Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/ severe combined immunodeficient (NOD/ SCID) repopulating cell (SRC). Coculture of human ABM CD34 ؉ cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34 ؉ cells, induced more than 95% of the CD34 ؉ CD38 ؊ subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34 ؉ cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34 ؉ cells to be 1 in 9.9 ؋ 10 5 cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 ؋ 10 5 cells. All mice that received transplants of HUBECcultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow. IntroductionThe development of ex vivo culture methods that promote the expansion of adult human bone marrow (ABM) stem cells would have direct application in clinical gene therapy and stem cell transplantation. However, results obtained from stroma-based 1 and stroma-free ex vivo culture systems [2][3][4][5] have been disappointing, owing to insufficient activation of primitive CD34 ϩ CD38 Ϫ cells, cell differentiation, and a loss of repopulating capacity following short-term culture. 6 Moreover, increased CD34 ϩ cell numbers, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) are not quantitative indicators of in vivo repopulating potential. [7][8][9][10] Therefore, the importance of evaluating ex vivo cultured cells in an in vivo repopulation model has been emphasized. 8 The nonobese diabetic/severe combined immunodeficient (NOD/ SCID) model system has been used to measure the long-term reconstitution potential of ex vivo-expanded human lymphohematopoietic stem cells. 7-10 SCID-repopulating cells (SRCs) are enriched in human cord blood (CB) as compared with adult ABM and mobilized peripheral blood 11,12 and are most highly concentrated within the CD34 ϩ CD38 Ϫ population. 8 SRCs are considered to be biologically more primitive than assayable LTC-IC and CFC progenitors, 1,9,10,13 which are found in both the CD34 ϩ CD38 ϩ and the CD34 ϩ...