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

A novel glass needle-mediated microinjection method for delivery of macromo lecules,including proteins and larger transgene DNAs, into the nuclei of bl ood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent inje ction of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34()/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment p rotocols were identified, which had no adverse effect on cell survival, pro genitor cell function (colony forming ability), or stem cell function (NOD/ SCID reconstituting ability). Delivery of fluorescent dextrans to stem/prog enitor 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 sur vival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Deli very of DNA constructs (currently less than or equal to 19.6 kb) and fluore scently labeled proteins into CD34(+) and CD34(+)/CD38(-) cells was achieve d with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle-mediated deliv ery of macromolecules into primitive hematopoietic cells is a valuable meth od 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 So ciety of hematology.