 2005), and fetal liver (Holyoake et al., 1999). Human HSC isolated from UCB and mobilized stem cells have been used to generate a human immune system in BALB/c-Rag2 null IL2rγ null, NOD/Lt-scid IL2rγ null, and NOD/Shi-scid IL2r null mice. While a direct comparison of these stem cell sources has not yet been reported, all appear capable of generating a human immune system following human HSC engraftment.
Obtaining human HSC for research purposes requires appropriate Institutional Review Board (IRB) approval. UCB is an abundant tissue source for HSC and is easiest to obtain for most investigators, as most umbilical cords are considered by IRBs as discarded tissue that does not require informed consent. Chapter 22 provides methods of tissue sample collection and HSC enrichment and isolation. Bone marrow aspirates and G-CSF mobilized peripheral blood samples are most often obtained from volunteers following informed consent, and they require controlled harvesting procedures necessitating coordination with a clinical trials unit or hematology department at a medical center. Finally, human tissue banks such as the NDRI (see Internet Resources) provide a nonprofit fee-for-service resource for obtaining human tissues and HSC.
Obtaining PBMC samples for engraftment into immunodeficient mice is relatively straightforward. With appropriate IRB approval, volunteers (following informed consent) can be enrolled to donate peripheral whole blood, which can be collected via standard venipuncture techniques by a trained phlebotomist (see APPENDIX 3F and UNIT 7.1). Multiple individuals can be enrolled, minimizing the need to perform repeated draws on the same donor. Approximately 150 ml of whole blood collected in a single draw will provide enough PBMC to engraft into 10 to 15 immunodeficient NOD/Lt-scid IL2rγ null mice using a dose of 20 × 106 cells/mouse. Each recipient should receive PBMC from an individual donor.
This protocol describes the generation of HSC-engrafted immunodeficient mice using a simple method: the intravenous injection of T cell–depleted UCB containing at least 3 × 104 CD34+ hematopoietic stem cells into irradiated adult immunodeficient mice. The mouse strain used for all basic protocols in this unit is the NOD/Lt-scid IL2rγ null strain. HSC engraftment of newborn mice is described in Alternate Protocol 1.
This protocol describes engraftment of NOD/Lt-scid IL2rγ null mice with PBMC via the intravenous route. Previous generations of immunodeficient host strains did not support human PBMC engraftment following intravenous injection but were readily engrafted following intraperitoneal injection of human PBMC. The ability of NOD/Lt-scid IL2rγ null mice to support PBMC engraftment via intravenous injection allows the injection of PBMC directly into the host’s circulation as opposed to requiring migration of the engrafted PBMC from the peritoneal cavity to the circulation. Alternate Protocol 2 describes an additional method for delivering PBMC directly into the spleen to achieve PBMC engraftment in the circulation of NOD/Lt-scid IL2rγ null and other immunodeficient strains of mice.
HSC engraftment of newborn mice is commonly performed and requires only modest increases in time, resources, and technical proficiency. The protocol can use one of a number of different engraftment routes that include intrahepatic, intravenous via the facial vein, intravenous via the intracardiac route, and intraperitoneal injections. This protocol will describe two of the most technically simple newborn engraftment routes that lead to robust human HSC engraftment: intrahepatic and intracardiac injection.
NOD-scid/Lt IL2rγ null mice support equivalent levels of PBMC engraftment via intravenous, intraperitoneal, or intrasplenic routes, so investigators can choose the most appropriate route of injection for their studies. The procedures for delivering PBMC via the intrasplenic route is provided in this protocol.
Flow cytometry is a convenient method for evaluating human cell engraftment in humanized mice. The unique aspects of the method for achieving interpretable results in chimeric humanized mice are highlighted in this protocol. All anti-human antibodies are potentially useful for evaluating human leukocyte engraftment in humanized mice, with the only limitation being the ability of the cell type bearing a particular antigen to engraft and develop in immunodeficient mice. All antibodies should be first titered on a 1:5 mixture of human PBMC and splenocytes from a nonengrafted NOD/Lt-scid IL2rγ null mouse to ensure specificity and non-cross-reactivity with mouse cells. The protocol below outlines a four-color analysis of whole blood, but staining other lymphoid/hematopoietic tissues and scale-up to six parameter or greater analysis can easily be accomplished. Antibodies specific for murine CD45 are used for the single color controls to ensure a strong signal when calibrating the flow cytometer. The approach for flow cytometric analysis of human cell engraftment is described in detail for assessment of chimerism in the blood. Similar methodology can be used to determine human cell engraftment in the spleen, bone marrow, thymus, and lymph nodes of the engrafted mice.
