Enrichment of hematopoietic progenitor cells (CFUC and BFUE) from human peripheral blood.
Academic Article
Overview
abstract
Granulocytic (CFUC) and erythroid (BFUE) progenitor cells have been rapidly purified from human peripheral blood approximately 140 fold by combining centrifugation on a density cushion and immunoadherence cell separation methods. An initial light density (d less than 1.071 g/cm3) mononuclear cell fraction, enriched for progenitor cells, was obtained by centrifugation of whole blood on a modified Ficoll-Hypaque density cushion. Cultures of the light-density cells gave cloning efficiencies (defined as the percentage of total cells plated) of 0.008% and 0.015% for CFUC and BFUE respectively. Further purification was achieved by negative selection whereby selective populations of immunocompetent cells were removed. Thus, B cells and monocytes (as well as up to 50% high affinity Fc receptor bearing cells) were simultaneously depleted by immunoadherence to plastic petri dishes coated with rabbit anti-human IgG. Leu-3a positive (helper) and Leu-2a positive (suppressor) T cells were then simultaneously depleted by an indirect "panning" method, whereby the T cell subsets were coated with the corresponding murine monoclonal antibodies prior to their removal by immunoadherence to plastic petri dishes coated with goat anti-mouse IgG. The final cell fraction, which contained approximately 2% of the initial light density cells were highly enriched for CFUC and BFUE, having cloning efficiencies of 0.37% (+/- 0.30) and 0.27% (+/- 0.24) respectively. Overall, the purification procedure used in the present study is relatively rapid, simple and reproducible. As such, it should provide a viable and convenient alternative approach to previously published methods for purifying hematopoietic progenitor cells from human peripheral blood.
