Platelet-poor Plasma-derived Serum Research Articles

Differentiation of Human ES Cell into the Hemangioblast.

We hypothesize that during early human embryo development a common endothelial/hematopoietic precursor is formed. This precursor, named the hemangioblast, is an early endothelial/hematopoietic precursor responsible for angiogenesis and both primitive and definite hematopoiesis. The aims of this work are 1) to establish the ES/EB model system for genotypic studies of early vasculo/hematopoiesis. 2) to phenotype human hemangioblast and establish standard culture/storage condition that will stabilize the phenotype of these cells for a defined period and permit consistent, predictable outcomes of experiments. Human ES (H-1 line) cells (NIH code WA01) has been maintained on g -irradiated MEF cells supplemented with DMEM/F12 media containing 4ng/ml b-FGF and 15% knockout serum by changing media everyday and passaging cells every 3–4 days. EBs were generated either in liquid or 1% methylcellulose cultures (1 x 104 ES cells per 35-mm Petri dish) in medium containing Iscove modified Dulbecco medium (IMDM), 15% FCS, 2 mM glutamine, 450 μM MTG, 50 μg/mL ascorbic acid, and 20% BIT (1% bovine serum albumin (BSA), 10 μg/mL insulin, and 200 μg/mL transferrin. In order to induce EB formation two days old hES cells were plated in 1% methyl cellulose in IMDM media supplemented with 50mg/ml ascorbic acid, 10mg/ml insulin, 200 mg/ml transferrin, 20% Bits, 15% FCS, 450mM MTG, 2mM-L-glutamine for 3days. To generate hemangioblasts, 1 x 104/mL EB cells were replated in 1% methylcellulose in the presence of IMDM, 10% PDS (bovine platelet-poor plasma-derived serum; Biomedical Technologies, Stoughton, MA), 2 mM glutamine, 450 μM MTG, 25 μg/mL ascorbic acid, 20% BIT, 5 ng/mL human vascular endothelial growth factor (hVEGF), 50 ng/mL SCF, 10 ng/mL human fibroblast growth factor 2 (hFGF-2), and 2 U/mL hEPO. RNA from day 0, 2, 3 and 4 days old EBs was collected for gene regulation studies. We have chosen to study the key regulatory genes (FLK-1, Runx-1, SCL, HEX, GATA-1) during this process. RNA collected from undifferentiated hES cells has been used as control. We observed expression of stemness genes: Sox-2 and Oct-4, in the undifferentiated ES cells while there was an upregulation in expression of Brachury on day 2 and FLK-1 and Hex genes in day 3 of differentiation. In hemangioblast forming cells (BL-CFC), we observed upregulation of FLK-1, SCL, Runx and downregulation of Brachury and Hex. This sptio-temporal expression profile of genes suggest that during hemangioblast formation and at the initial stages of differentiation hemangioblast fate is decided by Hex, while after day 2 the control is taken by FLk-1, SCl and Runx genes. Ongoing studies will define signaling pathways involved in this process. Understanding of molecular processes involved in hemangioblast formation will allow directed generation of this progenitor from human ES cells in the future.

Plasma-derived serum from hypertensive rats differentially increases growth of cultured arteriolar smooth muscle

A role for humoral factors in the arterial hypertrophy accompanying hypertension has been suggested, but direct evidence is lacking. We measured [3H]thymidine incorporation of growth-arrested smooth muscle cells, cultured from renal preglomerular arterioles of normal rats and exposed for 12 h to 10% platelet-poor plasma-derived serum (PDS) from male rats with early, benign, one-kidney, one-clip hypertension and paired one-kidney normotensive control rats. In the presence of low background concentrations (1-2%) of fetal calf serum, PDS from hypertensive rats increased [3H]thymidine incorporation up to 117% more than PDS from normotensive rats (P < 0.0001). Results were similar for the < 10,000 mol wt fraction of PDS and for PDS from rats drinking saline vs. water or eating a 7% (high) vs. a 0.45% NaCl diet. PDS from hypertensive rats also differentially increased smooth muscle cell proliferation and c-fos mRNA expression. Platelet-derived growth factor levels in PDS were undetectable (< 0.17 ng/ml). This study provides evidence for altered concentrations of sodium chloride-unresponsive plasma factor or factors in hypertension that result in enhanced growth of arteriolar smooth muscle cells.

