Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 437-444
Glass needle-mediated microinjection of macromolecules and
transgenes into primary human blood stem/progenitor cells
Brian R. Davis,
Judith Yannariello-Brown,
Nicole
L. Prokopishyn,
Zhongjun Luo,
Mark R. Smith,
Jue Wang,
N. D. Victor Carsrud, and
David B. Brown
From Sealy Center for Oncology and Hematology, Department of
Microbiology and Immunology, Department of Human Biological Chemistry
and Genetics, University of Texas Medical Branch, Galveston; Gene-Cell,
Inc, Houston, TX; and Frederick Cancer Research and Development Center,
National Cancer Institute, Frederick, MD.
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+/CD38cells 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.