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.
The ability to engraft mice with human hematopoietic tissues was first made possible by the discovery of the scid mutation in the CB17 strain mice over 20 years ago (Bosma et al., 1983). These mice were found to be B and T cell deficient as a result of a mutation in the gene encoding the DNA repair enzyme protein kinase, DNA-activated, catalytic polypeptide (Prkdcscid, hereafter referred to as scid). However, CB17-scid mice supported only low levels of human hematopoietic cell engraftment due to a robust innate immune system (Shultz et al., 2007). The intervening twenty-plus years of research in humanized mice has focused on improving human hematopoietic engraftment by decreasing levels of innate immunity, particularly in immunodeficient NOD strains (Shultz et al., 2007). Creation of scid, Rag1 null, or Rag2null mice that also lack the IL-2rγ subunit (γc, CD132) are the new “gold standard” as immunodeficient hosts for human cell and tissue engraftment. This is because they support higher levels and greater multilineage development of human hematopoietic cells than any of the previous generations of immunodeficient hosts (Ito et al., 2002; Traggiai et al., 2004; Ishikawa et al., 2005; Shultz et al., 2005; Watanabe et al., 2006). The different strains of immunodeficient mice that have been cited in the published literature and are available for “humanized” mouse studies are be discussed below.
Humanized mice are of great interest to immunologists who are focused on translating basic research findings into clinical applications. The need for humanized mice evolved from several key needs: first, human and rodent immune systems are different, and results obtained in animal models have not always translated into human therapies; additionally, there are a number of human diseases that don’t have appropriate animal models, or the animal models that do exist have significant differences from the human counterpart. Examples of these situations include viral infections such as dengue and human immunodeficiency virus, which do not replicate in murine tissues, but have been reported to infect humanized mice (Hesselton et al., 1995; Bente et al., 2005; Watanabe et al., 2006). While many exciting preliminary reports have demonstrated the utility of humanized mice, it is important to remember that humanized mouse models are still under development, and efforts continue to improve the models to facilitate human cell engraftment and function.
NOD/Lt-scid IL2rγ null, NOD/Shi-scid IL2rγ null, and BALB/c-Rag2 null IL2rγ null mice represent a technological leap over previous generations of immunodeficient hosts as recipients of human hematolymphoid cells. Most importantly, these IL2r null stocks of immunodeficient mice support high levels of de novo human T cell development from stem cell precursors, previously a rare event in other immunodeficient strains of mice. Additionally, in earlier models of hu-PBMC-SCID mice, reliable engraftment required much higher cell doses, and engraftment via the intravenous route was not routinely achieved.
Humanized immunodeficient mice can be used to study a number of immunologic and hematologic processes. However, it is important to note that the protocols presented in this unit are specifically directed towards the engraftment of NOD/Lt-scid IL2rγ null mice with a human immune system for the generation of functional T and B cell responses. Other uses of this model, including engraftment with human stem cells of nonhematopoietic origin or human tumor cells, may require additional optimization.
The two basic humanized mouse models presented in this chapter offer the investigator a choice when developing an experimental model system. The decision of which model to use must be based on the particular experimental objective and the hypothesis to be tested. The hu-PBL-SCID model is well suited for examining the function of mature cells of the immune system; it has been employed to study islet and skin allograft rejection (Turgeon et al., 2003; Banuelos et al., 2004). The hu-PBL-SCID model system also has utility for the study of recall responses to vaccinations or infection (Lim et al., 2007) and for investigation of hematological malignancies (Fujii et al., 2007). The HSC-engrafted humanized mouse model (hu-SRC-SCID) is useful for studying questions related to immune system development or hematopoiesis, and virtually all areas of immune function (Shultz et al., 2007). In many cases, it may be advantageous for investigators to pursue testing their hypothesis in both model systems.
