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 autologous transplantation is based on two rationales: (1) elimination of clonogenic tumor cells, and/or (2) gene transfer into indefinitely self-replicating hematopoietic stem cells. Primitive CD34⁺ stem cells can be separated from more mature stem cells, or probably from clonogenic tumor cells, by differences in HLA-DR surface antigen expression. The objective of this study was to establish a large-scale technique for purification of CD34⁺, DR^- progenitor cells from a large volume marrow 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 sorting to separate DR⁺ and DR^- cells, which resulted in the generation of between 1.75 and 11.3 x 10⁵ CD34⁺, DR^- cells. The purity of DR^- cells increased from between 0.5% and 4.3% in the immunoadsorbed fraction up to 99% in the DR^- sorted fraction. As shown in a single experiment, the purity of CD34⁺, DR^- cells immediately after thawing increased from 0.01% to 94.3% while losing 99% of those early progenitor cells during the multistep purification procedure. We were able to physically separate one CD34⁺, DR^- cell from up to 8000 nucleated cells in the prepurified cell suspension. One million highly purified 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.