| This thesis is a comparative study of variables affecting differentiation and proliferation of progenitor cells from various embryonic and fetal tissue sites as they differentiate into mast cells. Embryonic stem cells from gestation day 4.5 (d4.5) differentiated exclusively into mast cells when cultured in semi-solid media with stem cell factor (SCF) and Interleukin-3 (IL-3). The same was true of d8–11 yolk sac cells and d8–11 cells from the embryo itself. However, culture with SCF and IL-3 of cells from d14 fetal livers differentiated into mast cells, cells of the erythrocyte lineage and a few “macrophages” which may have been similar to the stromal cell found in the bone marrow that supplies iron to and removes the nucleus from erythrocytes. An erythrocyte-nurturing role for the fetal liver “macrophages” was substantiated by removing cells expressing Mac1, and sorting for cells expressing the early mast cell marker, Kit, resulting in cells that differentiated only into mast cells with SCF and IL-3. The variables affecting proliferation that were studied were brief incubation at very high cell density, and co-culture with fibroblasts. In addition, a few experiments were done to determine the mechanism of the effect of these variables on proliferation. High cell density incubation increased proliferation of yolk sac, fetal liver and bone marrow cells. The increased proliferation of yolk sac and bone marrow cells was attenuated by an inhibitor of the sodium-ion/hydrogen-ion exchanger, 5-(N,N-hexamethylene) amiloride (HMA). The exchanger was also found to play a role in the subsequent proliferation of mast cells from bone marrow cells co-cultured with fibroblasts since, if HMA was present during the first thirty minutes of contact, mast cell proliferation decreased by 75%. Using fluorescent imaging, activation of this exchanger was monitored by an increase in intracellular pH. Altogether these results demonstrate a growing concept in developmental biology regarding the normal stem cell in its niche (tissue site) and the affects of exogenous variables on it. They substantiate the concept that the differentiation path and proliferation of a stem cell depends both on its past history and on its future circumstances. |
