A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells

Abstract

Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. To evaluate optimization of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (GFP) was explored as a reporter. We first improved sensitivity of detection >100-fold over that achieved previously by using a novel retroviral vector (termed MGIN) expressing a high level of an enhanced GFP gene. Primitive human hematopoietic cells bearing the CD34 surface antigen and lacking lineage differentiation markers (CD34⁺Lin⁻) were transduced with the MGIN vector using a clinically applicable supernatant procedure. Under the conditions employed, >75% of the target cells retained the CD34⁺Lin⁻ primitive phenotype after 4–5 days in culture; of those ⩾25% expressed a high level of GFP detectable by both flow cytometric analysis and fluorescence microscopy. When transduced cells were cultured in clonogenic progenitor assays, GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and not detrimental to the differentiative potential of the transduced CD34⁺Lin⁻ cells. We conclude that GFP is effective as a vital marker to quantify retrovirus-mediated gene transfer into human hematopoietic and perhaps other types of stem/progenitor cells, and monitor gene expression during their subsequent cell lineage determinations.