Characterization of Retroviral Gene Transfer into Highly Purified Human CD34− Cells with Primitive Hematopoietic Capacity
Authors: Barbara Murdoch, Lisa Gallacher, Kristin Chadwick, Mickie Bhatia
Journal: Molecular Therapy (2002)

Primitive human hematopoietic cells have recently been identified within a rare subfraction of CD34− lineage-depleted (Lin−) cells and further characterized by their restriction to a rarer subset expressing AC133. Here we show that CD34−AC133+Lin− cells can be transduced by retrovirus at a comparatively higher efficiency than either CD34−AC133−Lin− or CD34+CD38−Lin− cells. Subpopulations were transduced by enhanced green fluorescent protein (eGFP)-containing retrovirus in serum-free conditions. During the culture period, both CD34−AC133+Lin− and CD34+CD38−Lin− subfractions expanded, whereas CD34−AC133−Lin− cells could not be sustained. Fluorescent microscopic examination of progenitors assayed by colony-forming units (CFU) derived from CD34−AC133+Lin− cells revealed expression of eGFP, with the presence of provirus confirmed by clonal PCR analysis. Flow cytometry detecting eGFP revealed that cultures seeded with CD34−AC133+Lin− cells had a greater than threefold higher frequency of eGFP+ cells compared with transduced cultures of CD34+CD38−Lin− cells. Our results demonstrate that retroviral transduction efficiency and level of transgene expression into CD34−AC133+Lin− cells is distinct to either CD34−AC133−Lin− or CD34+CD38−Lin− cells. This study represents the first evaluation of retroviral transduction into this population of primitive CD34− cells, and therefore provides the basis for optimization of gene transfer protocols to examine the role of gene-marked CD34− stem cells in a clinical setting. Primitive human hematopoietic cells have recently been identified within a rare subfraction of CD34− lineage-depleted (Lin−) cells and further characterized by their restriction to a rarer subset expressing AC133. Here we show that CD34−AC133+Lin− cells can be transduced by retrovirus at a comparatively higher efficiency than either CD34−AC133−Lin− or CD34+CD38−Lin− cells. Subpopulations were transduced by enhanced green fluorescent protein (eGFP)-containing retrovirus in serum-free conditions. During the culture period, both CD34−AC133+Lin− and CD34+CD38−Lin− subfractions expanded, whereas CD34−AC133−Lin− cells could not be sustained. Fluorescent microscopic examination of progenitors assayed by colony-forming units (CFU) derived from CD34−AC133+Lin− cells revealed expression of eGFP, with the presence of provirus confirmed by clonal PCR analysis. Flow cytometry detecting eGFP revealed that cultures seeded with CD34−AC133+Lin− cells had a greater than threefold higher frequency of eGFP+ cells compared with transduced cultures of CD34+CD38−Lin− cells. Our results demonstrate that retroviral transduction efficiency and level of transgene expression into CD34−AC133+Lin− cells is distinct to either CD34−AC133−Lin− or CD34+CD38−Lin− cells. This study represents the first evaluation of retroviral transduction into this population of primitive CD34− cells, and therefore provides the basis for optimization of gene transfer protocols to examine the role of gene-marked CD34− stem cells in a clinical setting.