The bone marrow hematopoietic compartment (BMHC) is a complex tissue where hematopoietic cells interact with a heterogeneous microenvironment consisting of diverse hematopoietic and stromal cells, cytokines, extracellular matrix, and variable physiological conditions, such as pO2 and pH levels [1,, 2]. While much is known about the roles of stromal cells and cytokines [3,, 4], little is known about the effects of pH on megakaryocytic (Mk) differentiation and maturation. Hematopoietic stem and progenitor cells, including Mk progenitor cells, reside in the core of the BMHC located the farthest away from the sinuses, while Mk cells mature adjacent to bone marrow sinus walls, whereby they may release platelets directly into circulation through gaps of the sinusoid wall , or they may enter the circulation and release platelets in the pulmonary microvasculature . As cells differentiate, they move closer to the sinus lining. BM microphotographs [2,, 7,, 8] show that there are as few as four to eight to as many as 16-20 cells tightly packed between two sinuses in any one direction. Thus, many hematopoietic cells (and especially the most primitive stem and progenitor cells) are two to eight cells (or 20 to 80 μm, assuming a conservative 10 μm cell diameter) away from the closest sinus. Another measure of the likely distances of cells from sinuses is the size (80 to 1,200 μm in diameter) of the lymphocyte nodules, in which lymphocytes are organized in the human BMHC . Along with pO2 gradients, it has been demonstrated experimentally that pH drops from 7.35 to around 7.1 within about 25 μm from a blood vessel in normal subcutaneous tissue , and that the lowest pH (and pO2) values are observed the farthest away from the vessel wall . Furthermore, it has been shown that arterial blood has a pH of 7.4, while venous blood has a pH of 7.35 . These observations suggest that there exist spatial pH variations in the BMHC, and that primitive stem and progenitor cells are exposed to and differentiate in a lower (≤7.1) pH environment, while differentiated Mk cells mature at a higher pH (7.35-7.40) environment.
The effects of oxygen tension (pO2) on the granulocytic (G), erythroid (E), and Mk lineages have been extensively studied in our laboratory [13–, 17]. We reported that these effects were physiologically relevant and consistent with the expected exposure of the various cell types to variable pO2 levels in the BMHC. In addition to pO2, pH is also a powerful regulator of cell proliferation, differentiation, and cytokine secretion . We reported that culture pH was a potent proliferation and differentiation factor of E and G lineages, and that the cloning efficiencies of primitive erythroid progenitors (BFU-E) were ninefold higher at low pH (7.1) compared with high pH (7.6) [19,, 20]. A small pH increase of 0.2 units over the physiological value (7.4) yielded significant reductions (42%-85%) in cloning efficiencies for all E and G progenitor types and cytokine combinations tested. Differentiation of BFU-E in semisolid assays progressively increased as pH was raised from 6.95 (no colonies detected) to 7.4 (maximum colonies detected) to 7.6 (maximum hemoglobin content). In liquid cultures of peripheral blood (PB) CD34+ cells, E differentiation proceeded faster at high pH and was blocked at an intermediate stage by low pH . In G cultures, cell expansion was enhanced at pH 7.1-7.25, with twice as many total cells and G precursors produced as at pH 7.4 . For T-cell proliferation, a greater than threefold increase in the proliferation capacity was reported in pH 7.0 and 7.2 cultures compared with pH 7.4 cultures . Rich  has shown that optimal proliferation of murine macrophages occurs at pH 7.6-8.0, and that erythropoietin secretion by macrophages varies with pH.
The aim of this study is to understand the effects of pH on Mk differentiation, maturation, and apoptosis, and to test the hypothesis that pH regulates these processes in a physiologically relevant manner. It is expected that Mk progenitors will differentiate under conditions of low pH (and pO2), while maturation and platelet release will take place under high pH (and pO2) conditions. We cultured mobilized PB CD34+ cells in a serum-free medium supplemented with interleukin-3 (IL-3), Flt-3 ligand (Flt-3), and thrombopoietin (TPO) at three different pH levels (pH 7.2, 7.4, and 7.6). As in prior studies , the high pH value of 7.6 was used to enhance any possible pH effects and to contrast such effects with those at the low and physiologically relevant pH values of 7.2 and 7.4. Furthermore, the antiapoptotic agent Z-Val-Ala-Asp(Ome)-FMK (zVAD) was used to explore the relationship between Mk maturation (polyploidization) and apoptosis.