There are many publications reporting the study of immunodeficient mice engrafted with human cells and tissues. However, many different strains of mice have been used in these reports, and to reproduce these data, investigators must take notice of the precise model system used. Even though the immunodeficient IL2rγ null mouse models have gained in popularity, many studies continue to use other immunodeficient mouse hosts, including CB17-scid and NOD/Lt-scid mice. Furthermore, there are different genetic stocks of IL-2rγ null immunodeficient hosts, including those that differ in host background (NOD/Shi-scid versus NOD/Lt-scid versus BALB/c-Rag2 null) and the targeted mutation used to disrupt the IL-2rγ subunit, the fully null IL2rgtm1Wjl (Cao et al., 1995) and the truncated IL2rgtm1Sug (Ito et al., 2002). A caveat regarding the use of NOD/Lt-scid IL2rγ null mice is that the strain is still being characterized; adapting many protocols (e.g., treatment with anti-CD122 monoclonal antibody) devised using other host strains (e.g., NOD-scid) has not in all cases been thoroughly investigated. However, previous generations of immunodeficient hosts remain accessible, and an excellent overview of immunodeficient hosts available for humanized mouse studies can be obtained from the Jackson Laboratory Web site (see Internet Resources). On the other hand, given the high levels of human cell engraftment that are achieved in NOD-scid IL2rγ null mice, this strain is strongly recommended as the strain of choice for studying virtually all aspects of human immune function in humanized mice.
When deciding between using newborn or adult HSC engraftment model systems, there are additional practical considerations. First, there is a cost advantage to newborn engraftment methods, because newborns stay with parents until weaning, thus reducing per diem housing fees. Second, an additional advantage of the newborn engraftment protocol is that a humanized immune system is developed at a younger absolute age (≥ 12 weeks of age versus 17 to 24 weeks of age in mice engrafted as adults).
The major drawback to the newborn engraftment protocol is the need for increased resource management. For example, the constant monitoring of breeder cages for new litters can be labor intensive, especially if multiple litters are needed for experiments within a narrow time frame. One potential solution to this problem is to set up timed matings, which has the benefit of allowing for more precise scheduling of experiments. The protocol for timed matings has been described by Nagy (2003).
Another drawback to using the newborn protocol is the timing of newborn litters with availability of the human HSC for injection. However, HSC from umbilical cord blood or bone marrow can be T cell depleted, frozen in aliquots in liquid nitrogen, and thawed immediately prior to injection. This approach overcomes the major logistical problem of coordinating the timing of obtaining and preparing HSC samples and having recipient mice ready for engraftment.
In immunodeficient NOD-scid IL2rγ null mice, excellent engraftment with mature human T cells in the hu-PBL-SCID model has been observed (King et al., 2008). Because of this, in the HSC engraftment model, extensive T cell depletion of cord blood must be achieved to prevent the engraftment and expansion of mature human T cells that could lead to GVHD. This phenomenon is readily detectable by flow cytometry, where up to 90% of the CD45+ cells can be CD3+ mature T cells in mice engrafted with non/T cell–depleted cord blood HSC populations. Mice exhibiting a mature T cell outgrowth often appear ill, with hunched posture and weight loss characteristic of GVHD. To minimize GVHD, efficient T cell depletion of HSC should be verified by flow cytometry prior to injection of the HSC into mice.
In many cases, it is desirable to inject HSC populations that are highly enriched for CD34+ cells rather than injecting T cell-depleted HSC populations. For example, an experimental protocol may involve transduction of human CD34+ hematopoietic stem cells with adenovirus or lentivirus prior to engraftment in immunodeficient hosts. For these experiments, enrichment of the CD34+ cell population can be achieved using lineage depletion or positive selection methodology such as Miltenyi or Stem Cell Technologies cell separation methodology (see Internet Resources). These methodologies are widely used protocols for the positive selection and enrichment of human CD34+ cells. Enrichment of CD34+ cells would reduce the number of cells that will need to be transduced. This will reduce the amount of virus required for efficient transduction; high titer stocks of these viruses are often difficult to generate and are expensive. In these experiments, the absolute number of human CD34+ cells to be injected should be increased to 1 × 105/mouse to achieve optimal engraftment.
