EVALUATION OF THE CEPRATE LC34 AFFINITY SYSTEM FOR THE ENRICHMENT OF CD34(+) HEMATOPOIETIC STEM-CELLS FROM CORD-BLOOD

Umbilical cord blood (CB) has been shown to contain sufficient hematopoietic stem cells for allogeneic bone marrow transplant (BMT) and to contain progenitor cells susceptible to retrovirally mediated gene transduction. Enrichment of CD34(+) cells from fresh unseparated or thawed unseparated CB could offer several advantages for (1) the storage of CB samples in an unrelated stem cell bank, which includes a decrease in volume and thus less storage space and (2) gene transfer into these cells. Cord blood was collected from the umbilical cord vein immediately after vaginal full-term delivery and samples of 5-12 ml (total leucocytes: 100 + 50 x 10(6)) of fresh unseparated (n = 6) and thawed unseparated (n = 8) CB were processed to enrich CD34(+) cells using the Ceprate LC Biotin-Avidin affinity system. CD34(+) cells, present at a frequency of 1.2 +/- 0.8% among leukocytes from CB (calculated by immunophenotyping and fluorescence-activated cell sorter (FAGS) analysis), can be rapidly enriched to 54.4 +/- 12.3% (range, 38.6-87.1%) from fresh unseparated CB and 44.6 +/- 28% (range, 13.6-76%) from thawed unseparated CB. Hematopoietic progenitor assays for unseparated (cell concentration 3 x 10(4), 1 x 10(5), 3 x 10(5)) and CD34-enriched cells (cell concentration 1 x 10(3), 3 x 10(3), 1 x 10(4)) were performed in the presence of 5 U human interleukin-3 (hu IL-3), 30 U hu-IL-6, 10 U human granulocyte colony-stimulating factor, 6 U erytlhropoietin, and 15 ng human stem cell factor. For unseparated fresh CB, the yield of CD34(+) cells after separation was 76 +/- 28%, the yield of colony-forming cells (CFCs) was 65 + 23%; however, for thawed unseparated CB, the yield of CD34(+) cells after separation was only 33.5 +/- 20%, and the yield of CFCs was only 31.4 +/- 16%. Our results demonstrate (1) that CB CD34(+) cells can be highly enriched from fresh unseparated but also from thawed unseparated CB, so that CB can be frozen unseparated and used later for the enrichment of CD34(+) cells for ex vivo expansion of progenitor cells as well as in a gene therapy setting (2) and that in unseparated fresh CB samples, the loss of CD34(+) cells after separation is 24% and that of CFCs is 35%, so unseparated freezing of CB is preferred.