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Stem Cells in Human Peripheral Blood: A Method for Histologic Analysis of Colony Formation in Plasma Clot Diffusion Chambers

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Diffusion Chamber Culture

Abstract

The purpose of the present study was to define some of the quantitative and qualitative characteristics of colonies formed by normal human hemopoietic stem cells from peripheral blood in plasma clot diffusion chambers.

The chambers were implanted IP into 650 rad irradiated NMRI mice, reimplanted IP into newly irradiated mice after 7 days, and at different intervals thereafter harvested and completely embedded in plastic (methyl-methacrylate). Because this technique circumvents clot manipulation, exact impressions of colony formation could be obtained.

Three hundred sections of 3.0 μm each were made throughout the midportion of each chamber, and at intervals of 15 μm the sections were scored for different types of cell aggregates (CA) of ⩾ 3 cells. At day 13 of culture, myeloid and megakaryocytic CA predominated, and single erythroid CA were rare. Small numbers of purely monocytic and eosinophilic CA were also found, but no lymphoid CA.

Expressed as a percentage of the total number of CA per chamber, 5% mixed myeloid and megakaryocytic, 2.5% myeloid and erythroid, 0.58% myeloid, erythroid, and megakaryocytic, and 0.53% megakaryocytic and erythroid CA were found.

Mixed myelo-monocytic or neutrophilic-eosinophilic CA were not observed.

Quantitatively, the total number of CA per chamber increased with time after implantation, and on day 13 the mean (± SEM) number of myeloid CA per section was higher (p > 0.05) than for megakaryocytic CA. Analysis of the distribution pattern of myeloid and megakaryocytic CA showed that they were not always homogeneously localized in the midportion of the chamber. From several whole colonies the serial sections of 5 μm each were analyzed and three-dimensional impressions constructed. This revealed that small and large colonies were simultaneously present. Small colonies were perfect spheres, whereas larger colonies could have bizarre shapes with large cell protrusions and evidence of cell migration.

Myeloid cells were mostly megaloblastic and synchronized in their stages of differentiation, with a normal granulation pattern. Polymorph-nucleated cells tended to migrate to the surroundings of the colony. Eosinophilic colonies were very compact, without evidence of cell migration. Megakaryocytes showed abundant bleb formation and evidence of platelet production, as well as signs of cell migration.

Single erythroid colonies of pro-erythroblasts showed definite signs of dyserythropoiesis, while the majority of the erythroid cells were found in mixed colonies with neutrophilic or megakaryocytic cell lines.

In some colonies consisting of normoblasts, erythrocyte production was also observed.

This technique could contribute to a better understanding of proliferation and differentiation of hemopoietic stem cells in various hematologic disorders.

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Authors
  1. F. E. Zwaan
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  2. J. te Velde
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  3. A. Puliga
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  4. H. Goselink
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  5. R. Lobatto
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Editor information

Editors and Affiliations

  1. Medical Department, Brookhaven National Laboratory, Upton, N.Y., USA

    Eugene P. Cronkite MD  & Arland L. Carsten PhD  & 

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© 1980 Springer-Verlag Berlin Heidelberg

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Zwaan, F.E., te Velde, J., Puliga, A., Goselink, H., Lobatto, R. (1980). Stem Cells in Human Peripheral Blood: A Method for Histologic Analysis of Colony Formation in Plasma Clot Diffusion Chambers. In: Cronkite, E.P., Carsten, A.L. (eds) Diffusion Chamber Culture. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-67644-4_15

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  • DOI: https://doi.org/10.1007/978-3-642-67644-4_15

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-540-10064-5

  • Online ISBN: 978-3-642-67644-4

  • eBook Packages: Springer Book Archive

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