Heparin augments osteoclast resorption‐stimulating activity in serum

Increased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long-term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet-poor plasma-derived serum, heparin, in the range 25-100 micrograms/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption-stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor-beta, platelet-derived growth factors, insulin-like growth factors I or II, acidic or basic fibroblast growth factors, fibronectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin-binding factor. The ability of glycosaminoglycans (GAGs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin-2-sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor involved in the local and systemic regulation of osteoclastic bone resorption.

Regulation of Granulosa Cell Proliferation: Facilitative Roles of Platelet-Derived Growth Factor and Low Density Lipoprotein*

Recent studies suggest that epidermal growth factor (EGF) and/or transforming growth factor-alpha (TGF-alpha) and insulin-like growth factor-I (IGF-I) act synergistically to promote granulosa cell proliferation in vitro suggesting a similar role in vivo. Using a serum-restricted, monolayer culture system containing very low levels of platelet-poor plasma-derived serum (PPPDS), the facilitative roles of platelet-derived growth factor (PDGF) and low density lipoprotein (LDL) with respect to growth factor-stimulated granulosa cell proliferation were investigated. In nutrient medium containing only 0.1% PPPDS, PDGF (1-25 ng/ml) had no effect upon granulosa cell proliferation. When combined with EGF, which alone does not stimulate granulosa cell proliferation, PDGF dose-dependently increased cell proliferation to levels obtained with 10% fetal calf serum (2.4-fold increase relative to controls, P less than 0.05). When combined with EGF and IGF-I, a combination which does stimulate mitosis in granulosa cells, PDGF again dose-dependently enhanced proliferation (P less than 0.05). The extent of proliferation obtained with EGF + IGF-I + PDGF was consistently greater than that obtained with 10% fetal calf serum (P less than 0.05) but significantly less than that obtained with EGF + fetal calf serum, a treatment which stimulates rapid granulosa cell proliferation. LDL has been shown to greatly enhance granulosa cell steroidogenesis by providing exogenous cholesterol. However, cholesterol is also required for plasma membrane biosynthesis and cell growth. LDL alone, had no effect upon porcine granulosa cell proliferation relative to media controls (0.1% PPPDS) nor did it synergize with any single growth factor to induce mitosis. When combined with EGF + IGF-I, and EGF + PDGF, but not PDGF + IGF-I, LDL dose-dependently (1-25 micrograms/ml) enhanced proliferation (P less than 0.05) to levels equivalent to that obtained with 10% fetal calf serum. When combined with EGF, IGF-I, and PDGF, LDL at 10 micrograms/ml enhanced proliferation to an extent equivalent with EGF + fetal calf serum (a 5.4-fold increase relative to media controls). High density lipoprotein did not itself stimulate proliferation nor did it facilitate proliferation mediated by growth factors. When maintained in medium alone (0.1% PPPDS), the cell population doubling time was 8.0 +/- 0.5 days. In the presence of EGF, IGF-I, PDGF, and LDL (10, 10, and 5 ng/ml, and 10 micrograms/ml, respectively) the doubling time was reduced to 2.0 +/- 0.1 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased plasma levels of platelet-derived growth factor activity in patients with progressive systemic sclerosis.

We measured mitogenic activity of whole blood serum and platelet-poor plasma-derived serum of a group of 10 patients with progressive systemic sclerosis and of 8 controls. Mitogenic activity of plasma-derived serum was greater in patients than in controls, in the absence of other signs of platelet activation. This increased activity was inhibited by specific antibodies, anti-platelet derived growth factor, suggesting that circulating levels of platelet-derived growth factor may be present in progressive systemic sclerosis patients. Platelet-derived growth factor, released either by platelets or by monocytes, might play a role in the pathogenesis of scleroderma.

Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor I, and transforming growth factor-beta on porcine granulosa cell deoxyribonucleic acid synthesis and cell proliferation.

Recent studies have suggested that the mammalian ovary synthesizes epidermal growth factor (EGF), somatomedin-C/insulin-like growth factor I (Sm-C), and transforming growth factor-beta (TGFb) and that these growth factors may in part form a basis for intraovarian regulation of granulosa cell proliferation and differentiation. The studies described herein were initiated to determine to what extent EGF, Sm-C, and TGFb function to regulate DNA synthesis and granulosa cell proliferation during primary monolayer culture. EGF, but neither Sm-C nor TGFb, alone consistently stimulated, in a dose-dependent manner, [3H]thymidine incorporation by porcine granulosa cells under defined conditions (P less than 0.01). Sm-C (10 ng/ml) and TGFb (1 ng/ml) both enhanced EGF-stimulated [3H]thymidine incorporation (56% and 300%, respectively; P less than 0.05). The levels of incorporation obtained with EGF plus TGFb were equal to or greater than those obtained using fetal bovine serum alone. When EGF, Sm-C, and TGFb were combined, [3H]thymidine incorporation was equivalent to that obtained with EGF plus 10% fetal bovine serum, heretofore the most potent stimulatory combination for [3H]thymidine incorporation. Thus, under defined conditions, EGF, Sm-C, and TGFb act synergistically to promote DNA synthesis in primary cultures of porcine granulosa cells. Although DNA synthesis is a requisite step for but is not an accurate measurement of cell proliferation per se, we investigated whether the observed effects of EGF, Sm-C, and TGFb on DNA synthesis were realized in terms of actual cell proliferation. This was accomplished using platelet-poor plasma-derived serum (PPPDS; 0.1-2.5%), which contains reduced levels of endogenous growth factors but not components needed for cell attachment. EGF (P less than 0.05), but neither Sm-C nor TGFb, alone consistently stimulated, in a dose-dependent manner, granulosa cell proliferation, an effect directly related to the PPPDS concentration. Sm-C consistently and significantly (P less than 0.05) enhanced EGF-stimulated cell proliferation in a dose-dependent manner. The facilitative effect of Sm-C was inversely related to the PPPDS concentration, ranging from a 76 +/- 15% increase at 0.1% PPPDS to a 14% increase at 1.0% PPPDS. TGFb exhibited a bifunctional effect on granulosa cell proliferation. At low levels of PPPDS (0.1% and 0.25%) and in the absence of Sm-C, TGFb enhanced EGF-stimulated cell division, an effect which, although small and variable (24 +/- 16%), was consistent.(ABSTRACT TRUNCATED AT 400 WORDS)

Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: Its relationship to type β transforming growth factor (TGF‐β)

Whole blood serum (WBS) and platelet-poor plasma-derived serum (PDS) from the same normal subject were compared for their abilities to support human megakaryocyte (MK) colony formation. In all cases, PDS promoted the growth of a higher number (20-50%) of MK colonies than did WBS. Increasing amounts of WBS decreased the number of colonies, whereas increasing concentration of PDS had no marked effects. Crude platelet extracts or platelet secretory products from thrombin-activated platelets also elicited an inhibition of MK colony formation in a dose-dependent manner. A complete inhibition was found for a dose equivalent to 1.10(9) platelets/ml and a 50% inhibition in a range of 1.10(7)-1.10(8) platelets/ml. These platelet products were also inhibitory for erythroid progenitor growth. Platelets from two patients with gray platelet syndrome elicited only a minor inhibition of MK growth, suggesting that the platelet alpha granule is the origin of this inhibition. When platelet extracts were acid-treated, the biological activity of the inhibitor on CFU-MK and CFU-E growth was 20-50-fold higher. In addition, a potent stimulatory activity on the growth of day 7 CFU-GM was observed. The enhancement of biological activities by acid treatment suggests that type beta transforming growth factor (TGF-beta) could be involved in this platelet inhibitory activity. The homogeneous native TGF-beta (from 1 pg to 1 ng/ml) produced the same effects previously induced by platelet products. It totally inhibited CFU-MK growth (at a 500 pg/ml), it inhibited CFU-E growth, and it stimulated growth of day 7 CFU-GM in the presence of a colony-stimulating factor. The inhibition of CFU-MK growth was also observed on purified progenitors. In conclusion, these results suggest that TGF-beta may be implicated in negative autocrine regulation of megakaryopoiesis. However, since this molecule has ubiquitous biological activities, its physiologic relevance as a normal regulator of megakaryopoiesis requires further investigation.

