Tnf-alpha-dependent increase of adenoviral transduction in cd34(+) hematopoffitic progenitor cells correlates with capping of the adenovirus at the cell surface

Anja Moldenhauer, Philip L. Leopold, Jae Huang Shieh, Ronald Crystal, Malcolm A S Moore

Research output: Contribution to journalArticle

Abstract

This study was initiated in order to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34(+) hematopoietic progenitor cells. CD34(+)-cells (0.5-1 x 105) from cord or mobilized peripheral blood were incubated with TNFa (0.01 to 1 mg/ml) for 2 hours. After removal of TNF, the cells were infected with an adenoviral vector encoding for GFP (MOI 500, 18 hours). Two days later, the viable cells were counted and analyzed for GFP and CD34 by flow cytometry. Plating efficiencies pre- and post-transduction were evaluated by methylcellulose assay. To visualize vectorial trafficking, CD34(+) cells were incubated with fluorophore-conjugated Ad. In a dose dependent manner, pretreatment with TNFa increased transduction efficiency more than 2fold (39.2 versus 15.5%) and improved survival of CD34(+)-cells resulting in a 6fold increase of CD34(+)GFP(+)-cells (24.4 versus 4.2 x 103), which was maintained for five days. Induction of apoptosis was excluded by Annexin and propidium iodide. Plating efficiency, especially CFU-GM formation was enhanced by TNF. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface ( caps ) in infected cells of the TNFtreated group (8±2.4% versus 18.1±5%; p< 0.05). No difference was found in the relative number of infected cells. Time course experiments showed that the effect of TNF could be observed with initial incubation times as short as 10 minutes. FACS-analysis did not indicate a significant increase in adenoviral receptors (CAR, av-integrins). Short-term incubation of CD34(+)-cells with TNFa before adenoviral infection improves transduction and plating efficiency as well as cell survival. This finding correlated with increased Ad capping at the cell surface but not Ad receptor expression suggesting that Ad trafficking may be altered.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART I
Publication statusPublished - 1 Dec 2000
Externally publishedYes

Fingerprint

Adenoviridae
Stem Cells
Plating
Annexins
Methylcellulose
Flow cytometry
Fluorophores
Propidium
Integrins
Cell Survival
Assays
Blood
Cells
Apoptosis
Granulocyte-Macrophage Progenitor Cells
Hematopoietic Stem Cells
Flow Cytometry
Cell Count
Experiments
Incidence

ASJC Scopus subject areas

  • Hematology

Cite this

Tnf-alpha-dependent increase of adenoviral transduction in cd34(+) hematopoffitic progenitor cells correlates with capping of the adenovirus at the cell surface. / Moldenhauer, Anja; Leopold, Philip L.; Shieh, Jae Huang; Crystal, Ronald; Moore, Malcolm A S.

In: Blood, Vol. 96, No. 11 PART I, 01.12.2000.

Research output: Contribution to journalArticle

@article{0b046c62b1e14c68b068b8f51cd9201c,
title = "Tnf-alpha-dependent increase of adenoviral transduction in cd34(+) hematopoffitic progenitor cells correlates with capping of the adenovirus at the cell surface",
abstract = "This study was initiated in order to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34(+) hematopoietic progenitor cells. CD34(+)-cells (0.5-1 x 105) from cord or mobilized peripheral blood were incubated with TNFa (0.01 to 1 mg/ml) for 2 hours. After removal of TNF, the cells were infected with an adenoviral vector encoding for GFP (MOI 500, 18 hours). Two days later, the viable cells were counted and analyzed for GFP and CD34 by flow cytometry. Plating efficiencies pre- and post-transduction were evaluated by methylcellulose assay. To visualize vectorial trafficking, CD34(+) cells were incubated with fluorophore-conjugated Ad. In a dose dependent manner, pretreatment with TNFa increased transduction efficiency more than 2fold (39.2 versus 15.5{\%}) and improved survival of CD34(+)-cells resulting in a 6fold increase of CD34(+)GFP(+)-cells (24.4 versus 4.2 x 103), which was maintained for five days. Induction of apoptosis was excluded by Annexin and propidium iodide. Plating efficiency, especially CFU-GM formation was enhanced by TNF. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface ( caps ) in infected cells of the TNFtreated group (8±2.4{\%} versus 18.1±5{\%}; p< 0.05). No difference was found in the relative number of infected cells. Time course experiments showed that the effect of TNF could be observed with initial incubation times as short as 10 minutes. FACS-analysis did not indicate a significant increase in adenoviral receptors (CAR, av-integrins). Short-term incubation of CD34(+)-cells with TNFa before adenoviral infection improves transduction and plating efficiency as well as cell survival. This finding correlated with increased Ad capping at the cell surface but not Ad receptor expression suggesting that Ad trafficking may be altered.",
author = "Anja Moldenhauer and Leopold, {Philip L.} and Shieh, {Jae Huang} and Ronald Crystal and Moore, {Malcolm A S}",
year = "2000",
month = "12",
day = "1",
language = "English",
volume = "96",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "11 PART I",

