Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells

A novel glass needle-mediated microinjection method for delivery of macromolecules,including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34+ cells and 42% +/- 14% of CD34(+)/CD38(-) cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently less than or equal to 19.6 kb) and fluorescently labeled proteins into CD34(+) and CD34(+)/CD38(-) cells was achieved with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle-mediated delivery of macromolecules into primitive hematopoietic cells is a valuable method for studies of stem cell biology and a promising method for human blood stem cell gene therapy, (Blood 2000;95:437-444) (C) 2000 by The American Society of hematology. [References: 36]

See More