Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells

Maria Valeria Corrias, Francesca Scuderi, Mirella Pasino, Paola Biglino, Paola Bocca, Fernando Marotta, Edoardo Figini, Vito Pistoia, Pier Giorgio Mori

Research output: Contribution to journalArticle

Abstract

Background and Objective. The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retrovital vectors. Design and Methods. Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Results. The results obtained indicated that the amount of transduced cells increased with the length of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. Interpretation and Conclusions. These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

Original languageEnglish
Pages (from-to)580-586
Number of pages7
JournalHaematologica
Volume83
Issue number7
Publication statusPublished - 1998

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Fetal Blood
Blood Cells
Intercellular Signaling Peptides and Proteins
Interleukin-3
Infection
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Appointments and Schedules
Polymerase Chain Reaction
Stem Cell Factor
Neomycin
Retroviridae
Genetic Therapy
Genes
Extracellular Matrix
Interleukin-6
Cell Culture Techniques

Keywords

  • CD34 cells
  • Hematopoietic growth factors
  • PCR
  • Retrovirus-mediated gene transfer
  • Umbilical cord blood

ASJC Scopus subject areas

  • Hematology

Cite this

Corrias, M. V., Scuderi, F., Pasino, M., Biglino, P., Bocca, P., Marotta, F., ... Mori, P. G. (1998). Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells. Haematologica, 83(7), 580-586.

Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells. / Corrias, Maria Valeria; Scuderi, Francesca; Pasino, Mirella; Biglino, Paola; Bocca, Paola; Marotta, Fernando; Figini, Edoardo; Pistoia, Vito; Mori, Pier Giorgio.

In: Haematologica, Vol. 83, No. 7, 1998, p. 580-586.

Research output: Contribution to journalArticle

Corrias, MV, Scuderi, F, Pasino, M, Biglino, P, Bocca, P, Marotta, F, Figini, E, Pistoia, V & Mori, PG 1998, 'Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells', Haematologica, vol. 83, no. 7, pp. 580-586.
Corrias, Maria Valeria ; Scuderi, Francesca ; Pasino, Mirella ; Biglino, Paola ; Bocca, Paola ; Marotta, Fernando ; Figini, Edoardo ; Pistoia, Vito ; Mori, Pier Giorgio. / Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells. In: Haematologica. 1998 ; Vol. 83, No. 7. pp. 580-586.
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AU - Corrias, Maria Valeria

AU - Scuderi, Francesca

AU - Pasino, Mirella

AU - Biglino, Paola

AU - Bocca, Paola

AU - Marotta, Fernando

AU - Figini, Edoardo

AU - Pistoia, Vito

AU - Mori, Pier Giorgio

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N2 - Background and Objective. The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retrovital vectors. Design and Methods. Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Results. The results obtained indicated that the amount of transduced cells increased with the length of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. Interpretation and Conclusions. These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

AB - Background and Objective. The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retrovital vectors. Design and Methods. Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Results. The results obtained indicated that the amount of transduced cells increased with the length of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. Interpretation and Conclusions. These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

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