Retroviral vector-mediated gene transfer into umbilical cord blood CD34brCD38−CD33− cells

In this report, we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially, we found that fresh UCB isolated with the CD34brCD38−CD33− phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition, following ex vivo gene transfer, this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38−CD33− cells. Proliferation of the CD34brCD38−CD33− cells was found to be a prerequisite for efficient transduction. However, in all conditions tested, proliferation of the CD34brCD38−CD33− cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression, an increase in CD38/CD33 expression, and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38−CD33− cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report.