Abstract Abstract 815 We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only <10% of the total CFCs were CFU-Mix. The phenotypic and functional characterizations of these 18 Lin−CD34− cells were then further investigated by cocultures with the HESS-5 murine stromal cell line in the presence of a cocktail of cytokines, such as SCF, flt3 ligand, TPO, IL-3, IL-6, and G-CSF. After 7 days of coculture, the total number of cells was observed to expand by 20 to 30 folds, 40 to 60% of which were consisted of CD34+ cells. Next, we investigated the SRC activity of these 18 Lin−CD34− cells using NOD/Shi-scid mice. When 4×104 18 Lin−CD34− cells were transplanted using IBMI, all 4 mice were highly repopulated with human CD45+ cells, including CD19+ B-lymphoid and CD33+ myeloid cells. In addition, the level of human cell engraftment in the bone marrow (BM) ranged from 93.3 to 97.5% (median, 96.8%), at 12 weeks after the transplantation. Interestingly, the BM cells obtained from the primary engrafted NOD/ Shi-scid mice that received transplants of 1,000 to 2,000 18 Lin−CD34− cells showed a secondary repopulating capacity. Furthermore, a limiting dilution analysis demonstrated the frequency of CD34− SRCs in these 18 Lin−CD34− cells to be approximately 1/1,000. The next approach to characterize the CD34− SRCs with respect to the self-renewal potential as well as the long-term repopulating potential, was to serially analyze the kinetics of engraftment for 24 weeks in the NOD/Shi-scid/IL-2Rgcnull (NOG) mice that received transplants of 2,000 18 Lin−CD34− cells containing only 2 CD34− SRCs (estimated number). All 6 mice showed signs of human cell repopulation (0.4 to 28.9%, median 2.5%) at 5 weeks after the transplantation at the contra-lateral sites of IBMI. The repopulation rates gradually increased, and reached a high level of repopulation (47.0 to 87.9%, median 72.0%) at 18 weeks after the transplantation. These results indicated that CD34− SRCs could thus sustain a long-term human cell repopulation in NOG mice, thereby suggesting that CD34− SRCs are a distinct class of primitive HSCs in comparison to the previously reported CD34+ SRCs. Disclosures: No relevant conflicts of interest to declare.