}

TY - JOUR

T1 - Tnf-alpha-dependent increase of adenoviral transduction in cd34(+) hematopoffitic progenitor cells correlates with capping of the adenovirus at the cell surface

AU - Moldenhauer, Anja

AU - Leopold, Philip L.

AU - Shieh, Jae Huang

AU - Crystal, Ronald

AU - Moore, Malcolm A S

PY - 2000/12/1

Y1 - 2000/12/1

N2 - This study was initiated in order to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34(+) hematopoietic progenitor cells. CD34(+)-cells (0.5-1 x 105) from cord or mobilized peripheral blood were incubated with TNFa (0.01 to 1 mg/ml) for 2 hours. After removal of TNF, the cells were infected with an adenoviral vector encoding for GFP (MOI 500, 18 hours). Two days later, the viable cells were counted and analyzed for GFP and CD34 by flow cytometry. Plating efficiencies pre- and post-transduction were evaluated by methylcellulose assay. To visualize vectorial trafficking, CD34(+) cells were incubated with fluorophore-conjugated Ad. In a dose dependent manner, pretreatment with TNFa increased transduction efficiency more than 2fold (39.2 versus 15.5%) and improved survival of CD34(+)-cells resulting in a 6fold increase of CD34(+)GFP(+)-cells (24.4 versus 4.2 x 103), which was maintained for five days. Induction of apoptosis was excluded by Annexin and propidium iodide. Plating efficiency, especially CFU-GM formation was enhanced by TNF. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface ( caps ) in infected cells of the TNFtreated group (8±2.4% versus 18.1±5%; p< 0.05). No difference was found in the relative number of infected cells. Time course experiments showed that the effect of TNF could be observed with initial incubation times as short as 10 minutes. FACS-analysis did not indicate a significant increase in adenoviral receptors (CAR, av-integrins). Short-term incubation of CD34(+)-cells with TNFa before adenoviral infection improves transduction and plating efficiency as well as cell survival. This finding correlated with increased Ad capping at the cell surface but not Ad receptor expression suggesting that Ad trafficking may be altered.

AB - This study was initiated in order to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34(+) hematopoietic progenitor cells. CD34(+)-cells (0.5-1 x 105) from cord or mobilized peripheral blood were incubated with TNFa (0.01 to 1 mg/ml) for 2 hours. After removal of TNF, the cells were infected with an adenoviral vector encoding for GFP (MOI 500, 18 hours). Two days later, the viable cells were counted and analyzed for GFP and CD34 by flow cytometry. Plating efficiencies pre- and post-transduction were evaluated by methylcellulose assay. To visualize vectorial trafficking, CD34(+) cells were incubated with fluorophore-conjugated Ad. In a dose dependent manner, pretreatment with TNFa increased transduction efficiency more than 2fold (39.2 versus 15.5%) and improved survival of CD34(+)-cells resulting in a 6fold increase of CD34(+)GFP(+)-cells (24.4 versus 4.2 x 103), which was maintained for five days. Induction of apoptosis was excluded by Annexin and propidium iodide. Plating efficiency, especially CFU-GM formation was enhanced by TNF. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface ( caps ) in infected cells of the TNFtreated group (8±2.4% versus 18.1±5%; p< 0.05). No difference was found in the relative number of infected cells. Time course experiments showed that the effect of TNF could be observed with initial incubation times as short as 10 minutes. FACS-analysis did not indicate a significant increase in adenoviral receptors (CAR, av-integrins). Short-term incubation of CD34(+)-cells with TNFa before adenoviral infection improves transduction and plating efficiency as well as cell survival. This finding correlated with increased Ad capping at the cell surface but not Ad receptor expression suggesting that Ad trafficking may be altered.

UR - http://www.scopus.com/inward/record.url?scp=33748663731&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748663731&partnerID=8YFLogxK

M3 - Article

VL - 96

JO - Blood

JF - Blood

SN - 0006-4971

IS - 11 PART I

ER -