Periodicity of Differentiation and Self-Renewal Suggest Homeostatic Feedback Control of Commitment in a Hematopoietic Progenitor Cell Line.

FDCPmix cells are karyotypically normal, non-leukemic, murine multipotent progenitors which undergo extensive self-renewal in the presence of a high concentration of interleukin-3 (IL-3), and progressive commitment and differentiation along all myelo-erythroid lineages upon substitution with the appropriate cytokines. Although clonal, the FDCPmix cells are characterised by a variable degree of heterogeneity in the population. This heterogeneity is apparent firstly from the variable background of morphologically differentiated cells which accumulate in self-renewal (high IL-3) conditions, and secondly from the low and variable incidence of colony-forming cells (CFU) which are capable of extensive self-renewal in semi-solid media and which are generally assumed to be the founder cells of the population. In order to define the variability in more detail, early passage FDCPmix cells were thawed into standard culture conditions and passaged back to 6x10⁴ cells per ml every 2 days over a period of nine months. The degree of background differentiation and the incidence of colony forming cells were determined at weekly intervals. Based on our unpublished observations that glucose concentration can influence progenitor cell fate, parallel cultures were maintained in IMDM (4g/L glucose) and DMEM (1g/L glucose) and all CFU assays were performed using both IMDM and DMEM based semi-solid media. In both IMDM and DMEM cultures, the level of background differentiation increased from 0 to an initial peak of 15% (IMDM) to 30%(DMEM) over the first 5 weeks, followed thereafter by further peaks at 3–4 week intervals. There was no corresponding pattern in the CFU^IMDM frequency. However, a similar 3–4 week periodicity (displaced from that of the background differentiation) was evident in both the frequency of CFU^DMEM and particularly in the ratio of CFU^DMEM to CFU^IMDM. The high frequency of CFU^DMEM and the low frequency of differentiated cells over the first 5 weeks of culture corresponded to the period in which the FDCPmix cells underwent a homogenous and rapid differentiation in response to erythroid or myeloid growth factors. These patterns of periodicity in both IMDM and DMEM cultures are suggestive of a delayed feedback phenomenon in which the accumulation of differentiating cells causes a decrease in the ratio of commitment-to-self-renewal of the founder cells, and subsequent decrease in differentiating cell numbers, with the delay between commitment and differentiation being responsible for overshoot, and the relatively long periods of phasing observed. Since the cultures were diluted regularly into fresh media, the basis of such a feedback is likely to involve cell-cell contact rather than diffusible factors. In vivo, a local cell census taken in this way may represent a means of restricting the local concentration of stem cells in a given niche.