Introduction
Humanized mice are exceptional models for preclinical testing of cell and gene therapies. Standard mouse models can provide an approximation of human systems, but the murine immune system has important differences from humans. To overcome these limitations, mouse models have been developed with a functioning human immune system to allow human relevant responses. Mice with an engrafted human immune system allow researchers to study cancer therapeutics, immune diseases, infectious diseases, and more in the presence of a human immune system and in an in vivo setting. The generation of highly immunodeficient mice strains like NSG and its derivatives have provided an optimized platform for generation of a variety of humanized models. The term “humanized mice” is used to refer to a several different models. One model is human peripheral blood lymphocyte (hu-PBL) mice. Hu-PBL mice are generated by injecting human peripheral blood mononuclear cells (PBMCs) into immunodeficient mice. PBMCs contain a mixture of different immune cells, including T cells, B cells, and natural killer (NK) cells. Hu-PBL mice tend to have engraftment and expansion of human T cells, with more limited amounts of B cells and other lineages. Another popular model is human hematopoietic stem cell (hu-HSC) mice. These mice are generated by transplanting human CD34+ hematopoietic stem cells into immunodeficient mice. After engraftment, the human stem cells then differentiate into various immune cell types, leading to the development of a more complete and stable human immune system compared to the hu-PBL model.
Noble Life Sciences (Noble) has GLP-compliant animal facilities for preclinical studies and provides humanized mouse models for preclinical research of new immunotherapeutic agents. The flow cytometry facility at Noble has capabilities for multi-color flow cytometric analysis of immune cell type profiles in blood and tissues (e.g., bone marrow, spleen, etc.) with human hematopoietic stem cell engraftment, intracellular cytokine/protein expression in immune/cancer cells, cancer stem cell populations, etc. Other flow cytometry services include (but not limited to) apoptotic assays (e.g., Annexin V/7-AAD), cell cycle analysis, cell proliferation analysis (e.g., BrdU incorporation assay), etc. Noble also offers custom-designed flow cytometry services by designing and validating FACS assays according to the client’s need. Noble offers the full service packages for preclinical studies using hu-PBL or hu-HSC humanized mouse models.
To demonstrate Noble’s capability in the establishment of humanized mouse models, we engrafted two different strains of immunocompromised mice with human CD34+ hematopoietic stem cells. After cell injection, we performed in-house flow cytometry assays to analyze the human cell engraftment and stem cell differentiation over time.
Experimental Design
The experimental design is illustrated in Figure 1. Human CD34+ stem cells used in the study were isolated immunomagnetically from umbilical cord blood using positive selection and cryopreserved in serum-free medium. Cryopreserved cells were thawed, washed with Iscove’s MDM media with 2% FBS and subsequently with HBSS buffer, and finally resuspended in 0.2 ml of HBSS for animal injection. For this study, six female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were irradiated at 175 cgy and then injected intravenously with human CD34+ cells via the animal tail vein. There were two groups of three mice each. The first group was dosed with 5 X 105 cells, and the second group was dosed with 1 X 106 cells. Prior to engraftment, human CD34+ cells were analyzed by flow cytometry assays for examining the expression states of hCD34, hCD45, hCD3, and hCD19. Human PBMCs were also stained to validate the antibodies used to target human immune cell markers. Non-terminal peripheral blood was collected 4, 8, and 12 weeks after engraftment. Animal blood was analyzed for human chimerism, defined as %hCD45/(%hCD45+%mCD45). Mice dosed with 1 X 106 cells were terminated at 6 weeks after engraftment. Mice dosed with 5 X 105 cells were terminated after 18 weeks. At termination, animal bone marrow and spleen samples were collected and analyzed for mouse CD45 and human immune cell markers including CD45, CD3, CD19, CD34, and CD33.

Figure 1. The Diagram for Illustrating the Experimental Study Design.
Results
Characterization of Human CD34+ Stem Cells Using Flow Cytometry Assays
To test whether the antibodies used would show genuine staining signals with the desired cell markers, antibody validation was performed using the human CD34+ cells as well as human peripheral blood mononuclear cells (PBMCs). After staining human CD34+ cells with the PerCP-Cy5 conjugated hCD34 antibody and the APC conjugated hCD45 antibody, the cells showed strong positivity for both CD34 and CD45 markers, consistent with the hematopoietic stem cell phenotype (Figure 2A). There were two cell populations present in isolated human CD34+ cells, hCD34highhCD45+ and hCD34mediumhCD45+ populations (Figure 2A). Both cell populations together accounted for approximately 94%. The hCD34highhCD45+ cell population was predominant in isolated cells, accounting for 86.5% (Figure 2A). 99.5% of hCD34highhCD45+ cells were negative for hCD3 (the T lymphocyte marker) and hCD19 (the B lymphocyte marker) (Figure 2B), indicating that the cells were undifferentiated prior to injection. To test antibodies for human immune cell lineage markers, human PBMCs were stained and analyzed by flow cytometry assays. The PE-Cy7 conjugated hCD19 antibody showed a positive staining signal specific for human B cells (hCD45+hCD19+ cells), and the FITC conjugated hCD3 antibody showed a positive staining signal specific for human T cells (hCD45+hCD3+ cells). The PE conjugated hCD33 antibody showed a positive staining signal specific for human myeloid cells (hCD45+hCD33+ cells). These antibody validation assays have demonstrated that these antibodies used in the study have validated specificities to their respective cell antigen markers.

