Mice
NOG [1] (NOD.Cg-prkdcscidil2rgtm1Sug/Jic) and NOG-EXL [3] (NOG-hIL-3/GM-CSF Tg; NOD.Cg-PrkdcscidIl2rgtm1SugTg (SRa-IL3, CSF2)/Jic) mice were previously established in the Central Institute for Experimental Animals (CIEA). NOGW (NOD.Cg-PrkdcscidIl2rgtm1SugKitem1(V831M)Jic) mice were established by genome editing using the transcription activator-like effector nuclease technique [8]. NOG-EXL mice were backcross-mated with NOGW mice to generate NOGW-EXL mice. The mice had access to sterilized food and water ad libitum and were used for human cell transplantation studies at 6–12 weeks of age.
Generation of humanized mice
X-ray irradiation was performed using the MBR-1520R-4 model (Hitachi Power Solutions Co., Ltd., Ibaraki, Japan) to generate humanized mice. Commercially available human cord blood CD34+ cells, purchased from StemExpress LLC (Folsom, CA, USA), were used; the cells were prepared according to the manufacturer’s instructions. Briefly, a cryopreserved CD34+ cell vial was thawed in a water bath at 37°C and immediately transferred to RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), containing 10% fetal calf serum and DNase I (Roche Diagnostics, Basel, Switzerland). The viability of CD34+ HSCs was measured by 2.5% trypan blue staining, and cells with > 90% viability were used for transplantation. Subsequently, these human HSCs were intravenously injected into 1.5-Gy irradiated NOG and NOG-EXL mice, non-irradiated or 1-Gy irradiated NOGW mice, and non-irradiated NOGW-EXL mice.
Flow cytometry
Human or mouse immune cells in the PB, BM, and spleen were analyzed by flow cytometry and stained with anti-human or anti-mouse antibodies. The total cell number in tissues was determined using a Microsemi LC662 hematology analyzer (HORIBA Ltd., Kyoto, Japan). Cells were prepared using BD Pharm-Lyse (BD Biosciences, San Jose, CA, USA) to remove red blood cells and incubated in the dark for 20 min at 4°C with a mixture of fluorescently labeled monoclonal antibodies. After washing with phosphate-buffered saline, the cells were suspended in propidium iodide/RNase staining buffer (BD Biosciences), subjected to multicolor flow cytometry with the LSR Fortessa X-20 instrument (BD Biosciences), and analyzed using FlowJo version 10.6.2 software (BD Biosciences). The engraftment ratio of human cells was expressed as the percentage of human CD45+ cells relative to the total number of leukocytes (mouse plus human), excluding erythrocytes and/or debris. The following antibodies against cell-surface molecules were used: anti-human CD66b-fluorescein isothiocyanate (FITC) (clone G10F5), CD56-FITC (clone HCD56), CD33-phycoerythrin (PE) (clone WM53), CD38-PE (clone HB-7), CD34-PE-Cy7 (clone 581), CD41-PE-Cy7 (clone HIP8), CD16-allophycocyanin (APC) (clone B73.1), CD201-APC (clone RCP-401), CD19-APC-Cy7 (clone HIB19), CD45RA-APC-Cy7 (clone HI100), CD3-Brilliant Violet (BV) 421 (clone UCHT1), CD10-BV421 (clone HI10a), CD45-BV510 (clone HI30), hLineage cocktail-FITC (CD3, CD14, CD16, CD19, CD20, CD56) (clone UCHT1, HCD14, 3G8, HIB19, 2H7, HCD56) (BioLegend, San Diego, CA), and CD14-Brilliant Ultraviolet (BUV) 737 (clone M5-E2) and anti-mouse CD45-BUV395 (clone 30-F11) (BD Bioscience).
Serial BM transplantation
Briefly, 1 × 107 BM cells were isolated from the femurs of humanized mice at 16 weeks, reconstituted with 2.5 × 104 human CD34+ cells, and serially transplanted into 1.5-Gy irradiated NOG mice via the tail vein. The frequency of engrafted human hematopoietic cells in the PB and BM were analyzed by flow cytometry.
Immunohistochemistry
Bone, liver, and lung tissues were harvested, fixed overnight in 10% buffered formalin (FujiFilm Wako, Osaka, Japan), and embedded in paraffin. Tissue sections (3 µm thick) were placed on aminosilane-coated glass slides (Muto Pure Chemicals, Tokyo, Japan) and immunostained using the Bond-Max automated stainer and a BOND Polymer Refine Detection system (Leica biosystems K.K., Tokyo, Japan). After deparaffinization, sections were incubated with an anti-human CD61 antibody (clone; Y2/51) (Agilent Technologies Inc., Santa Clara, CA, USA) for 30 min, and then incubation with polymer for 30 min followed by DAB chromogen for 10 min. To visualize nuclei, immunostained sections were counterstained with hematoxylin (Leica biosystems K.K).
Statistics
Data are presented as the mean ± standard deviation. The significance of differences was evaluated by two-tailed Student’s t-tests or one-way analyses of variance. Statistical analysis was performed using Excel (Microsoft Corp., Redmond, WA, USA) or GraphPad Prism 7 (GraphPad, San Diego, CA, USA). A p-value of < 0.05 was considered statistically significant. The statistical details are shown in the respective figure legends.