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

Abstract

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-1lo 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 ≤ 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.