Figure 2. Validation of antibodies targeting human hematopoietic lineage-specific cell markers. Human CD34+ cells and peripheral blood mononuclear cells (PBMCs) were stained with fluorescent dye-conjugated antibodies for human CD34, CD45, CD3, CD19, and CD33. (A) Gated single human CD34+ cells were analyzed for CD34 and CD45. (B) Gated hCD34+hCD45+ cells shown in A were analyzed for hCD3 (the human T lymphocyte marker) and hCD19 (the human B lymphocyte marker). (C) Gated hCD45+ human PBMCs were analyzed for hCD3 and hCD19. (D) Gated hCD45+ human PBMCs were analyzed for hCD33, a marker for myeloid-lineage cells.
Short-Term Analysis of the Human CD34+ Stem Cell Engraftment in NSG Mice
The animal group (n = 3) dosed with the higher number of human CD34+ cells (1 X 106) was designed for short-term monitoring of human CD34+ cell engraftment in NSG mice. Human cell engraftment was measured in the blood at 4 weeks after injection, and in the bone marrow at termination. Samples were stained with fluorescence-conjugated antibodies for human CD45 (hCD45) and mouse CD45 (mCD45). Human cell engraftment was defined as the percentage of human CD45+ cells divided by the percentage of total mouse and human CD45+ cells according to the following equation:
For the high-dose mouse group shown in Figure 3, human cell engraftment in animal blood was 9.00 ± 6.27% (the error is standard deviation) at 4 weeks post-injection. When the high-dose mice were euthanized at 6 weeks post-injection, human cell engraftment in the bone marrow was 36.81 ± 11.56% (Figure 3).

Figure 3. Short-term engraftment analysis. (A) Three NSG mice injected with 1 X 106 of human CD34+ cells were subjected to blood collection at 4 weeks post-engraftment and their blood samples were analyzed for mouse CD45 (mCD45) and human CD45 (hCD45) using flow cytometry assays. A representative flow dot plot from a mouse is shown here. (B) These three mice dosed with 1 X 106 human CD34+ cells were euthanized at 6 weeks post-engraftment and their bone marrow samples were collected from femur bones, which were analyzed for mCD45 and hCD45 using flow cytometry assays. A representative flow dot plot from a mouse is shown here. (C) The bar graph analysis of human CD45+ cell engraftment in animal blood and bone marrow. The error bars indicate standard errors (SE).
Long-Term Analysis of the Human CD34+ Stem Cell Engraftment in NSG Mice
For three NSG mice dosed with 5 X 105 human CD34+ cells, human CD45 cell engraftment was measured in the blood at 4, 8, and 12 weeks after cell injection. Peripheral blood was collected from the retroorbital sinus and stained for hCD45 and mCD45. As shown in Figure 4A, human CD45 cell engraftment in blood was stable from 4-8 weeks and then decreased significantly between 8 and 12 weeks.

Figure 4. Long-term monitoring analysis of human immune cell engraftment in NSG mice. (A) Human CD45 cell engraftment in animal blood. The engraftment percentages of human CD45+ cells in peripheral blood collected at 4, 8, and 12 weeks post-engraftment from three mice dosed with 5 X 105 cells are analyzed in the bar graph. (B, C) Human immune cell engraftment in animal bone marrows and spleens. The mice of the low-dosed group were terminated at 18 weeks post-injection and their bone marrows and spleens were harvested. After tissue cell dissociation processing, single tissue cells were stained for mCD45, hCD45, hCD34, hCD3, hCD19, and hCD33. The human CD45 cell engraftment data based on staining of mCD45 and hCD45 are presented in the bar graph as shown in (B). The percentages of three different immune cell lineages (hCD45+hCD3+ for T cells, hCD45+hCD19+ for B cells, and hCD45+hCD33+ for myeloid cells) in the gated human CD45+ cell population obtained from assessing bone marrow and spleen cell samples after immunostaining assays are presented in the grouped bar graph as shown in (C). The data represents the average of three mice with standard error (SE) bars. The symbol (**) indicates the p value < 0.01.
Mice dosed with 5 X 105 human CD34+ cells were terminated 18 weeks after injection. Bone marrow and spleen samples were collected, and their prepared single tissue cells were stained with antibodies for detecting mCD45, hCD45, hCD34, hCD3, hCD19, and hCD33. The gated human CD45+ cells were analyzed for hCD3, hCD19 and hCD33 to show the differentiation of human immune cell lineages in the mice from human CD34+ cells. The results from flow cytometry analyses showed that human CD45 cell engraftment tended to be higher in the spleen than the bone marrow after 18 weeks although it is not statistically significant due to the small sample size (Figure 4B). Analyses of differentiated immune cell lineages in the gated human CD45+ cell population showed that the spleen tended to mainly contain human T (44.93 ± 26.94%) and B (31.35 ± 12.57%) lymphocytes, whereas the bone marrow tended to contain all three immune cell lineages for T (11.80 ± 10.38%), B (45.44 ± 11.25%) and myeloid (31.87 ± 4.00%) cells (Figure 4C and Figure 5).

