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Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential.
Blood. 1998 May 01; 91(9):3487-93.Blood

Abstract

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.

Authors+Show Affiliations

Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9558409

Citation

Conneally, E, et al. "Efficient Retroviral-mediated Gene Transfer to Human Cord Blood Stem Cells With in Vivo Repopulating Potential." Blood, vol. 91, no. 9, 1998, pp. 3487-93.
Conneally E, Eaves CJ, Humphries RK. Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. Blood. 1998;91(9):3487-93.
Conneally, E., Eaves, C. J., & Humphries, R. K. (1998). Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. Blood, 91(9), 3487-93.
Conneally E, Eaves CJ, Humphries RK. Efficient Retroviral-mediated Gene Transfer to Human Cord Blood Stem Cells With in Vivo Repopulating Potential. Blood. 1998 May 1;91(9):3487-93. PubMed PMID: 9558409.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. AU - Conneally,E, AU - Eaves,C J, AU - Humphries,R K, PY - 1998/5/23/pubmed PY - 1998/5/23/medline PY - 1998/5/23/entrez SP - 3487 EP - 93 JF - Blood JO - Blood VL - 91 IS - 9 N2 - Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols. SN - 0006-4971 UR - https://www.unboundmedicine.com/medline/citation/9558409/Efficient_retroviral_mediated_gene_transfer_to_human_cord_blood_stem_cells_with_in_vivo_repopulating_potential_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-4971(20)55260-9 DB - PRIME DP - Unbound Medicine ER -