title: 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-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 </= 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)
authors.affiliation: Sealy Center for Oncology and Hematology, Department of Microbiology and Immunology, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
authors.forename: B R
authors.initials: BR
authors.lastname: Davis
authors.forename: J
authors.initials: J
authors.lastname: Yannariello-Brown
authors.forename: N L
authors.initials: NL
authors.lastname: Prokopishyn
authors.forename: Z
authors.initials: Z
authors.lastname: Luo
authors.forename: M R
authors.initials: MR
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: N D
authors.initials: ND
authors.lastname: Carsrud
authors.forename: D B
authors.initials: DB
authors.lastname: Brown
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000097:blood; D000943:Antigens, Differentiation / Q000032:analysis; D002448:Cell Adhesion; D002455:Cell Division; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D004247:DNA / Q000235:genetics; D005109:Extracellular Matrix / Q000502:physiology; D005312:Fetal Blood / Q000166:cytology*; D015316:Genetic Therapy; D005898:Glass; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D007231:Infant, Newborn; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008845:Microinjections / Q000295:instrumentation / Q000379:methods*; D009244:NAD+ Nucleosidase / Q000032:analysis; D009339:Needles; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018800:Thy-1 Antigens / Q000032:analysis; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D014183:Transplantation, Heterologous
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins; D004247:DNA; D010735:Phosphoglycerate Kinase; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1