MULTIPARAMETER-FLUORESCENCE ACTIVATED CELL SORTING ANALYSIS OF RETROVIRAL VECTOR GENE-TRANSFER INTO PRIMITIVE UMBILICAL-CORD BLOOD-CELLS

Retroviral vector gene transfer strategies are currently being develop ed to treat a variety of hematopoietic disorders. To date, genetic mod ification of human pluripotent hematopoietic stem cells has been ineff icient. In the present study we developed reagents and procedures for rapidly screening retroviral vector gene transfer conditions using a m ultiparameter fluorescence-activated cell sorting (FACS) assay. To ide ntify transduced cells using FAGS analysis, we developed a retroviral vector, termed MN, which stably expressed high levels of a truncated v ersion of the low-affinity nerve growth factor receptor (LNGFR). In ad dition, procedures were developed for enriching CD34(+) cells from cry opreserved umbilical cord blood. These cells were transduced with MN a nd evaluated using multiparameter FAGS analysis for expression of CD34 , CD38, and LNGFR. Stem cell maintenance was determined by measuring t he CD34(hi) and CD34(hi)CD38(lo/-) cells remaining after ex vivo gene transfer. Gene transfer into these cells was measured by evaluating ce lls expressing high levels of LNGFR. Initial studies with this assay a nd with in vitro functional assays indicated that retroviral gene tran sfer following pre-incubation with a variety of cytokines in serum con taining conditions resulted in 1) poor maintenance of hematopoietic st em cells and 2) gene transfer predominantly in relatively mature cells . When gene transfer in serum-free conditions was performed, some impr ovement was observed in tile maintenance of cells retaining primitive immunophenotypes with no reduction in the gene transfer efficiency. Th e MN vector and multiparameter FAGS analysis will be useful in efficie ntly screening ongoing efforts designed to improve stem tell gene tran sfer.