Successful Short-Term Ex Vivo Expansion of NOD/SCID Repopulating Ability and CAFC Week 6 from Umbilical Cord Blood.

In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34 + selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34 + CD38 − subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34 + , CD34 + CD38 − cell numbers and CFC content was, respectively, 102 ± 76, 24 ± 19, 190 ± 202 and 53 ± 37 for stroma-free and 315 ± 110, 25 ± 3, 346 ± 410 and 53 ± 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34 + CD38 − cells (27 ± 18-fold) and nc (6 ± 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34 + UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.