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 Citations (Scopus)

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
StatePublished - Jan 15 2000

Fingerprint

Microinjections
Stem cells
Macromolecules
Transgenes
Needles
Glass
Blood Cells
Blood
Stem Cells
Cells
Cytology
Gene therapy
DNA
Green Fluorescent Proteins
Dextrans
Proteins
Immobilization
Injections
Cell- and Tissue-Based Therapy
Fetal Blood

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

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

Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells. / Davis, Brian R.; Yannariello-Brown, Judith; Prokopishyn, Nicole L.; Luo, Zhongjun; Smith, Mark R.; Wang, Jue; Victor Carsrud, N. D.; Brown, David B.

In: Blood, Vol. 95, No. 2, 15.01.2000, p. 437-444.

Research output: Contribution to journalArticle

Davis, BR, Yannariello-Brown, J, Prokopishyn, NL, Luo, Z, Smith, MR, Wang, J, Victor Carsrud, ND & Brown, DB 2000, 'Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells', Blood, vol. 95, no. 2, pp. 437-444.
Davis BR, Yannariello-Brown J, Prokopishyn NL, Luo Z, Smith MR, Wang J et al. Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells. Blood. 2000 Jan 15;95(2):437-444.
Davis, Brian R. ; Yannariello-Brown, Judith ; Prokopishyn, Nicole L. ; Luo, Zhongjun ; Smith, Mark R. ; Wang, Jue ; Victor Carsrud, N. D. ; Brown, David B. / Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells. In: Blood. 2000 ; Vol. 95, No. 2. pp. 437-444.
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