Irradiation doses will vary with the strain used and even the dose needed within the same strain may vary between investigators and facilities. A dose of 240 cGy represents a conservative dose for preconditioning adult NOD/Lt-scid IL2rγ null mice, as levels of whole body irradiation (WBI) up to 325 cGy have been reported in this strain (Shultz et al., 2005). Rag1 null or Rag2 null mice are more radioresistant than scid mice and require higher irradiation doses. Indeed, newborn BALB/c-Rag2 null IL2rγ null mice withstand 400 cGy of split dose WBI for HSC engraftment (Traggiai et al., 2004) while 100 cGy of WBI is sufficient for HSC engraftment in newborn NOD/Lt-scid IL2rγ null mice (Ishikawa et al., 2005).
Finally, the protocols in this chapter describe only the generation of humanized mice and their phenotypic characterization by flow cytometry. However, this only represents the jumping off point for humanized mouse studies. Many protocols for improving human PBMC and HSC engraftment have been reported using previous generations of humanized mice. These protocols include depletion of mouse macrophages or granulocytes prior to engraftment, treatment of engrafted mice with human cytokines, or co-engraftment with mesenchymal stem cells (Pearson et al., 2008). In addition, other routes of engraftment have been reported in previous models of humanized mice to enhance engraftment (Shultz et al., 2007). However, the use of these alternative engraftment routes have not yet been described in immunodeficient IL2rγ null mice. Each investigator must determine what scientific questions he or she wishes to address and then tailor the assays and functional readouts to specific needs. This aspect of humanized mouse studies represents the real challenge (but also potential benefit) of this system.
In PBMC-engrafted NOD/Lt-scid IL2rγ null mice, a single injection (intravenous, intraperitoneal, or intrasplenic) of 2 × 107 cells typically results in 15% to 45% human CD45+ cells in the peripheral blood within 4 weeks. This percentage of human CD45+ cells in the blood will correspond to approximately 50% human CD45+ or 2–3 × 107 total human CD45+ cells engrafted in the spleen at 4 weeks. Virtually all PBMC injected NOD/Lt-scid IL2rγ null mice engraft, unless the injection itself is a technical failure. The majority of the human CD45+ cells will be CD3+ T cells, with a memory/effector CD45RO phenotype and a CD4:CD8 ratio of ~1:1 at 4 weeks post-engraftment. At earlier time points, the CD4:CD8 ratio will be higher, ~2 to 3:1 but reaches a stable 1:1 ratio by 3 weeks post-engraftment. It is currently unknown if this represents an attrition of CD4+ T cells or the late preferential expansion of CD8+ T cells. B cells are detectable in the spleens of PBMC-engrafted mice, but only at low levels (0.5 to 1.0%). B cell engraftment is confirmed by the persistence of human Ig in the serum of engrafted mice. The serum Ig levels in PBMC-engrafted NOD/Lt-scid IL2rγ null mice remains high over the 4-week period of study (unpublished data).
PBMC-engrafted NOD/Lt-scid IL2rγ null mice will begin to exhibit symptoms of xeno-GVHD, such as weight loss and hunched posture sometime after 4 weeks following injection of human PBMC. The development of GVHD is a major limiting factor for long-term studies using the hu-PBL-SCID model. Engraftment of cell lineages in addition to B and T cells is poor and/or brief in PBMC-engrafted NOD/Lt-scid IL2rγ null mice. While some antigen presenting cells are a component of the initial PBMC inoculum, they are undetectable using flow cytometry by 4 weeks after PBMC injection.
Previous generations of hu-PBL-SCID mice were hindered by high levels of variability in engraftment from donor to donor. The NOD/Lt-scid IL2rγ null stock appears to have overcome this problem, with uniformly high engraftment in the spleen 4 weeks after IV injection of PBMC, regardless of the donor used. It is important to note that engraftment in the peripheral blood of NOD/Lt-scid IL2rγ null mice may show some donor-to-donor variability, but splenic human PBMC engraftment appears to be extremely reproducible between individual mice and between different donors. In summary, the NOD/Lt-scid IL2rγ null mouse is a much more robust host for human PBMC engraftment than previously available immunodeficient strains of mice.