Stimulatory activity on prostacyclin production decreases in sera from streptozotocin-induced diabetic rats

We recently reported that serum stimulatory activity on prostacyclin (PGI2) production by cultured bovine aortic endothelial cells decreased in noninsulin-dependent diabetic patients. In the present study, this activity was compared in streptozotocin-induced (STZ) diabetic rats and controls. Platelet-poor plasma-derived serum (PDS) from Wistar male rats stimulated 6-keto-PGF1 alpha production (a stable metabolite of PGI2) by cultured bovine aortic endothelial cells, rat lung fibroblasts, and rat aortic rings in a time- and dose-dependent manner. Namely, PDS from rats has a stimulatory activity on PGI2 production (PGI2 stimulatory activity; PSA). Furthermore, PSA in PDS from STZ diabetic rats (n = 12) significantly decreased as compared with that from control rats (n = 10) using three types of in vitro systems. The reduction in PDS-stimulated PGI2 production by the vascular wall may lead to platelet hyperaggregation and thrombus formation in diabetics, which is considered to be involved in the pathogenesis of diabetic macro- or microangiopathy.

Interactions of insulin, insulinlike growth factor II, and platelet-derived growth factor in erythropoietic culture.

To examine the influence of insulin and insulinlike growth factor (IGF) on erythropoiesis, we tested their effects in human bone marrow cultures prepared with biochemically defined medium or a platelet-poor plasma-derived serum (PDS) that was depleted of hormones by adsorption to activated charcoal. Erythroid colony formation was enhanced two- to threefold by 10 ng/ml of electrophoretically pure IGF-II and 100 ng/ml of highly purified insulin (P less than 0.05). Dose-response curves for IGF-II were parallel to and shifted by one to two orders of magnitude to the left relative to those for insulin. When added together to culture, IGF-II and insulin expressed additive activities. In contrast, their activities were synergistic with those of erythropoietin and burst-promoting activity. The erythropoietic actions of IGF-II and insulin were similar in PDS and whole blood serum (WBS) containing cultures. Furthermore, when added to cultures with electrophoretically pure platelet-derived growth factor, their respective activities were synergistic. We conclude that insulin and IGF-II potentiate human marrow erythropoiesis in vitro. Their activities appear to be mediated by a similar receptor or postreceptor system.

Platelet-derived growth factor enhances in vitro erythropoiesis via stimulation of mesenchymal cells.

The growth of erythroid colonies (from erythroid colony-forming cells) and erythroid bursts (from burst-forming cells [BFU-E]) is enhanced in the presence of serum as compared with plasma. A significant proportion of the enhanced growth is due to the platelet release product, platelet-derived growth factor (PDGF). Colony growth in cultures of whole marrow cells in platelet-poor plasma-derived serum (PDS) and erythropoietin was enhanced in a dose-dependent fashion by increasing concentrations of purified human PDGF with optimal enhancement at 12.5 ng/ml. However, no effect of platelet-release products or PDGF was observed on nonadherent human marrow cells or peripheral blood BFU-E, suggesting that an accessory cell population was required for the effect of PDGF on hematopoietic progenitors. In a two-layer culture system, pure populations of fibroblasts or smooth muscle cells, known to be present in the marrow microenvironment, restored the response of nonadherent marrow cells in the overlayer to PDGF and also conferred responsiveness to peripheral blood BFU-E. Endothelial cells in the two-layer culture system and macrophages, in contrast, lacked the ability to restore the enhancing effect of PDGF. Because other platelet-release mitogenic products are also found in serum, a monospecific anti-PDGF IgG preparation was added to cultures grown in platelet rich plasma-derived serum. Only partial reduction in colony and burst growth was seen, suggesting that other platelet-release products were acting in this system. These results demonstrate that PDGF enhancement of human hematopoietic progenitor cell growth requires mesenchymal cells, and provide an example and mechanism by which growth factors may influence hematopoietic progenitors via cells of the marrow microenvironment.

