LARGE-SCALE PREPARATION OF HIGHLY PURIFIED, FROZEN THAWED CD34(+), HLA-DR(-) HEMATOPOIETIC PROGENITOR CELLS BY SEQUENTIAL IMMUNOADSORPTION (CEPRATE SC) AND FLUORESCENCE-ACTIVATED CELL SORTING - IMPLICATIONS FOR GENE TRANSDUCTION AND/OR TRANSPLANTATION/

The purification of early hematopoietic progenitor cells for autologou s transplantation is based on two rationales: (1) elimination of clono genic tumor cells, and/or (2) gene transfer into indefinitely self-rep licating hematopoietic stem cells. Primitive CD34(+) stem cells can be separated from more mature stem cells, or probably from clonogenic tu mor cells, by differences in HLA-DR surface antigen expression. The ob jective of this study was to establish a large-scale technique for pur ification of CD34(+), DR(-) progenitor cells from a large volume marro w harvest. In five different experiments, CD34(+) cells were purified to between 76% and 91% by avidin-biotin immunoadsorption (CEPRATE SC) as a first step. This was followed by fluorescence-activated cell sort ing to separate DR(+) and DR(-) cells, which resulted in the generatio n of between 1.75 and 11.3 X 10(5) CD34(+), DR(-) cells. The purity of DR(-) cells increased from between 0.5% and 4.3% in the immuno-adsorb ed fraction up to 99% in the DR(-) sorted fraction. As shown in a sing le experiment, the purity of CD34(+), DR(-) cells immediately after th awing increased from 0.01% to 94.3% while losing 99% of those early pr ogenitor cells during the multistep purification procedure. We were ab le to physically separate one CD34(+), DR(-) cell from up to 8000 nucl eated cells in the prepurified cell suspension. One million highly pur ified CD34(+), DR(-) progenitor cells is potentially an adequate cell dose for autologous transplantation equivalent to what is contained in an unselected and functioning marrow autograft.