RED-BLOOD-CELL DEPLETION AND ENRICHMENT OF CD34(-CORD BLOOD USING SOYBEAN AGGLUTININ AND CD34 IMMUNOSELECTION() HEMATOPOIETIC PROGENITOR CELLS FROM HUMAN UMBILICAL)

Realization of the full potential of human umbilical cord blood (HUCB) as a source of transplantable hematopoietic progenitor cells (HPC) wi ll be contingent on the development of a reliable method for ex vivo c ell processing and stem cell enrichment. A first step in stem cell enr ichment protocols involves depletion of the red blood cells (RBC) in t he sample. We have compared several RBC depletion methods on the basis of recovery of total nucleated cells. It was found that 3% gelatin wa s effective at depleting RBC with a nucleated cell recovery of 72.4 7.3% (mean ri: standard deviation [SDI]) in the HUCB samples. Several lectins were also used for processing HUCB and compared for their abil ity to remove RBC. Of these, soybean agglutinin (SBA) was found to rem ove RBC without substantial loss of HPC. Moreover, sequential treatmen t of HUCB with 3% gelatin and the AIS MicroCELLector (R) SBA resulted in a product with <1% hematocrit, 57% reduction in T cells, 35% nuclea ted cell recovery, and relatively high yields of burst-forming units-e rythroid (BFU-E) (61%) and colony-forming units-granulocyte/macrophage (CFU-GM) (58%) and -mixed (CFU-GEMM) (66%). In a separate series of s tudies, enrichment of the CD34(+) subset in HUCB was accomplished by p rocessing HUCB with Ficoll followed by sequential treatment with the A IS MicroCELLector (R) SBA and AIS MicroCELLector CD34. The CD34(+) fra ction was enriched for BFU-E (14-fold), CFU-GM (ninefold), and CFU-GEM M (five-fold) with an overall purity greater than or equal to 92% CD34 (+) by flow cytometry. This report demonstrates that 3% gelatin and th e AIS MicroCELLector (R) SBA can be combined as an ex vivo processing technique to reduce the volume of the HUCB product by depleting RBC an d T cells while still maintaining a high recovery of HPC. Moreover, se paration of highly enriched CD34(+) cells from HUCB is achievable and opens up the possibility of using CD34(+) cells from HUCB for ex vivo progenitor cell expansion for transplantation, transfusion, and gene t herapy.