Diversity of human granulopoietic precursor cells: Separation of cells that form colonies in diffusion chanbers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation

N. Jacobsen, Hal Broxmeyer, E. Grossbard, M. A S Moore

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32 Citations (Scopus)

Abstract

Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d). A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells (3) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 0.9 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar culture, were a heterogeneous population, possibly derived from both day 7 and day 14 CFU-c. Mixing experiments did not show evidence of cell interaction that could explain the observed velocity differences. The velocity sedimentation profiles of colony-forming cells in DNA synthesis (S phase) and non-S phase were determined by assessment of the proportion of separated cells sensitive to pulse treatment with high specific activity 3H-thymidine (3H-Tdr) prior to culture. The results excluded the possibility that the assays simply detected identical cells in various stages of the mitotic cycle. Rather, three different populations of precursor cells were distinguishable by the culture techniques employed. In a series of five unseparated normal bone marrow samples, neutrophilic CFU-d had a small proportion in S phase (7% ± 10%) as measured by 3H-TdR suicide experiments. The average proportion of CFU-c in S phase was 21% ± 7% and 48% ± 4% for day 14 and day 7 CFU-c, respectively.

Original languageEnglish (US)
Pages (from-to)221-232
Number of pages12
JournalBlood
Volume52
Issue number1
StatePublished - 1978
Externally publishedYes

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Cell Separation
Sedimentation
Cells
Agar
Bone
Population
S Phase
Experiments
Bone Marrow Cells
Fibrin
Thymidine
Culture Diffusion Chambers
Assays
In Vitro Techniques
Culture Techniques
DNA
Cell Communication
Suicide
Bone Marrow

ASJC Scopus subject areas

  • Hematology

Cite this

@article{b637ff2c59fd4debb07fda14768e22c4,
title = "Diversity of human granulopoietic precursor cells: Separation of cells that form colonies in diffusion chanbers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation",
abstract = "Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d). A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells (3) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 0.9 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar culture, were a heterogeneous population, possibly derived from both day 7 and day 14 CFU-c. Mixing experiments did not show evidence of cell interaction that could explain the observed velocity differences. The velocity sedimentation profiles of colony-forming cells in DNA synthesis (S phase) and non-S phase were determined by assessment of the proportion of separated cells sensitive to pulse treatment with high specific activity 3H-thymidine (3H-Tdr) prior to culture. The results excluded the possibility that the assays simply detected identical cells in various stages of the mitotic cycle. Rather, three different populations of precursor cells were distinguishable by the culture techniques employed. In a series of five unseparated normal bone marrow samples, neutrophilic CFU-d had a small proportion in S phase (7{\%} ± 10{\%}) as measured by 3H-TdR suicide experiments. The average proportion of CFU-c in S phase was 21{\%} ± 7{\%} and 48{\%} ± 4{\%} for day 14 and day 7 CFU-c, respectively.",
author = "N. Jacobsen and Hal Broxmeyer and E. Grossbard and Moore, {M. A S}",
year = "1978",
language = "English (US)",
volume = "52",
pages = "221--232",
journal = "Blood",
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T1 - Diversity of human granulopoietic precursor cells

T2 - Separation of cells that form colonies in diffusion chanbers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation

AU - Jacobsen, N.

AU - Broxmeyer, Hal

AU - Grossbard, E.

AU - Moore, M. A S

PY - 1978

Y1 - 1978

N2 - Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d). A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells (3) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 0.9 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar culture, were a heterogeneous population, possibly derived from both day 7 and day 14 CFU-c. Mixing experiments did not show evidence of cell interaction that could explain the observed velocity differences. The velocity sedimentation profiles of colony-forming cells in DNA synthesis (S phase) and non-S phase were determined by assessment of the proportion of separated cells sensitive to pulse treatment with high specific activity 3H-thymidine (3H-Tdr) prior to culture. The results excluded the possibility that the assays simply detected identical cells in various stages of the mitotic cycle. Rather, three different populations of precursor cells were distinguishable by the culture techniques employed. In a series of five unseparated normal bone marrow samples, neutrophilic CFU-d had a small proportion in S phase (7% ± 10%) as measured by 3H-TdR suicide experiments. The average proportion of CFU-c in S phase was 21% ± 7% and 48% ± 4% for day 14 and day 7 CFU-c, respectively.

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