Transduction of human c-kit cdna into single hematopotic stem/progenitor cells from cord blood enhances erythroid cell containing colony formation in the absence of steel factor

L. Lu, Y. X. Li, L. Wane, M. C. Heinrich, Hal Broxmeyer

Research output: Contribution to journalArticle

Abstract

The c-kit receptor tyrosine kinase and its cognate ligand, steel factor (SLF), are known to be critical for hematopoiesis. In the present study, we evaluated whether transduciion of single hematopoietic stem/progenitor cells with a retrovirus encoding the human c-kit cDNA could influence progenitor cell proliferation and differentiation. MACS separated CD34+ cells with 90 - 95% purity were further sorted for subsets, kit, kit and kit' of CD34W cells as 1 cell/well directly into 96 well plates in the presence of methylcellulose culture medium containing Epo, IL-3, GM-CSF with or without SLF for 2 days, and then incubated with retroviruses for gene transduction. Cells were further cultured for 14 and 21 days. Results from 3 separate experiments showed that in the presence of Epo, GM-CSF and IL-3 without SLF, colony formation by BFU-E and CFU-GEMM was significantly enhanced in ckit transduced cells from all three subsets compared To the mock transduced cells. The greatest effect was noted in CD344~Mkit" cells transduced with c-kit, in which colony formation by BFU-E and CFU-GEMM was enhanced from 2.6 to 9.9% (p<0.05) and 0,5 to 3.6% (p<0.05), respectively. Transduction of c-kit had no significant influence on colony formation by CFU-GM and HPP-CFC. No difference in % erythroid-cell containing wells could be detected between c-kit- and mocktransduced cells when SLF was added to the growth factor combination. In order to determine the possible mechanism involved in this enhancing activity by introduction of c-kit, MACS separated CD34cells were incubated with IL~3, GMCSF and Epo, without SLF, after gene transduction, for 24 hrs and evaluated for apoptosis by tunnel assay. The results showed that addition of SLF to mocktransduced cells decreased apoptosis from 27.4% to 15-18%. Transduction of c-kit by itself in the absence of SLF, but in the presence of Epo, GM-CSF and IL-3, decreased apoptotic cells to 6.9%, suggesting that transduced c-kit increased cell survival. Our results with transduction of c-kit into single progenitors suggest that accessory cells may not be playing a significant role in these effects and that transduced c-kit might be involved in the survival of the celts. There might also be cross tafk between c-kit and other cytokine receptors, such as EpoR, GM-CSFR and /or IL-3R in the single cell initiated cell cultures.

Original languageEnglish
Pages (from-to)764
Number of pages1
JournalExperimental Hematology
Volume26
Issue number8
StatePublished - 1998

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Erythroid Cells
Stem Cell Factor
Fetal Blood
Stem Cells
Interleukin-3
Granulocyte-Macrophage Colony-Stimulating Factor
Myeloid Progenitor Cells
Erythroid Precursor Cells
Retroviridae
Hematopoietic Stem Cells
Proto-Oncogene Proteins c-kit
Apoptosis
Granulocyte-Macrophage Progenitor Cells
Cytokine Receptors
Methylcellulose
Hematopoiesis
Protein-Tyrosine Kinases
Genes
Culture Media
Cell Differentiation

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

@article{129c9a5ed3d540108d60e4dba874a7bf,
title = "Transduction of human c-kit cdna into single hematopotic stem/progenitor cells from cord blood enhances erythroid cell containing colony formation in the absence of steel factor",
abstract = "The c-kit receptor tyrosine kinase and its cognate ligand, steel factor (SLF), are known to be critical for hematopoiesis. In the present study, we evaluated whether transduciion of single hematopoietic stem/progenitor cells with a retrovirus encoding the human c-kit cDNA could influence progenitor cell proliferation and differentiation. MACS separated CD34+ cells with 90 - 95{\%} purity were further sorted for subsets, kit, kit and kit' of CD34W cells as 1 cell/well directly into 96 well plates in the presence of methylcellulose culture medium containing Epo, IL-3, GM-CSF with or without SLF for 2 days, and then incubated with retroviruses for gene transduction. Cells were further cultured for 14 and 21 days. Results from 3 separate experiments showed that in the presence of Epo, GM-CSF and IL-3 without SLF, colony formation by BFU-E and CFU-GEMM was significantly enhanced in ckit transduced cells from all three subsets compared To the mock transduced cells. The greatest effect was noted in CD344~Mkit{"} cells transduced with c-kit, in which colony formation by BFU-E and CFU-GEMM was enhanced from 2.6 to 9.9{\%} (p<0.05) and 0,5 to 3.6{\%} (p<0.05), respectively. Transduction of c-kit had no significant influence on colony formation by CFU-GM and HPP-CFC. No difference in {\%} erythroid-cell containing wells could be detected between c-kit- and mocktransduced cells when SLF was added to the growth factor combination. In order to determine the possible mechanism involved in this enhancing activity by introduction of c-kit, MACS separated CD34cells were incubated with IL~3, GMCSF and Epo, without SLF, after gene transduction, for 24 hrs and evaluated for apoptosis by tunnel assay. The results showed that addition of SLF to mocktransduced cells decreased apoptosis from 27.4{\%} to 15-18{\%}. Transduction of c-kit by itself in the absence of SLF, but in the presence of Epo, GM-CSF and IL-3, decreased apoptotic cells to 6.9{\%}, suggesting that transduced c-kit increased cell survival. Our results with transduction of c-kit into single progenitors suggest that accessory cells may not be playing a significant role in these effects and that transduced c-kit might be involved in the survival of the celts. There might also be cross tafk between c-kit and other cytokine receptors, such as EpoR, GM-CSFR and /or IL-3R in the single cell initiated cell cultures.",
author = "L. Lu and Li, {Y. X.} and L. Wane and Heinrich, {M. C.} and Hal Broxmeyer",
year = "1998",
language = "English",
volume = "26",
pages = "764",
journal = "Experimental Hematology",
issn = "0301-472X",
publisher = "Elsevier Inc.",
number = "8",

