Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies

L. Lu, D. Walker, H. E. Broxmeyer, R. Hoffman, W. Hu

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Abstract

Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10+++DR+) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-α, recombinant human interferon-γ, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-α and interferon-γ and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions. These effects were at least as great in the highly enriched progenitor cell population, containing up to 47% colony-forming cells, as they were in the presorted population of nonadherent low density T-lymphocyte-depleted cells with a cloning efficiency of approximately 0.4% and strongly suggest that these factors can act directly on the progenitor cells themselves.

Original languageEnglish (US)
Pages (from-to)1823-1829
Number of pages7
JournalJournal of Immunology
Volume139
Issue number6
StatePublished - Nov 24 1987

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Histocompatibility
Color
Bone Marrow
Monoclonal Antibodies
Granulocyte-Macrophage Progenitor Cells
HLA-DR Antigens
Antigens
Erythroid Precursor Cells
Organism Cloning
Myeloid Progenitor Cells
Stem Cells
Alprostadil
T-Lymphocytes
Interferons
Population
Megakaryocyte-Erythroid Progenitor Cells
Macrophages
Phycoerythrin
Dye Lasers
Conditioned Culture Medium

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

@article{07bb94e276db4a43a77a8241f4712001,
title = "Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies",
abstract = "Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98{\%} of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01{\%}. In the antigen-positive sorted fractions, the CE was highest (up to 47{\%}) in the fractions of cells expressing high My10 and low DR (My10+++DR+) antigens and was lowest (2.5{\%}) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-α, recombinant human interferon-γ, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-α and interferon-γ and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions. These effects were at least as great in the highly enriched progenitor cell population, containing up to 47{\%} colony-forming cells, as they were in the presorted population of nonadherent low density T-lymphocyte-depleted cells with a cloning efficiency of approximately 0.4{\%} and strongly suggest that these factors can act directly on the progenitor cells themselves.",
author = "L. Lu and D. Walker and Broxmeyer, {H. E.} and R. Hoffman and W. Hu",
year = "1987",
month = "11",
day = "24",
language = "English (US)",
volume = "139",
pages = "1823--1829",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "6",

}

TY - JOUR

T1 - Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies

AU - Lu, L.

AU - Walker, D.

AU - Broxmeyer, H. E.

AU - Hoffman, R.

AU - Hu, W.

PY - 1987/11/24

Y1 - 1987/11/24

N2 - Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10+++DR+) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-α, recombinant human interferon-γ, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-α and interferon-γ and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions. These effects were at least as great in the highly enriched progenitor cell population, containing up to 47% colony-forming cells, as they were in the presorted population of nonadherent low density T-lymphocyte-depleted cells with a cloning efficiency of approximately 0.4% and strongly suggest that these factors can act directly on the progenitor cells themselves.

AB - Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR-My10-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR-My10- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10+++DR+) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10+DR+) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR+ and DR++ fractions, but most of the BFU-E and CFU-GEMM were found in the DR+ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR+ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR++ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-α, recombinant human interferon-γ, and prostaglandin E1. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-α and interferon-γ and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E1 in all antigen-positive fractions. These effects were at least as great in the highly enriched progenitor cell population, containing up to 47% colony-forming cells, as they were in the presorted population of nonadherent low density T-lymphocyte-depleted cells with a cloning efficiency of approximately 0.4% and strongly suggest that these factors can act directly on the progenitor cells themselves.

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