Figure 5. Flow cytometry analysis of Long-term engraftment and differentiation in a representative mouse dosed with 5 X 105 of human CD34+ cells. A, B, C are bone marrow. D, E, F are spleen. Analysis of human CD34+CD45+ cells is shown in A and D. Analysis of human CD45+CD3+ T cells and CD45+CD19+ B cells is shown in B and E. Analysis of human CD45+CD33+ myeloid cells is shown in C and F.
The comparison of human immune cell engraftments between NSG and NBSGW mouse models.
To demonstrate human immune cell engraftment in an alternative mouse strain, NOD.Cg-KitW-41JTyr+ PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice were also engrafted with human CD34+ cells at Noble. These mice do not need to be irradiated prior to injection, as they are more severely immunocompromised than NSG mice and therefore more tolerant to engraftment by human cells.
Table 1. Human immune cell engraftment comparison between NSG and NBSGW mouse strains after implantation for 18 weeks.
1The total engraftment percentage in the bone marrow and spleen is calculated according to the equation stated in the text.
2The percentage of B cells is defined as %hCD19+ of the gated hCD45+ cells.
3The percentage of T cells is defined as %hCD3+ of the gated hCD45+ cells.
4The percentage of myeloid cells is defined as %hCD33+ of the gated hCD45+ cells.
The NBSGW mice showed higher levels of human CD45 cell engraftment than their NSG counterparts (Table 1). However, flow cytometry analysis of the NBSGW bone marrow and spleen at termination showed that the implanted human CD34+ stem cells had differentiated primarily into CD19+ B cells, with a deficiency of T cells (Table 1). On the other hand, the implanted human CD34+ stem cells had been able to differentiate into various immune cell lineages including B lymphocytes, T lymphocytes and myeloid cell types in NSG mice (Table 1).
Conclusion
Through this study, we have demonstrated the process of creating a humanized mouse model by injecting irradiated NSG mice with human CD34+ stem cells. The high-dose group mice injected with 1 X 106 human CD34+ cells showed the greatest human cell engraftment over a shorter period of time. The low-dose group mice injected with 5 X 105 human CD34+ cells showed moderate engraftment which decreased after around 2 months. In the bone marrow of NSG mice, human CD34+ stem cells were able to differentiate into B and T lymphocytes, and myeloid cells. In contrast, human CD34+ stem cells preferentially differentiated into B and T lymphocytes within the spleen of NSG mice.
The NBSGW mouse strain is a convenient animal model offering a much higher engraftment efficiency for human CD34+ cell implantation when compared to the NSG mouse strain, consistent with findings from McIntosh et al. (2015). However, the implanted human CD34+ stem cells preferentially differentiate into B lymphocytes rather than T lymphocytes in the bone marrow and spleen, which renders this humanized mouse model not suitable for studies requiring the involvement of human T lymphocytes. Deficiency in NBSGW mice for human T cell differentiation has been reported elsewhere (McIntosh et al., 2015; Hess et al., 2020), which is mostly likely due to thymic atrophy in this immunocompromised mouse strain. In contrast, humanized NSG mice are a more effective model for studies investigating the maturation and development of human T cells.
References
Hess NJ, Lindner PN, Vazquez J, Grindel S, Hudson AW, Stanic AK, Ikeda A, Hematti P, Gumperz JE. Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source. Front Immunol. 2020 Oct 19;11:573406. doi: 10.3389/fimmu.2020.573406.
McIntosh BE, Brown ME, Duffin BM, Maufort JP, Vereide DT, Slukvin II, Thomson JA. Nonirradiated NOD,B6.SCID Il2rγ-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Reports. 2015 Feb 10;4(2):171-80. doi: 10.1016/j.stemcr.2014.12.005.