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 10(5) CD34+, DR- cells. The purity of DR- cells increased from between 0.5% and 4.3% in the immuno-adsorbed 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.
Körbling
M
M
University of Texas, M.D. Anderson Cancer Center, Department of Hematology, Houston 77030.
Drach
J
J
Champlin
R E
RE
Engel
H
H
Huynh
L
L
Kleine
H D
HD
Berenson
R
R
Deisseroth
A B
AB
Andreeff
M
M
D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000032:analysis*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007163:Immunosorbent Techniques; D014182:Transplantation, Autologous
D015703:Antigens, CD; D018952:Antigens, CD34; D006684:HLA-DR Antigens