The level of human hematopoietic development in HSC-engrafted NOD/Lt-scid IL2rγ null mice is dependent on several factors and will, in most cases, be more variable than PBMC engraftment. First, the CD34+ cell dose will have a large impact on total human CD45+ cell engraftment levels in the periphery of mice at 12 weeks after HSC injection. CD34+ cell inoculums as low as 3 × 104 per recipient give reliable, but highly variable, levels from less than 1% to greater than 30% human CD45+ in the peripheral blood at 12 weeks post-engraftment. Increasing the CD34+ dose will generally increase the total CD45 engraftment at the time of analysis. The 10- to 12-week time point is used because it is the earliest point at which reliable human T cell generation can be detected, but total CD45 levels and human B and T cell levels will continue to increase over the following weeks. Human CD45+ cells are detectable in the blood of HSC-engrafted mice by as early as 4 weeks post-engraftment, but at low levels, and CD3+ T cells are not present.
Head to head comparisons of NOD/Lt-scid IL2rγ null mice engrafted with the same HSC source as newborns and adults reveals that the total CD45+ cell levels are roughly equivalent. However, T cell development is superior in newborn engrafted mice as compared to comparably engrafted adult mice (T. Pearson, un-pub observ.). A comparison of NOD/Lt-scid IL2rγ null mice engrafted as newborns via the intracardiac or intrahepatic routes of injection revealed essentially similar levels of human CD45+ cell engraftment and T cell development (T. Pearson, unpub. observ.). Facial vein injection of newborns also can also be used. However, this method is technically more challenging.
Human CD45+ cell engraftment levels after HSC injection are also variable depending on the lymphoid tissue examined. Blood, while a convenient tissue for evaluating human cell engraftment, contains human cells at lower proportions than is observed in other hematopoietic lymphoid tissues. The highest engraftment of human cells is observed in the bone marrow, where human CD45+ cell levels of greater than 75% are commonly observed following an original injected cell dose of 3 × 104 CD34+ cells in a T cell–depleted UCB. Therefore, using human CD45+ levels in the blood as a tissue for evaluating engraftment in the immunodeficient mice will underestimate total engraftment levels and may yield some false negatives. Intermediate between blood and bone marrow is the spleen, with CD45+ cell engraftment levels typically ranging from 25% to 75% of nucleated splenocytes.
Multilineage hematopoietic development and stable engraftment is much more reliable in HSC-engrafted NOD/Lt-scid IL2rγ null, NOD/Shi-scid IL2rγ null and BALB/c-Rag2 null IL2rγ null mice than in any of the previous generations of immunodeficient hosts (Shultz et al., 2007). Importantly, human myeloid and plasmacytoid dendritic cells are generated, as well as T and B cell populations and other human leukocyte subsets. Table 15.21.3 provides a list of lineages that have been reported to be present in HSC-engrafted NOD/Lt-scid IL2rγ null mice. To date, no direct comparisons of HSC or PBMC engraftment between NOD/Lt-scid IL2rγ null and BALB/c-Rag2 null IL2rγ null mice have been reported. However, published studies suggest that all three strains are well suited as hosts for a human immune system generated by engraftment of HSC (Ito et al., 2002; Traggiai et al., 2004; Shultz et al., 2005; Ishikawa et al., 2006). Future studies should continue to reveal the optimal host and protocol for creating humanized mice.
The time required to engraft NOD/Lt-scid IL2rγ null mice with human cells (either PBMC or HSC) depends on the method of engraftment used and the degree of input cell preparation needed.
HSC engraftment of adult NOD/Lt-scid IL2rγ null mice using a standard intravenous injection (Basic Protocol 1) requires a 4-hr resting period for the recipient mice after WBI, which is the most important timing consideration for this protocol. The need for this resting period has been associated with increased expression of stromal derived factor-1 (SDF-1), a chemoattractant for HSC that is produced by bone marrow stromal cells and is up-regulated within 4 hr after irradiation (Ponomaryov et al., 2000).
The time for irradiation will vary as a function of the rate of γ emission of the 137Cs source. However, exposure to 240 to 325 cGy will take less than 10 min for most irradiators and commonly used dose rates. If T cell depleted CD34+ stem cells have been previously prepared and frozen (either in-house or from an outside source such as NDRI), the thawing and dilution of the stem cells can easily be accomplished during the 4-hr period between irradiation of the mice and the time for injection of the human HSC.
