Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

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.     

TGFbeta1 limits the expansion of the osteoprogenitor fraction in cultures of human bone marrow stromal cells

Currently, there is considerable interest in the possibility of using cultured human bone marrow stromal cells (BMSCs) for skeletal tissue engineering. However, the factors that regulate their ex vivo expansion and promote their osteogenic maturation remain poorly defined. Using BMSCs obtained from a large cohort of adult donors, the effects of transforming growth factor (TGF)beta1 on these processes have been determined. BMSCs were found to express TGFbeta receptors (TbetaRs) I, II, III (betaglycan) and CD105/endoglin. The expression of TbetaRs I and II, but not TbetaR III or endoglin, was linked to the cells' state of maturation. Treatment with TGFbeta increased the colony-forming efficiency (CFE) of marrow cell suspensions but reduced the median diameter of the colonies that formed and the number of cells harvested at the end of primary culture. Treatment with TGFbeta also resulted in a significant downregulation in the expression of the developmental markers alkaline phosphatase (AP) and STRO-1. The reduction in AP was due to a decrease in the absolute number of cells expressing this enzyme and in the level (sites/cell) at which it was expressed. Overall, the changes in the expression of STRO-1 and AP are consistent with TGFbeta acting to decrease the size of the osteoprogenitor fraction, and hence the potential clinical utility of the cultured cell population.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

培养骨髓基质细胞的尿嘧啶核苷二磷酸半乳糖-4-表异构酶活性

本实验用液体静置法体外培养小鼠骨髓基质细胞。用细胞化学法观察处于不同分化阶段基质细胞中的尿嘧啶核苷二磷酸半乳糖－－４－表异构酶（Ｕｒｉｄｉｎｅｄｉｐｈｏｓｐｈｏｇａｌａｃｔｏｓｅ－４－ｅｐｉｍｅｒａｓｅ,ＵＤＰＧａｌ,ＥＣ５．１．３．ａ）活性。结果在各类骨髓基质细胞中均可见到很强的酶反应。在由原始网状细胞向成熟星状细胞分化过程中,酶活性逐渐增强。随着培养时间和延长（培养５、７、１０天）,星状细胞和成纤维细胞中酶反应的阳性率和阳性度逐渐增高。这证明在体外培养骨髓基质细胞中由ＵＤＰＧａｌ催化的代谢非常活跃     

Effect of platelet-derived growth factor on DNA synthesis and gene expression in bone marrow stromal cells derived from adult and old rats

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast-like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum-starved stromal cells, PDGF stimulated [³H]-thymidine incorporation into DNA in a dose-dependent manner with a maximum stimulation of 15-fold at 500 ng/ml. By comparison, insulin-like growth factor (IGF-I) has a small effect on [³H] -thymidine incorporation. The effect of PDGF and IGF-I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1-, 2.3-, and 14-fold, respectively. Addition of PDGF to dexamethasone-treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)₂D₃. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF-I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF-I. Furthermore, PDGF attenuated the stimulatory effect of IGF-I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF-I increased [³H]-thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent. © 1995 Wiley-Liss, Inc.     

淫羊藿苷促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素

比较研究蛇床子素与淫羊藿苷处理对体外培养的大鼠骨髓间充质细胞(rat bone marrow stromal cell, rBMSC)成骨性分化的影响.从体外分离培养的大鼠骨髓间充质细胞,筛选出最佳的蛇床子素和淫羊藿苷处理的浓度为1×10-5 mol/L, 然后用最佳的浓度对体外培养的大鼠骨髓间充质细胞进行药物干预;在药物干预后的第3、6、9、12和15 d后测定碱性磷酸酶活性（alkaline phosphatase,ALP）和钙含量;第12 d 进行钙化结节茜素红染色;第12 h、24 h、48 h、72 h和96 h 对OXS、Runx-2、骨形态发生蛋白(bone morphogenetic protein,BMP-2)和collagen-I mRNA 表达水平进行real-time RT-PCR检测.结果显示,浓度为1×10-5 mol/L蛇床子素和淫羊藿苷干预均可提高体外培养的骨髓间充质细胞ALP活性,增加Ca含量,提高Runx、OXS、BMP-2和collagen-1 mRNA的表达水平.同时,淫羊藿苷在促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素.     

Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.     

Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity

By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes.     

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10⁻¹⁰ M with maximum values achieved at 10⁻⁸ M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)₂D₃ during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. © 1996 Wiley-Liss, Inc.     

The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.     

Utility of human placental alkaline phosphatase as a genetic marker for cell tracking in bone and cartilage

It was the aim of the current study to evaluate the utility of human placental alkaline phosphatase (hPLAP) as a genetic marker for cell tracking in bone and cartilage, using transgenic Fischer 344 rats expressing hPLAP under the control of the ubiquitous R26 promoter [F344-Tg(R26-hPLAP)]. hPLAP enzyme activity was retained during paraffin and methylmethacrylate (MMA) embedding, and was best preserved using 40% ethanol as fixative. Endogenous alkaline phosphatase activity could be completely blocked by heat inactivation in paraffin and MMA sections, allowing histochemical detection of hPLAP in the complete absence of background staining. In addition, sensitive detection of hPLAP was also possible using immunohistochemistry. F344-Tg(R26-hPLAP) rats demonstrated ubiquitous expression of hPLAP in hematopoietic bone marrow cells and stromal cells such as osteoblasts, osteocytes, and chondrocytes. Osteoclasts only weakly expressed hPLAP. In conclusion, hPLAP provides superb detection quality in paraffin and plastic sections, and constitutes an excellent genetic marker for cell tracking in hard and soft tissues.     

Salicylideneamino-2-thiophenol enhances osteogenic differentiation through the activation of MAPK pathways in multipotent bone marrow stem cell

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.     

Osteogenic imprinting upstream of marrow stromal cell differentiation

Five spontaneously transformed cell lines were established from a population of murine bone marrow stromal cells (BMSCs) and the expression profiles of phenotype-characteristic genes, patterns of in vitro differentiation, and osteogenic capacity after in vivo transplantation were determined for each. All the clones expressed stable levels of cbfa1, the osteogenic "master" gene, whereas the levels of individual phenotypic mRNAs were variable within each, suggestive of both maturational and phenotypic plasticity in vitro. Varying levels of collagen type I and alkaline phosphatase (AP) were expressed in all the clonal lines. The clonal lines with proven in vivo osteogenic potential (3 out of 5) had a high proliferation rate and expressed bone sialoprotein (BSP), whereas the two nonosteogenic clones proliferated more slowly and never expressed BSP. Bone nodules were only observed in 2 out of 3 of the osteogenic lines, and only 1 out of three formed cartilage-like matrix in vitro. There was no evidence of chondrogenesis in the nonosteogenic lines. By contrast, LPL was expressed in two osteogenic and in two nonosteogenic lines. These results demonstrate the presence of multipotential and restricted progenitors in the murine stromal system. cbfa1, collagen type I, and AP expression were common to all, and therefore presumably early, basic traits of stromal cell lines that otherwise significantly differ with respect to growth and differentiation potential. This finding suggests that an osteogenic imprinting lies upstream of diversification, modulation, and restriction of stromal cell differentiation potential.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.     

Simultaneous autoradiographic and histochemical analysis of bone formed in vitro

The simultaneous histochemical demonstration of alkaline phosphatase activity and autoradiographic demonstration of [3H]-thymidine uptake is valuable for study of bone cell kinetics in vivo or in vitro. By use of this technique, it has been possible to detect changes induced by a single dose of dexamethasone (10(-7) M) in the time course of alkaline phosphatase activity, the number of alkaline phosphatase-positive cells, and [3H]-thymidine labeling in bone formed in vitro.