Evidence for structured variation in self-renewal capacity within long-term bone marrow cultures.

TLDR

Bone marrow CFUs are pluripotent stem cells capable of proliferation and differentiation, with their proliferative capacity quantified by serial transplantability and the Rs metric from 14‑day transfers. This study aimed to measure the self‑renewal capacity of both adherent and nonadherent CFUs derived from long‑term bone marrow cultures. Cultures were set up so that nonadherent cells originated from the adherent layer, and the experimental design tracked changes in self‑renewal over time, revealing an initial proliferation of early stem cells followed by clonal succession that stabilized self‑renewal capacity. Both adherent and nonadherent CFUs formed spleen colonies, but adherent CFUs consistently displayed higher self‑renewal; their capacity initially declined with increasing CFU numbers, then stabilized after three weeks, supporting a two‑step proliferation model involving early stem‑cell expansion and later clonal maintenance.

Abstract

Bone marrow pluripotent stem cells (CFUs) demonstrate capacity for both proliferation and differentiation. The proliferative capacity of CFUs has been measured by serial transplantability and by the Rs, a measurement of CFU production in a single 14-day transfer. In the present study, the self-renewal capacity fo both adherent and nonadherent CFUs from long-term bone marrow cultures was measured. Culture conditions were established such that nonadherent cells were derived from the adherent cell layer. Both adherent and non-adherent cells produced spleen colonies, demonstrating that significant proliferative potential was present in both locations; however, at all times in culture, the CFUs within the adherent stromal cell layer had a significantly greater self-renewal capacity than did the nonadherent CFUs. During the initial establishment of the cultures, the self-renewal capacity of the adherent CFUs decreased as the total number of CFUs per flask increased. After 3 weeks in culture, the self-renewal potential of the adherent CFUs stabilized and was maintained. These results suggest two different mechanisms of stem cell proliferation. In order to increase the most primitive stem cell pool size, there was initial proliferation of early stem cells with a concomitant decrease in self renewal capacity. Once this pool was established, the self-renewal capacity of the adherent CFUs maintained for 13 weeks in culture suggests that CFU production and cell maintenance were achieved by clonal succession.