In Vitro Phenotypic Alteration of Human Melanoma Cells Induced by Differentiating Agents: Heterogeneous Effects on Cellular Growth and Morphology,Enzymatic Activity,and Antigenic Expression

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G₁ phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and -γ-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B863 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of -γ-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.     

In vitro phenotypic alteration of human melanoma cells induced by differentiating agents: heterogeneous effects on cellular growth and morphology, enzymatic activity, and antigenic expression.

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G1 phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and gamma-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B8G3 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of gamma-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.     

Acid phosphatase and phosphoamino acid phosphatases in murine erythroleukaemic cells

The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM).     

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0.5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.     

1,25-Dihydroxyvitamin D3 increases bone alkaline phosphatase isoenzyme levels in human osteogenic sarcoma cells

The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines, SAOS and TE85, after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3). Enzyme activity increased when the cells were incubated with concentrations of 1,25(OH)2D3 between 10(-9) and 10(-7) M and cell growth was not inhibited at these concentrations. The specific activity of alkaline phosphatase was 4- to 7-fold higher than that in the control cells after 5 to 7 days of continuous exposure to 1,25(OH)2D3. Immunochemical studies demonstrated that the enzyme from both control and 1,25(OH)2D3-treated cultures cross-reacted with antisera specific for the phosphatase isoenzyme produced by normal human bone, and did not cross-react with antisera specific for the placental alkaline phosphatase isoenzyme. The increased enzyme activity in cultures induced with 1,25(OH)2D3 correlated with an absolute increase in the number of bone-specific phosphatase molecules, as determined by radioimmunoassay. No effect on alkaline phosphatase activity was observed when the cells were treated with other vitamin D metabolites or with 5-bromo-2'-deoxyuridine. Comparative studies demonstrated that hydrocortisone, another steroid hormone, increased the phosphatase activity with a different time course than did 1,25(OH)2D3. High affinity cytoplasmic receptors for 1,25(OH)2D3 and hydrocortisone were found in the SAOS and TE85 cells.     

Divergent phenotypic patterns and commitment to apoptosis of Caco-2 cells during spontaneous and butyrate-induced differentiation

Caco-2 cells differentiate spontaneously when cultured in confluence and on exposure to the physiologically relevant short-chain fatty acid, butyrate. This study aimed to compare the phenotype induced by these pathways and their relations to cell turnover. Caco-2 cells were treated with butyrate at a nontoxic concentration of 2 mM for 3 days, or allowed to spontaneously differentiate for 0-21 days. Brush border hydrolase activities and carcinoembryonic antigen (CEA) expression, transepithelial resistance and dome formation, expression of components of the urokinase system, and cell turnover by flow cytometry, and the degree of DNA fragmentation were quantified. Butyrate induced increases in alkaline phosphatase activity and CEA expression but not the activities of other hydrolases, while culture alone induced progressive increases in the activities/expression of all markers. Butyrate induced a significantly greater increase in transepithelial resistance (TER) than occurred during culture alone but the densities of domes were similar. Butyrate induced a ninefold increase in urokinase receptor expression and twofold increase in urokinase activity, while culture alone induced a significantly smaller increase in receptor expression, an increase in plasminogen activator inhibitor-1 but no change in activity. While both stimuli induced cell cycle arrest, only butyrate increased the proportion of cells undergoing apoptosis. In conclusion, differentiation of Caco-2 cells can proceed along multiple pathways but does not necessarily lead to apoptosis. The phenotypic changes during spontaneous differentiation mimic those that occur in normal colonic epithelial cells in vivo during their migration from the crypt base to neck, while butyrate-induced effects more closely follow those occurring when normal colonic epithelial cells migrate from crypt neck to the surface compartment.     

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

野黄芩苷对内毒素抑制人牙周膜细胞碱性磷酸酶活性的影响

目的观察野黄芩苷对内毒素(LPS)抑制人牙周膜细胞的碱性磷酸酶活性的影响。方法原代培养人牙周膜细胞,采用酶动力学方法观察野黄芩苷对LPS抑制人牙周-膜细胞碱性磷酸酶活性的影响。结果100μg/mL LPS可显著抑制体外培养的人牙周膜细胞碱性磷酸酶活性。加入0.001-10μg/ml野黄芩苷干预后,对LPS抑制碱性磷酸酶活性有一定的拮抗作用,在1μg/ml时达到高峰。结果 野黄芩苷可能通过拮抗LPS抑制牙周膜细胞碱性磷酸酶的活性,促使牙周膜细胞向成骨细胞分化而利于牙周组织再生修复。     

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.     

Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.     

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K_m, pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.     

Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM β-glycerophosphate (βGP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM βGP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number or APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes. J. Cell. Biochem. 64:295–312. © 1997 Wiley-Liss, Inc.