If the T cell depletion of the HSC is being performed on the day of injection, the time required will vary according to the protocol being followed, but it should also be completed in about a half day (see Chapter 22 for additional time considerations regarding stem cell isolation).
Following stem cell preparation and the rest time following irradiation, the final time consideration is the time for performing intravenous injections, which will vary depending on individual investigator’s technical proficiency and the number of mice to be injected. In total, Basic Protocol 1 can be accomplished in ~6 hr.
For intravenous engraftment of adult NOD-scid IL2rγ null mice with PBMC (Basic Protocol 2), the major time considerations are the collection and preparation of the PBMC from whole blood, which can be accomplished in 2 to 3 hr, and the time to perform intravenous injections (as described above). Generally, 4 to 5 hr in total should be sufficient for Basic Protocol 2.
Engraftment of newborn NOD/Lt-scid IL2rγ null mice with HSC (by either the intracardiac or intrahepatic routes; Alternate Protocol 1) requires relatively less time to complete because the pups can be injected immediately after irradiation. Preconditioning of newborn BALB/c-Rag2 null IL2rγ null mice with 400 cGy in two split doses of irradiation separated by 3 to 4 hr, followed by a 4- to 12- hr wait before injection of the human HSC after completion of the last irradiation has been reported (Traggiai et al., 2004), suggesting that engraftment of newborn mice, similar to adult mice, may benefit from a 4-hr wait following irradiation before human HSC injection. However, initial experiments suggest that there are no differences in the engraftment levels of human HSC by injection of human HSC immediately after irradiation as compared to waiting for 4 hrs (T. Pearson, unpub. observ.). Immediate injection also eliminates the additional handling and separation of the newborns from their parents.
In addition, the injection techniques take less time to perform than a standard intravenous injection in adult mice. Therefore, the time needed to prepare the HSC for injection is a major time consideration, but can be minimized on the day of injection by having the cells previously prepared and frozen. If this is the case, Alternate Protocol 1 can be accomplished comfortably in a half day.
Intrasplenic injections (Alternate Protocol 2) are more time consuming than the standard intravenous route, with surgical proficiency of the individual investigator being the major consideration. After injection with ketamine/ xylazine, it will take several minutes for the recipient to be deeply anesthetized before the surgical procedure can begin. With practice, it is possible for a single investigator to perform surgeries and injections on several groups of mice in a single day, with each recipient requiring ~10 min, once anesthetized. Efficiency can be maximized when three or four mice can be anesthetized simultaneously and the procedure performed on each recipient before regaining consciousness. Thus, it is reasonable to collect and prepare PBMC from whole blood and perform 12 to 15 intrasplenic injections in a single day.
We thank Drs. Thomas Chase and Bonnie Lyons for critical review of this unit. Supported by the Beta Cell Biology Consortium and Autoimmunity Prevention Centers from NIH, the Juvenile Diabetes Research Foundation, International, the American Diabetes Association, the Diabetes Endocrinology Center, the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and the National Heart, Lung and Blood Institute of NIH. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
The Jackson Laboratory. This Web site provides detailed information on nomenclature, genotyping protocols and phenotypes for several of the immunodeficient strains described in this unit, and is the sole repository and provider of the NOD.Cg-Prkdcscid Il2rgtm1Wjll (NOD-scid IL2rγ null)stock.
Choosing an Immunodeficient Mouse Model, Jax Notes, Spring 2006
Taconic Farm Web sites. A comprehensive overview of immunodeficient hosts for HSC engraftment, including salient features of each strain, availability, and references. Taconic Farms is another supplier of immunodeficient mice, including CB17-scid, NOD-scid, BALB/c-Rag2 null, and “C57BL/6-Rag2 null IL2rgnull (C57BL/6J × C57BL/10SgSnAi)-[KO]γc-[KO]Rag2”
National Disease Research Exchange (NDRI) is a nonprofit resource providing access (through an application process) to human biomaterials for investigators’ research studies.
Miltenyi Biotec is a commercial supplier of cell separation technologies to isolate/enrich for human hematopoietic stem cells.
Stem Cell Technologies is a commercial supplier of cell separation technologies to isolate/enrich for human hematopoietic stem cells.