A quantitative in vitro study of fibroblast and endothelial cell migration in response to serum and wound fluid

Chemoattractant activity for irradiated and nonirradiated rabbit skin fibroblast and bovine aortic arch endothelial cells was assayed in rabbit wound fluid and sera using a modification of the agarose well method originally described for polymorphonuclear leukocytes. Both serum and wound fluid contained chemoattractants for fibroblasts and endothelial cells. Fibroblast migration was decreased by 70 to 80% when the serum or wound fluid was heated to 56 degrees C for 30 min while endothelial cell migration was reduced by 50 to 60%. Platelet-poor plasma-derived serum had no directive effect on the migration of either cell type.

Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro.

To investigate serum requirements for optimal erythropoiesis in vitro, we studied the response of erythroid progenitor cell proliferation in culture to platelet-derived growth factor (PDGF). Human bone marrow cells cultured with platelet-poor plasma-derived serum (PDS) form fewer erythroid colonies than do cells cultured with human whole blood serum or fetal calf serum (P less than 0.05). Treatment of washed platelets with thrombin releases a low molecular weight (less than 100,000) factor that enhances colony growth. This secreted factor appears to be PDGF, based upon the ability of partially purified and electrophoretically pure PDGF to restore colony-forming capacity of PDS-containing cultures to 70-96% of the level found in control cultures with whole blood serum or fetal calf serum. Enhancement of colony growth by PDGF was noted only in marrow cultures supplemented with erythropoietin and PDS. Presence of bioactive erythropoietin in PDGF preparations was excluded by assay in hypertransfused, polycythemic mice, and in fasted rats. Although PDGF stimulates erythroid burst formation in marrow cultures containing optimal concentrations of burst-promoting activity (BPA), it does not influence proliferation of circulating erythroid bursts, regardless of BPA concentration added to culture. We conclude that PDGF is a serum determinant of optimal erythroid progenitor cell proliferation in marrow culture. The activity of PDGF is distinct from that of the apparent erythroid specific growth factors erythropoietin and BPA.

Serum contains a platelet-derived transforming growth factor.

High concentrations of fetal bovine serum induced colony formation in soft agar by anchorage-dependent, nontransformed mouse AKR-2B and rat NRK cells. The colony-stimulating activity in fetal bovine serum was precipitated by 45% saturated ammonium sulfate and migrated in molecular sieve chromatography as a single peak of activity in the 10,000-15,000 molecular weight range. The colony-stimulating activity was heat and acid stable and was destroyed by trypsin and dithiothreitol, indicating the activity is due to a polypeptide that requires disulfide bonds for biological activity. No competition for binding to the epidermal growth factor receptor was associated with the colony-stimulating activity. Isoelectric focusing revealed activity in the pI 4-5 range. The colony-stimulating activity in serum appeared to be of platelet origin because platelet-poor plasma and platelet-poor plasma-derived serum contained little activity, whereas acid/ethanol extracts of bovine and human platelets had potent colony-stimulating activity. Chromatography of platelet extracts on Bio-Gel P-60 revealed peaks of AKR-2B colony-stimulating activity in the 12,000 and 20,000 molecular weight ranges. The other biological and chemical properties of the platelet colony-stimulating activity were the same as those for the serum activity. The data indicate the presence in serum of a platelet-derived growth factor(s) with properties similar to those of the transforming growth factors.