Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer

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.