NSGS, NSGW41, and MISTRG mice support high-level multilineage human hematopoietic development but show differential maintenance of functional HSCs. Newborn mice were preconditioned (80 cGy) or not (0 cGy), and fetal CD34⁺ cells were transplanted by intrahepatic injection. (A) Blood human CD45⁺ immune cell chimerism measured over time in successfully engrafted mice. The dotted line indicates 10% engraftment; mice below this threshold at 15 weeks were excluded from the analysis. Error bars indicate mean ± standard deviation (SD) (n = 7-23; ****P < .0001 for NSGS vs all groups of mice at week 10; **P = .0248 for NSGS vs MISTRG mice [80 cGy]; and P = .0046 for NSGS vs MISTRG mice [0 cGy] at week 15 using a repeated measure two-way analysis of variance [ANOVA] with Tukey’s multiple comparison test; no significant difference at week 22). (B) Frequency and (C) absolute numbers of hCD45⁺ cells in the BM of recipient mice. Each symbol represents an individual mouse, and the bars indicate (B) mean ± SD or (C) geometric mean ± geometric SD (NSGS mice, n = 4; irradiated MISTRG [80 cGy] mice, n = 12; NSGW41 mice, n = 9; nonirradiated MISTRG [0 cGy] mice, n = 9; one-way ANOVA with Tukey’s multiple comparison test). (D) Composition of human white blood cells 10 weeks after transplantation in the same mice as in panel A (n = 7-23; error bars indicate mean ± standard error of the mean [SEM]). (E) Red blood cell (RBC) counts in the blood of nonhumanized (nonirradiated and noninjected) or successfully humanized mice (from panel A) 10 weeks after transplantation (n = 3-7 for nonhumanized mice; n = 7-23 for humanized mice; unpaired Student t test comparing NSGS and NSGW41 nonhumanized with humanized mice; one-way ANOVA with Tukey’s multiple comparison for MISTRG mice). (F) Survival curves for successfully engrafted mice (from panel A; n = 7-23; log-rank Mantel-Cox test). The arrowheads indicate the time points of blood collection represented in panel A. (G) Representative flow cytometry analysis of human HSPCs (CD34⁺CD38^lo cells pregated on hCD45⁺Lin^– cells) in the BM of the indicated recipient mice. The numbers indicate percentage of total hCD45⁺ cells. (H) Absolute numbers of Lin^–CD34⁺ and Lin^–CD34⁺CD38^lo HSPCs in the BM of the indicated recipient mice. Each symbol represents an individual mouse, and the bars indicate geometric mean ± geometric SD (NSGS mice, n = 4; irradiated MISTRG mice, n = 12; NSGW41 mice, n = 9; nonirradiated MISTRG mice, n = 9; one-way ANOVA with Tukey’s multiple comparison test). (I-J) Analysis of human HSPC function in the BM of irradiated (80 cGy) NSGS and MISTRG recipients performed with total BM cells after mCD45⁺ cell depletion and determined by (I) in vitro CFU assay (n = 4; unpaired Student t test) or (J) by their capacity to repopulate preconditioned (80 cGy) MISTRG mice 19 weeks after secondary transplantation of 1.8 × 10⁶ cells (recipients of NSGS BM, n = 8; recipients of MISTRG BM, n = 9; unpaired Student t test; 1 human donor). To quantify the functional properties of HSPCs in primary recipients of each strain, we decided to transplant these in the same secondary recipients (ie, MISTRG mice, 80 cGy). (K) Serial transplantation every 18 to 21 weeks of CD34⁺ cells from primary to quinary recipient MISTRG (80 cGy) mice, measured by human CD45⁺ cell engraftment in blood. Results from primary to tertiary transplantation show data from 2 independent experiments (2 human cell donors); 1 experiment (1 human donor) thereafter. (L-M) Analysis of human HSPC function within the CD34⁺ cell populations purified from the BM of nonirradiated NSGW41 and MISTRG recipients, determined by (L) in vitro CFU assay (n = 4; unpaired Student t test) or (M) by their capacity to repopulate preconditioned (80 cGy) MISTRG mice 19 weeks after secondary transplantation of 1 to 2.5 × 10⁵ CD34⁺ cells (recipients of NSGW41 CD34⁺ cells, n = 6; recipients of MISTRG CD34⁺ cells, n = 5; unpaired Student t test; data combined from 2 independent experiments with 2 human CD34⁺ cell donors). For in vitro CFU assays, colonies that arose were characterized as burst forming unit-erythrocyte (BFU-E), CFU macrophage (CFU-M), CFU granulocyte (CFU-G), CFU granulocyte-macrophage (CFU-GM), or mixed granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte (CFU-GEMM).