Isolation of cd34-ac133+lin- cells from the human stem cell compartment reveals superior retroviral transduction to that of cd34+cd38-lin-

A major goal of gene therapy is to be able to transfer and express genes into hematopoietic stem cells. Clinically, stem cells have been defined by the expression of t le cell surface marker CD34, however stem cell isolation and gene transfer into tl .is repopulating fraction have been disappointing. Human hematopoietic stem cells have recently been identified within the CD34- population and we have further defined the se lineage depleted cells (Lin-) by the cell surface marker AC 133 (CD34-AC133+Lin-). H< re we show that CD34-AC133+ cells can be transduced by retrovirus at a much higler efficiency than either CD34-AC133- or CD34+CD38- cells. Subpopulations were isolated and underwent a 4 day retroviral transduction procedure using an enhanced green fluorescs nt protein(GFP)-containing vector, PG13-MIEV, in serum free conditions. During the culti re period, both CD34-AC133+ and CD34+CD38- cells were capable of cell expansian whereas the number of CD34-AC133- cells could not be sustained. All populations yielded CFCs after transduction, although the CD34-AC133- population demonstratec a dramatically reduced frequency. Fluorescent microscopy of the CFC derived from CD24AC133+ cells revealed expression of GFP and was confirmed by PCR for proviral integration. Gene transfer and expression analysis by flow cytometry revealed that t le CD34-AC133+ cells had almost a 4 fold increase in GFP+ cells compared to CD34+38cells. Phenotypic analysis of GFP+ subfractions demonstrated that retrovirus preferentia ly transduced CD34-CD38- cells from originally isolated CD34-AC133+ cells, whereas the CD34+CD38- population contained more mature (CD38+) GFP+ cells. Our results demonstrate that in contrast to either CD34-AC133- or CD34+CD38- CB cells, the CD34-AC133+ cells illustrate distinct retroviral transduction that results in superior gene transfer. Since retroviral transduction has yet to be evaluated using this unique population in the clinic, we suggest that targeted transduction of CD34-AC133+ cells may providit a therapeutic benefit in future stem cell gene therapy trials.