Cloning and frequency analysis of haemopoietic stem cells producing CFC over weeks in long-term marrow cultures.

Using adherent marrow cell layers devoid of stem cells, in vitro cloning and the determination of the frequency of murine haemopoietic stem cells were performed by means of limiting dilution. The presence of stem cells in individual microcultures was indirectly proven by the sustained presence of in vitro colony-forming cells (CFC). These cells increased in number as a function of the culture period, which seems to indicate that the CFC were, de novo, produced in vitro. Although stem cell frequencies comparable to the in vivo spleen colony assay were determined in some experiments, most of our frequency estimates suggested stem cell frequencies ranging from 5/10(6) to 90/10(6). After a period of approximately 4 weeks, the stem cell activity in the microcultures declined rapidly. This may have been caused either by an increased differentiation pressure governed by CSF and/or similar factors or by sub-optimal culture conditions.