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Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells.

Millington M, Arndt A, Boyd M, Applegate T, Shen S - PLoS ONE (2009)

Bottom Line: A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle.We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia.

ABSTRACT

Background: Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+) HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/principal findings: Using commercially available G-CSF mobilized peripheral blood (PB) CD34(+) cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/significance: This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.

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Related in: MedlinePlus

Transduction efficiency of CD34+ cells in X-vivo 10 versus Stemline II serum-free media in various cytokine combinations.(A) Transduction efficiency in the CD34+ cell population. * p<0.05, when comparing Stemline II/triple cytokines to X-vivo 10/triple cytokines (B) Cell expansion and viability following transduction.
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pone-0006461-g003: Transduction efficiency of CD34+ cells in X-vivo 10 versus Stemline II serum-free media in various cytokine combinations.(A) Transduction efficiency in the CD34+ cell population. * p<0.05, when comparing Stemline II/triple cytokines to X-vivo 10/triple cytokines (B) Cell expansion and viability following transduction.

Mentions: CD34+ cells were pre-stimulated for 48 h, in either X-vivo 10 or Stemline II, in the presence of various cytokine combinations (SCF, TPO and Flt3L, all of the three or all combinations of two, each at 50 ng/ml). Cells were then transduced with virus containing media (VCM) containing GFP lentivirus harvested in either X-vivo 10 or Stemline II at an MOI of 9. Transduction was performed on retronectin coated plates via spinoculation. Cells were analyzed by FACS analysis for EGFP expression and cell surface marker expression 48 h post transduction. A higher percentage of transduction was seen in cultures supplemented with the triple cytokine combination as compared to any combination of two (p<0.05), irrespective of the basal media (Figure 3A). This was especially evident in the Stemline II cultures, in which both the transduction efficiency and the MFI of GFP expression was significantly increased with the triple cytokine combination (p<0.05 compared to X-vivo 10) (Figure 3A). Although a higher percentage of transduction was observed in Stemline II cultures, X-vivo 10 appeared to be more robust as less fluctuation in the overall performance of the cells (transduction efficiency and viability) was seen when supported with various cytokine combinations (Figure 3A). The triple cytokine combination also resulted in increased cell numbers (expressed as fold expansion from input cells) in both culture media, while viability remained consistently above 60% across the majority of the culture conditions. The exception was Stemline II supplemented with SCF and Flt3L or Flt3L and TPO combinations, both of which were shown to have viabilities below 30% in one experiment (Figure 3B). No significant difference was observed in the expression of cell surface markers between the various culture conditions with the majority of cells remaining CD34 positive after 96 h in culture (84.4% to 97.4%, data not shown).


Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells.

Millington M, Arndt A, Boyd M, Applegate T, Shen S - PLoS ONE (2009)

Transduction efficiency of CD34+ cells in X-vivo 10 versus Stemline II serum-free media in various cytokine combinations.(A) Transduction efficiency in the CD34+ cell population. * p<0.05, when comparing Stemline II/triple cytokines to X-vivo 10/triple cytokines (B) Cell expansion and viability following transduction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2714083&req=5

pone-0006461-g003: Transduction efficiency of CD34+ cells in X-vivo 10 versus Stemline II serum-free media in various cytokine combinations.(A) Transduction efficiency in the CD34+ cell population. * p<0.05, when comparing Stemline II/triple cytokines to X-vivo 10/triple cytokines (B) Cell expansion and viability following transduction.
Mentions: CD34+ cells were pre-stimulated for 48 h, in either X-vivo 10 or Stemline II, in the presence of various cytokine combinations (SCF, TPO and Flt3L, all of the three or all combinations of two, each at 50 ng/ml). Cells were then transduced with virus containing media (VCM) containing GFP lentivirus harvested in either X-vivo 10 or Stemline II at an MOI of 9. Transduction was performed on retronectin coated plates via spinoculation. Cells were analyzed by FACS analysis for EGFP expression and cell surface marker expression 48 h post transduction. A higher percentage of transduction was seen in cultures supplemented with the triple cytokine combination as compared to any combination of two (p<0.05), irrespective of the basal media (Figure 3A). This was especially evident in the Stemline II cultures, in which both the transduction efficiency and the MFI of GFP expression was significantly increased with the triple cytokine combination (p<0.05 compared to X-vivo 10) (Figure 3A). Although a higher percentage of transduction was observed in Stemline II cultures, X-vivo 10 appeared to be more robust as less fluctuation in the overall performance of the cells (transduction efficiency and viability) was seen when supported with various cytokine combinations (Figure 3A). The triple cytokine combination also resulted in increased cell numbers (expressed as fold expansion from input cells) in both culture media, while viability remained consistently above 60% across the majority of the culture conditions. The exception was Stemline II supplemented with SCF and Flt3L or Flt3L and TPO combinations, both of which were shown to have viabilities below 30% in one experiment (Figure 3B). No significant difference was observed in the expression of cell surface markers between the various culture conditions with the majority of cells remaining CD34 positive after 96 h in culture (84.4% to 97.4%, data not shown).

Bottom Line: A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle.We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd., Eveleigh, New South Wales, Australia.

ABSTRACT

Background: Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+) HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/principal findings: Using commercially available G-CSF mobilized peripheral blood (PB) CD34(+) cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/significance: This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.

Show MeSH
Related in: MedlinePlus