Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-α and transforming growth factor-β1

Li Lu, Michael Heinrich, Li Sheng Wang, Mushui Dai, Amy J. Zigler, Lin Chai, Hal E. Broxmeyer

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34+ cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34+++ and different levels of c-kit (++, +, (Lo/-)), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34+++ kit(Lo/-) cells transduced with c- kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor- β1 and tumor necrosis factor-α, c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.

Original languageEnglish (US)
Pages (from-to)2319-2332
Number of pages14
JournalBlood
Volume94
Issue number7
StatePublished - Oct 1 1999
Externally publishedYes

Fingerprint

Stem Cell Factor
Transforming Growth Factors
Hematopoietic Stem Cells
Fetal Blood
Blood
Tumor Necrosis Factor-alpha
Genes
Proto-Oncogene Proteins c-kit
Stem Cells
Interleukin-3
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Interleukin-6
Apoptosis
Immunosorbents
Polymerase chain reaction
RNA-Directed DNA Polymerase
Hematopoiesis
Retroviridae
Neutralizing Antibodies

ASJC Scopus subject areas

  • Hematology

Cite this

@article{d4f61340b59c4c32a9663ac1c89c161d,
title = "Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-α and transforming growth factor-β1",
abstract = "The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34+ cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34+++ and different levels of c-kit (++, +, (Lo/-)), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34+++ kit(Lo/-) cells transduced with c- kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor- β1 and tumor necrosis factor-α, c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.",
author = "Li Lu and Michael Heinrich and Wang, {Li Sheng} and Mushui Dai and Zigler, {Amy J.} and Lin Chai and Broxmeyer, {Hal E.}",
year = "1999",
month = "10",
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language = "English (US)",
volume = "94",
pages = "2319--2332",
journal = "Blood",
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TY - JOUR

T1 - Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-α and transforming growth factor-β1

AU - Lu, Li

AU - Heinrich, Michael

AU - Wang, Li Sheng

AU - Dai, Mushui

AU - Zigler, Amy J.

AU - Chai, Lin

AU - Broxmeyer, Hal E.

PY - 1999/10/1

Y1 - 1999/10/1

N2 - The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34+ cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34+++ and different levels of c-kit (++, +, (Lo/-)), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34+++ kit(Lo/-) cells transduced with c- kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor- β1 and tumor necrosis factor-α, c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.

AB - The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34+ cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34+++ and different levels of c-kit (++, +, (Lo/-)), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34+++ kit(Lo/-) cells transduced with c- kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor- β1 and tumor necrosis factor-α, c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.

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