A quantitative assay that evaluates the capacity of human stromal cells to support granulomonopoiesis in situ

Stem Cells
E TamayoP Herve

Abstract

We describe an assay that makes possible the observation of granulomonocytic colonies grown on allogeneic stromal layers and the quantification of the stroma-adherent colony-forming cells (CFC). Stromal layers were generated from Stro-1 positive cells isolated from adherent layers of primary long-term marrow cultures using magnetic beads coated with the Stro-1 antibody. The stromal layers consisted mainly of myofibroblastic cells. Marrow fractions depleted of cells bearing receptors for soybean agglutinin (SBA) and enriched in CD34+ cells were obtained by panning. SBA-, CD34+ marrow cells were seeded onto stromal cells grown in 96-well plates. After four weeks, a mixture of cytokines was added (granulocyte-macrophage colony-stimulating factor [GM-CSF]: 25 U/ml, interleukin [IL]-3: 4 ng/ml, Steel factor: 5 ng/ml and growth factors provided by 3% conditioned medium from the 5637 cell line). Wells with large colonies (containing 10(3) to 10(4) cells) were scored after 14 days. Limiting dilution analysis of data revealed a Poisson distribution of the stroma-adherent CFC. There was an average of one stroma-adherent CFC per 167 CD34+ enriched marrow cells, which gave an estimated frequency of one CFC per 10(5) unfractionated bone mar...Continue Reading

References

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Citations

Jul 27, 2000·Journal of Cellular Physiology·F Deschaseaux, P Charbord
May 30, 2001·Experimental Hematology·C LoeuilletD Chalmers
May 17, 2000·Journal of Hematotherapy & Stem Cell Research·N BrouardB Péault
Feb 15, 2001·Journal of Hematotherapy & Stem Cell Research·P CharbordB Péault
May 22, 2001·Journal of Hematotherapy & Stem Cell Research·F DeschaseauxP Charbord
Feb 1, 1996·Journal of Hematotherapy·S Gronthos, P J Simmons
Jun 11, 1998·Journal of Hematotherapy·C E BulaboisP Charbord
Nov 27, 2007·Journal of Tissue Engineering and Regenerative Medicine·Xuechao YangJohn A Jansen

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