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

Umbilical cord blood (CB) has been shown to contain sufficient hematop oietic stem cells for allogeneic bone marrow transplant (BMT) and to c ontain progenitor cells susceptible to retrovirally mediated gene tran sduction. 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 i n volume and thus less storage space and (2) gene transfer into these cells. Cord blood was collected from the umbilical cord vein immediate ly after vaginal full-term delivery and samples of 5-12 ml (total leuc ocytes: 100 + 50 x 10(6)) of fresh unseparated (n = 6) and thawed unse parated (n = 8) CB were processed to enrich CD34(+) cells using the Ce prate LC Biotin-Avidin affinity system. CD34(+) cells, present at a fr equency of 1.2 +/- 0.8% among leukocytes from CB (calculated by immuno phenotyping and fluorescence-activated cell sorter (FAGS) analysis), c an be rapidly enriched to 54.4 +/- 12.3% (range, 38.6-87.1%) from fres h unseparated CB and 44.6 +/- 28% (range, 13.6-76%) from thawed unsepa rated CB. Hematopoietic progenitor assays for unseparated (cell concen tration 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 huma n granulocyte colony-stimulating factor, 6 U erytlhropoietin, and 15 n g 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 yie ld of CD34(+) cells after separation was only 33.5 +/- 20%, and the yi eld 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 u sed 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 separat ion is 24% and that of CFCs is 35%, so unseparated freezing of CB is p referred.