Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells

Helmut Hanenberg, Kimikazu Hashino, Haruko Konishi, Randy A. Hock, Ikunoshin Kato, David A. Williams

Research output: Contribution to journalArticle

169 Scopus citations


Efficient retroviral gene transfer into hematopoietic stem and progenitor cells can be achieved by co-localizing retrovirus and target cells on specific adhesion domains of recombinant fibronectin (FN) fragments. In this paper, we further optimize this technology for human CD34+ cells. Investigating the role of cytokine prestimulation in retrovirus-mediated gene transfer on plates coated with the recombinant FN CH-296 revealed that prestimulation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) CD34+ cells was essential to achieve efficient gene transfer into clonogenic cells. The highest gene transfer occurred by prestimulating PB CD34+ cells for 40 hr with a combination of stem cell factor (SCF), G-CSF, and megakaryocyte growth and development factor (MGDF) prior to retroviral infection on CH-296. Surprisingly, a prolonged simultaneous exposure of primary CD34+ PB cells to retrovirus and cytokines in the presence of CH-296 lowered the gene transfer efficiency. Gene transfer into cytokine prestimulated CD34+ bone marrow (BM) cells was not influenced by increasing the coating concentrations of a recombinant FN fragment, CH-296, nor was it adversely influenced by increasing the number of CD34+ target cells, suggesting that the amount of retroviral particles present in the supernatant was not a limiting factor for transduction of CD34+ BM cells on CH-296-coated plates. The polycation Polybrene was not required for efficient transduction of hematopoietic cells in the presence of CH-296. Furthermore, we demonstrated that repeated exposure of CH-296 to retrovirus containing supernatant, called preloading, can be employed to concentrate the amount of retroviral particles bound to CH-296. These findings establish a simple and short clinically applicable transduction protocol that targets up to 68% of BM or G-CSF-mobilized PB CD34+ cells and is capable of genetically modifying up to 17% of CD34+CD38-/dim PB cells.

Original languageEnglish (US)
Pages (from-to)2193-2206
Number of pages14
JournalHuman gene therapy
Issue number18
StatePublished - Dec 10 1997

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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    Hanenberg, H., Hashino, K., Konishi, H., Hock, R. A., Kato, I., & Williams, D. A. (1997). Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. Human gene therapy, 8(18), 2193-2206. https://doi.org/10.1089/hum.1997.8.18-2193