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SUMMARYSevere combined immunodeficient (SCID)-repopulating cells (termed SRC) with lymphohaematopoietic differentiation potential reside at an extremely low frequency in unmobilised adult human peripheral blood. Recently, an ex vivomethod of increasing the relative numbers of at least four distinct human stem cell classes, that include CD34+haematopoietic progenitor cells, in mononuclear cells (MNC) obtained from unmobilised adult human peripheral blood has been described. This process is triggered by a monoclonal antibody (mAb) against the human monomorphic region of the beta chain of HLA-DP, DQ and DR (clone CR3/43). Herein, we assess the ability of human male donor-derived MNC, following ex vivoculturing for 3 hr in haematopoietic-conducive conditions (HCC) (3-hr MNC/HCC), to form SRC in female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. All 3-hr MNC/HCC-recipient animals exhibited significant levels (>0.5%) of human cell engraftment in thebone marrow, thymus and spleen when compared to animals receiving MNC cultured in the absence of CR3/43. Phenotypic characterisation of the bone marrow cell populations of engrafted mice demonstrated significant levels of human lymphohaematopoietic cell lineages, comprised of T lymphocytes, monocytes, erythrocytes and megakaryocytes, including platelets. In addition, significant levels of clonogenic human CD34+cells were also detected by in vitrosurrogate assay. The thymi of engrafted animals contained maturating human thymocytes, while the spleen consisted mainly of T lymphocytes. Fluorescence in situhybridisation (FISH) further identified the presence of human male X and Y chromosomes at engrafted sites, whilst the human origin of the cells was confirmed by a specific PCR assay for the human Cart-1 gene. In conclusion, the conversion of MNC to SRC in response to treatment with CR3/43 for 3 hr could have far-reaching clinical implications especially where time and donor-histocompatibility are limiting factors.