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Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture.

Abstract
We have investigated the ability of several cytokine combinations to improve retrovirus-mediated transduction of human primitive hematopoietic progenitors (PHPs) from mobilized peripheral blood (MPB). Retroviral infection of CD34+ cells was performed by culture on fibronectin fragment CH-296 (RetroNectin, RN), using the truncated human nerve growth factor receptor (NGFR) as the transgene reporter. Transgene expression among progeny of PHPs was assayed by FACS analysis after long-term stromal culture (LTC). Transgene delivery to PHPs was assessed by PCR of individual stromal culture-derived methylcellulose colonies (LTC-CFCs). Compared with interleukin 3 (IL-3), IL-6, and leukemia inhibitory factor (LIF), the combination of thrombopoietin (TPO), Flt3 ligand (FL), and Kit ligand (KL) effected a 73-fold increase in NGFR expression among CD34+ cells (to 14%) and a 14-fold increase in NGFR expression among total cells (to 10%) after LTC. In addition, a 2.4-fold increase in neo gene marking of LTC-CFCs was observed. A preclinical study comparing the effect of high-speed centrifugation ("spinoculation") or culture on RN during exposure to retroviral particles in teflon cell culture bags showed no difference in the efficiency of transduction of PHPs between these two methods.
AuthorsL Murray, K Luens, R Tushinski, L Jin, M Burton, J Chen, S Forestell, B Hill
JournalHuman gene therapy (Hum Gene Ther) Vol. 10 Issue 11 Pg. 1743-52 (Jul 20 1999) ISSN: 1043-0342 [Print] United States
PMID10446914 (Publication Type: Journal Article)
Chemical References
  • Antigens, CD34
  • Cytokines
  • Fibronectins
  • Membrane Proteins
  • Recombinant Proteins
  • Stem Cell Factor
  • flt3 ligand protein
  • retronectin
  • Thrombopoietin
  • Nerve Growth Factor
Topics
  • Antigens, CD34 (metabolism)
  • Cells, Cultured
  • Cytokines (immunology)
  • Fibronectins (metabolism)
  • Flow Cytometry
  • Genetic Therapy
  • Genetic Vectors
  • Hematopoietic Stem Cell Mobilization
  • Hematopoietic Stem Cells (metabolism, physiology, virology)
  • Humans
  • Membrane Proteins (metabolism)
  • Nerve Growth Factor (genetics, metabolism)
  • Recombinant Proteins (metabolism)
  • Retroviridae (genetics)
  • Stem Cell Factor (metabolism)
  • Stromal Cells
  • Thrombopoietin (metabolism)
  • Transduction, Genetic

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