Higher pH Promotes Megakaryocytic Maturation and Apoptosis

Abstract

Megakaryocytic (Mk) cells mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. In contrast, primitive stem and Mk progenitor cells reside the furthest away from the BM sinus walls. The existence of pH gradients in the BM raises the question of whether pH affects Mk maturation and differentiation. We generated Mk cells from peripheral blood CD34 + cells in a serum-free medium at different pH levels (7.2, 7.4, and 7.6) and found that higher pH resulted in an earlier and higher polyploidization of CD41 + Mk cells and an earlier onset of Mk-cell apoptosis. The peak day of high ploidy was correlated well with the onset day of Mk apoptosis, thus suggesting that a decline in the fraction of high-ploidy Mk cells at the late culture stage is caused by Mk-cell apoptosis. We further explored the relationship between Mk-cell maturation and apoptosis by employing an antiapoptotic agent Z-Val-AlaAsp(Ome)-FMK (zVAD). Addition of zVAD led to an average 30% higher and 2.8-day delayed polyploidization, while apoptosis was delayed by 2.4 days. Faster depletion of CD34 + cells and an earlier peak in the fraction of larger colony-forming Mk cells (BFU-Mks) were also observed at higher pH. Taken together, these data suggest that higher pH promotes Mk-cell differentiation, maturation, and apoptosis.

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…Our previous work demonstrated the importance of pH in Mk maturation and consistently showed that culturing primary Mks at higher pH in the range from 7.2 to 7.6 increased production of high-ploidy ( > 4N) Mks. 9,29 pH also modulates the differentiation of granulocytes, 41,59,60 erythrocytes, 60,61 and BM stromal cells. 62 Hematopoietic stem cells reside close to the bone surface and migrate toward the sinuses during maturation (Fig.…”

Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis

“…Ploidy and apoptosis assays were performed using previously published methods with minor modifications (22,23). For analysis of murine cell cultures, Mk cells were first labeled with fluorescein isothiocyanate-conjugated rat antimouse CD41 IgG 1 (BD Biosciences).…”

Review:Ex VivoEngineering of Living Tissues with Adult Stem Cells

“…74 Similarly, rat BMSCs cultivated at 5% oxygen instead of 20% oxygen exhibit more rapid proliferation and greater expression of key markers of osteogenesis such as alkaline phosphatase activity and calcium content. 75 A high pH of 7.6 promotes the maturation and apoptosis of human megakaryocytic cells better than physiological conditions do, 76 whereas a subvascular pH of 7.07 to 7.21 enhances granulopoiesis proliferation better than pH 7.38.…”