}

TY - JOUR

T1 - Transduction of human c-kit cdna into single hematopotic stem/progenitor cells from cord blood enhances erythroid cell containing colony formation in the absence of steel factor

AU - Lu, L.

AU - Li, Y. X.

AU - Wane, L.

AU - Heinrich, M. C.

AU - Broxmeyer, Hal

PY - 1998

Y1 - 1998

N2 - The c-kit receptor tyrosine kinase and its cognate ligand, steel factor (SLF), are known to be critical for hematopoiesis. In the present study, we evaluated whether transduciion of single hematopoietic stem/progenitor cells with a retrovirus encoding the human c-kit cDNA could influence progenitor cell proliferation and differentiation. MACS separated CD34+ cells with 90 - 95% purity were further sorted for subsets, kit, kit and kit' of CD34W cells as 1 cell/well directly into 96 well plates in the presence of methylcellulose culture medium containing Epo, IL-3, GM-CSF with or without SLF for 2 days, and then incubated with retroviruses for gene transduction. Cells were further cultured for 14 and 21 days. Results from 3 separate experiments showed that in the presence of Epo, GM-CSF and IL-3 without SLF, colony formation by BFU-E and CFU-GEMM was significantly enhanced in ckit transduced cells from all three subsets compared To the mock transduced cells. The greatest effect was noted in CD344~Mkit" cells transduced with c-kit, in which colony formation by BFU-E and CFU-GEMM was enhanced from 2.6 to 9.9% (p<0.05) and 0,5 to 3.6% (p<0.05), respectively. Transduction of c-kit had no significant influence on colony formation by CFU-GM and HPP-CFC. No difference in % erythroid-cell containing wells could be detected between c-kit- and mocktransduced cells when SLF was added to the growth factor combination. In order to determine the possible mechanism involved in this enhancing activity by introduction of c-kit, MACS separated CD34cells were incubated with IL~3, GMCSF and Epo, without SLF, after gene transduction, for 24 hrs and evaluated for apoptosis by tunnel assay. The results showed that addition of SLF to mocktransduced cells decreased apoptosis from 27.4% to 15-18%. Transduction of c-kit by itself in the absence of SLF, but in the presence of Epo, GM-CSF and IL-3, decreased apoptotic cells to 6.9%, suggesting that transduced c-kit increased cell survival. Our results with transduction of c-kit into single progenitors suggest that accessory cells may not be playing a significant role in these effects and that transduced c-kit might be involved in the survival of the celts. There might also be cross tafk between c-kit and other cytokine receptors, such as EpoR, GM-CSFR and /or IL-3R in the single cell initiated cell cultures.

AB - The c-kit receptor tyrosine kinase and its cognate ligand, steel factor (SLF), are known to be critical for hematopoiesis. In the present study, we evaluated whether transduciion of single hematopoietic stem/progenitor cells with a retrovirus encoding the human c-kit cDNA could influence progenitor cell proliferation and differentiation. MACS separated CD34+ cells with 90 - 95% purity were further sorted for subsets, kit, kit and kit' of CD34W cells as 1 cell/well directly into 96 well plates in the presence of methylcellulose culture medium containing Epo, IL-3, GM-CSF with or without SLF for 2 days, and then incubated with retroviruses for gene transduction. Cells were further cultured for 14 and 21 days. Results from 3 separate experiments showed that in the presence of Epo, GM-CSF and IL-3 without SLF, colony formation by BFU-E and CFU-GEMM was significantly enhanced in ckit transduced cells from all three subsets compared To the mock transduced cells. The greatest effect was noted in CD344~Mkit" cells transduced with c-kit, in which colony formation by BFU-E and CFU-GEMM was enhanced from 2.6 to 9.9% (p<0.05) and 0,5 to 3.6% (p<0.05), respectively. Transduction of c-kit had no significant influence on colony formation by CFU-GM and HPP-CFC. No difference in % erythroid-cell containing wells could be detected between c-kit- and mocktransduced cells when SLF was added to the growth factor combination. In order to determine the possible mechanism involved in this enhancing activity by introduction of c-kit, MACS separated CD34cells were incubated with IL~3, GMCSF and Epo, without SLF, after gene transduction, for 24 hrs and evaluated for apoptosis by tunnel assay. The results showed that addition of SLF to mocktransduced cells decreased apoptosis from 27.4% to 15-18%. Transduction of c-kit by itself in the absence of SLF, but in the presence of Epo, GM-CSF and IL-3, decreased apoptotic cells to 6.9%, suggesting that transduced c-kit increased cell survival. Our results with transduction of c-kit into single progenitors suggest that accessory cells may not be playing a significant role in these effects and that transduced c-kit might be involved in the survival of the celts. There might also be cross tafk between c-kit and other cytokine receptors, such as EpoR, GM-CSFR and /or IL-3R in the single cell initiated cell cultures.

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