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, David B. Brown

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stern/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 stern/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.

Original languageEnglish (US)
Pages (from-to)437-444
Number of pages8
JournalBlood
Volume95
Issue number2
DOIs
StatePublished - Jan 15 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Fingerprint Dive into the research topics of 'Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells'. Together they form a unique fingerprint.

  • Cite this

    Davis, B. R., Yannariello-Brown, J., Prokopishyn, N. L., Luo, Z., Smith, M. R., Wang, J., Victor Carsrud, N. D., & Brown, D. B. (2000). Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells. Blood, 95(2), 437-444. https://doi.org/10.1182/blood.v95.2.437