Development of a high-resolution purification method for precise functional characterization of primitive human cord blood–derived CD34–negative SCID-repopulating cells

Objective We have successfully identified human cord blood (CB)–derived CD34-negative (CD34 − ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid repopulating ability using the intrabone marrow injection method. In our previous study, a limiting dilution analysis demonstrated the frequency of CD34 − SRCs in CB-derived 13lineage-negative (Lin − ) CD34 − cells to be approximately 1/25,000. In this study, we intended to develop a high-resolution purification method to obtain highly purified CD34 − SRCs. Materials and Methods The pooled CB-derived Lin − cells were stained with 13 reported Lin monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA, and CD127. Then 18Lin − CD34 high , 18Lin − CD34 − , and 13Lin − CD34 high CD38 − cells were sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractions of cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of this new purification method. Results A limiting dilution analysis demonstrated the frequency of CD34 − SRCs in these 18Lin − CD34 − cells to be approximately 1/1,000, which is associated with a seeding efficiency 25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/IL-2Rγc null mice that received transplants of only two CD34 − SRCs were highly engrafted with human lymphomyeloid cells at 24 weeks after primary transplantation and showed secondary multilineage repopulating abilities. Conclusions We succeeded to highly purify the CD34 − SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification method is indispensable to precisely characterize a distinct class of primitive human CB-derived CD34 − hematopoietic stem cells.