Cell Cycle Influence of Stem Cell Differentiation in Culture.

Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. To address whether the cell cycle plays a role in the differentiation of stem cells, we co-cultured murine bone marrow Lin- Sca-1+ cells, at different points in their cycle, with the OP9-DL1 system. OP9-DL1 stromal cell layer has been transduced to allow T-cell differentiation in culture. We first induced cell cycle synchrony by exposing the isolated cells to a cytokine cocktail of TPO, Flt-3 and Stem Cell Factor. The cells were exposed to this primary culture for 0, 6, 24, 32 and 40 hours and were subsequently cultured on an OP9-DL1 stromal cell layer grown in 6-well plates. Cells were co-cultured for 8 days and 21 days, in the presence of IL-7 and Flt-3. Cultured cells were evaluated for CD4, CD8, B220, CD19, NK1.1, and Mac-1 surface markers, using flow cytometry. On Day 8, we found a significant hotspot at 32-hours (early-S phase) for B220+ cells (34.3 %), while Mac-1 positive cells demonstrated a 24-hour hotspot (18.1 %). As expected, terminal T and B-cell differentiation (CD 4, CD8, and CD19) was undetectable at 8 days. Three separate short-term (8 day) experiments have confirmed these data. Cells in culture for 21 days similarly show variation in differentiation outcome. CD4 cells demonstrate a peak at the 40 hour time point (mid-S phase) (69.9%), while CD8 positive cells were significantly increased at the 32 hour time point (34.4%). These data indicate both B and T cells show reversible differentiation fluxes linked to cell cycle. This work supports previous evidence that marrow hematopoiesis at the stem cell level is regulated on a continuum and that stem cells have reversible, cycle-related differentiation capacity.