The effects of glycosaminoglycans on thrombopoietin-induced megakaryocytopoiesis.

BACKGROUND AND OBJECTIVES: The extracellular matrix plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAG) are major components of the matrix. In this study, the effects of various GAG on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. DESIGN AND METHODS: CD34+ cells were highly purified from steady-state human peripheral blood. The GAG tested were hyaluronic acid (from humans, pigs and roosters), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale, shark or squid cartilage) and dermatan sulfate (DS). RESULTS: When used alone, none of the GAG supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin, human hyaluronic acid, whale chondroitin sulfate and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by thrombopoietin alone after 9-12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. INTERPRETATION AND CONCLUSIONS: DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.

Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34+ megakaryocytic progenitor cells from human placental and umbilical cord blood.

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34(+) cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34(+) CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.

Effects of the combination of thrombopoietin with cytokines on the survival of X-irradiated CD34(+) megakaryocytic progenitor cells from normal human peripheral blood.

Combinations of thrombopoietin and cytokines that act on megakaryocyte development (stem cell factor, IL3, IL6, IL11, flt3 ligand (now known as FLT3LG), erythropoietin, GM-CSF and G-CSF were evaluated for their ability to enhance clonal growth in vitro of X-irradiated CD34(+) megakaryocytic progenitor cells (CFU-megakaryocytes) purified from normal human peripheral blood. These data were compared with corresponding results described previously for CD34(+) CFU-megakaryocytes from human placental/umbilical cord blood (I. Kashiwakura, Radiat. Res. 153, 144-152, 2000). All cytokines, except IL3, promoted thrombopoietin-induced colony formation, but they resulted in exponential radiation survival curves. No significant differences in the D(0) (46-61 cGy) and extrapolation number n (1.00-1.04) were observed between thrombopoietin alone and in combination with these cytokines. IL3 did not promote colony formation, but marked shoulders were observed on the survival curves (D(0) = 91 cGy, n = 2.83). Flow cytometric analysis of cells harvested from cultures of X-irradiated cells stimulated with thrombopoietin plus IL3 showed no significant differences in the expression of surface antigens and DNA ploidy distribution of megakaryocytes from the control. These findings suggest that IL3 plays a key role in promoting the survival of X-irradiated CD34(+) CFU-megakaryocytes from peripheral blood as well as those from cord blood, though the former are more radiosensitive.

Inhibitory effect of murine kidney extracts on mast cells derived from human umbilical cord blood.

We examined the effect of aqueous murine kidney extract (MKE) on the growth of mast cells prepared from the liquid culture of human umbilical cord blood CD34+ cells in the presence of a combination of recombinant human stem cell factor (SCF) and interleukin-6 (IL-6). Cultured cells were mature mast cells that expressed CD117 antigen on their surface, a specific marker for human mast cell, and they contained 6.53 pg histamine per cell. Adding MKE resulted in a 53% inhibition of mast cell growth and a 40% decrease in histamine content in mast cells in a serum-free liquid culture stimulated by SCF and IL-6. The inhibitory molecule for the growth of human mature mast cells was estimated at about 30 kDa of protein from gel-filtration HPLC. This fraction also inhibited the growth of murine peritoneal cells-derived mast cells. These results suggest that MKE contains regulator(s) that suppress the growth of mast cells and histamine synthesis. and that act beyond species specificity.

Effects of amifostine on the proliferation and differentiation of megakaryocytic progenitor cells.

This study investigated the effects of amifostine, a clinically usable radioprotector or chemoprotector, on the proliferation and differentiation of normal and X-irradiated cluster of differentiation 34 positive (CD34+) megakaryocytic progenitor cells (colony-forming unit in megakaryocytes, CFU-Meg) from human placental and umbilical cord blood (CB) in vitro. Amifostine significantly accelerated megakaryocyte colony formation in a plasma clot culture supplemented with recombinant human thrombopoietin because of an increase in immature CFU-Meg-derived large megakaryocyte colony formation. An analysis of the cells that were harvested from the culture showed that amifostine induced a 70- and an 83-fold increase in the total cell and CFU-Meg numbers, respectively, and produced hyperploid megakaryocytes of more than 8 N ploidy. The radioprotective effect of amifostine on the clonal growth of X-irradiated CD34+ CFU-Meg was observed by treatment before or after irradiation. These findings suggest that the action of amifostine extends from immature CFU-Meg to the terminal differentiation of megakaryopoiesis, and its radioprotective effect is shown in megakaryopoiesis and thrombopoiesis.