We did not observe any significant GVHD in transplanted mice (supplemental Figure 2)

We did not observe any significant GVHD in transplanted mice (supplemental Figure 2). Engraftment of human lymphocytes in the brain after in utero transplantation The ability to transplant human cells prior to formation of the bloodCbrain barrier, which occurs at E15.5 in mice,31 may present an opportunity to engraft human cells within the brain. cells in a nonirradiated host. NSG (NOD-SCID IL2R-null) mice, which are developed with SCID and IL-2R-null chain mutations, are a robust platform for the engraftment of human AMG232 hematopoietic cells because they have no endogenous T, B, or natural killer cells.19-22 In this study, we used IUHCT of human CD34+ cells in NSG mice to create a reproducible mouse model to study stem cell engraftment, differentiation, and systemic repopulation after IUHCT. Methods Mice NSG mice were obtained from The Jackson Laboratory. All procedures were performed according to a University of California, San Francisco Institutional Animal Care and Use CommitteeCapproved protocol. Study design Human cord blood CD34+ cells were either purchased from AllCells (Alameda, CA) or purified from cord blood collected at time of delivery from normal-term infants. CD34+ cells were isolated and transplanted into fetal mice at embryonic day 13.5 (E13.5) or E14.5 as previously described23 with or without preconditioning with ACK2. 17 Chimerism levels were checked at 4-week intervals and at harvest using flow cytometry and immunohistochemistry. Please see supplemental Methods for a more detailed description of the methods used. Results and discussion Stable, multilineage chimerism in humanized mice after in utero transplantation of human cord blood CD34+ cells We first determined whether IUHCT in NSG mice would result in efficient engraftment and injected fetal NSG mice at E14.5 with 25?000 to 50?000 human cord blood CD34+ cells per pup using an intrahepatic injection method that we previously described utilizing murine hematopoietic stem cells23 (Figure 1A). A total of 57% of injected dams delivered viable pups; 74 of 206 transplanted pups survived to birth (36%), with 49 CD1D pups surviving to wean (24%). This survival rate is lower than we previously reported in wild-type mice24 and likely represents the fragility of the immunodeficient mouse model compared with BALB/c or B6 dams. Overall, 25 of 49 (51%) mice were found to be engrafted with human hematopoietic cells. The levels of human cells in peripheral blood (chimerism) were determined by flow cytometry every 4 weeks for 16 to 24 weeks (Figure 1A-B). Peripheral blood chimerism increased over time in 23 of 25 mice from the initial analysis at the time of 4 weeks after birth to the time of harvest (Figure 1C). Open AMG232 in a separate window Figure 1. Stable, multilineage chimerism in humanized mice after in utero transplantation of human cord blood CD34+cells. (A) Experimental design for in utero transplantation timing and measurements of blood chimerism levels. Arrows indicate time points for peripheral blood chimerism bank checks. (B) Representative gating strategy of peripheral blood to detect human being CD45+ cells. (C) Percentage of AMG232 human being CD45+ cells in peripheral blood over time (n = 25, * .05, ** .01 by College student test comparing chimerism levels each week to the initial 4-week level). (D) Representative gating strategy for lineage-specific chimerism, FoxP3+ cells, and confirmation of the presence of CD34+ cells within the bone marrow. Chimeric mice shown different relative proportions of granulocytes, monocytes, B cells, and T cells. (E) Compiled lineage data in 10 individual mice with evidence of CD34+ cells in the bone marrow (n = 10). CB, wire blood;.

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