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Enzymatic treatment of long-term human marrow cultures reveals the
preferential location of primitive hemopoietic progenitors in the adherent
layer
L Coulombel, AC Eaves and CJ Eaves
Recent studies with long-term mouse marrow cultures have indicated the
importance of the adherent layer as a primary reservoir of the most
primitive stem cells, from which derivative stem cells and more
differentiated progenitors are continuously generated. We have now examined
the role of the adherent cell layer in long-term human marrow cultures from
this point of view. Prerequisite to such an undertaking was the development
of a nontoxic and reproducible method for detaching the adherent layer and
making it into a single-cell suspension suitable for characterization by
colony assays. Both trypsin and collagenase could be used to obtain
suspensions that met these criteria. Lack of toxicity was demonstrated by
the preservation of CFU-E, BFU-E, and CFU- C plating efficiency in fresh
human marrow cell suspensions exposed to the same enzymatic treatments.
Collagenase treatment of long-term marrow culture adherent layers was
considered superior because it freed all hemopoietic colony-forming cells
but left some of the other cells still adherent. Using this method, we
found that CFU-C, BFU-E, and CFU- G/E were consistently detectable in the
adherent layer for at least 8 wk, with the majority of the BFU-E and
CFU-G/E being located in the adherent layer (70%-75% after 2-3 wk and more
than 90% by 7-8 wk). Although corresponding numerical differences in
adherent and nonadherent CFU-C populations were not observed, the colonies
derived from them showed marked differences in the size they achieved; the
adherent layer being the exclusive site of CFU-C, with a very high
proliferative capacity. These findings emphasize the importance of
assessing the progenitor content of the adherent layer of long-term human
marrow cultures and provide an appropriate methodology.
Volume 62,
Issue 2,
pp. 291-297,
08/01/1983
Copyright © 1983 by The American Society of Hematology
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