Multilineage engraftment of human CB MNC in NSG mice.

<p>In this study, NOD/SCID or NSG mice received <i>iv</i> busulfan conditioning followed by <i>iv</i> injection of CB MNC or CD34⁺ cells that were previously cultured <i>in vitro</i> for 7 days, in the presence or absence of BME. This figure represents NSG mice which received <i>iv</i> injection of 3×10⁶ CB MNC according to the same protocol. Mice were sacrificed and bone marrow cells were harvested from femurs, tibia and pelvis and examined for multilineage engraftment by flow cytometry according to the following gating strategy: <b>A</b>) Live cells were first gated using forward scatter versus side scatter plots (R1 region). The three plots represent mice injected with saline (negative control), CB, or CB+BME; respectively. <b>B</b>) Human leucocytes were then gated using human CD45 staining (pan-leukocyte marker, R2 region). <b>C</b>) From CD45⁺ gate (R2 region), cells were then examined for multi-lineage engraftment defined by the presence of separate lymphoid (CD45⁺CD19⁺, R3 region) and myeloid (CD45⁺CD33⁺, R4 region) populations. <b>D</b>) The population of CD45⁻ cells were gated in order to determine the erythroid populations. Indeed, erythroid populations made up of mature RBCs (CD45⁻CD36⁻CD235a⁺, R5 region) or immature erythroblasts (CD45⁻CD36⁺CD235a⁺, R6 region) were also determined. It’s important to note that the CD36⁺CD235a⁻ population, which is not present in the controls, does not represent immature erythroblasts but are CD45+ mature cells. The percent engraftment was defined as the total number of leucocytes and immature erythroblasts (CD45⁺ and CD45⁻CD36⁺CD235a⁺ cells).