Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There is widespread interest in the use of bone marrow stromal cells (BMSC) for tissue reconstruction and repair and for gene therapy. BMSC represent the differentiated progeny of CFU-F, which however comprise a developmentally heterogeneous population as is reflected in the cellular heterogeneity of the cell populations to which they give rise. We have compared the efficacy of monoclonal antibodies recognising a series of stromal antigens, viz. STRO-1, HOP-26, CD49a and SB-10/CD166, as tools for the enrichment of CFU-F prior to culture and as developmental markers for culture-expanded BMSC. In freshly isolated bone marrow mononuclear cells (BMMNC), the proportion of antigen-positive cells was 27%, 46%, 5% and 19% for STRO-1, HOP-26, CD49a and CD166, respectively. All CD49a(+) cells co-expressed STRO-1. The degree of CFU-F enrichment obtained with anti-CD49a (approximately 18-fold) by a one-pass immunoselection strategy was significantly greater than that of all other antibodies tested. BMSC expressed higher levels of all antigens investigated (except for HOP-26) compared with BMMNC. Expression of STRO-1 and CD49a remained restricted to a subset of BMSC, whereas all BMSC were SB-10/CD166 positive. Treatment with dexamethasone (10 nM), which promotes the differentiation and further maturation of cells of the osteogenic lineage in this cell culture system, increased the expression of CD49a and HOP-26. The CD49a(+) and HOP-26(+) fractions of BMSC were further subdivided by dual-labelling with anti-STRO-1 and B4-78 (an antibody recognising the B/L/K isoform of the enzyme alkaline phosphatase), respectively. By using a variety of criteria, the HOP-26 antigen was identified as CD63, a member of the tetraspanin family of proteins thought to modulate integrin compartmentalisation and signalling.
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PMID:STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro. 1288 98

Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D(3) in bone formation and mineralization has been established in several osteoblast cell culture studies. The aim of the present study was to evaluate the role of this hormonal combination alone and in the presence of bone morphogenetic protein-4 (BMP-4) or-6 (BMP-6) in inducing osteogenic differentiation of human MSC. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D(3) in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D(3) induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D(3). Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D(3). The present results revealed a novel potent effect of PTH and vitamin D(3) plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development.
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PMID:The role of BMP-6, IL-6, and BMP-4 in mesenchymal stem cell-dependent bone development: effects on osteoblastic differentiation induced by parathyroid hormone and vitamin D(3). 1518 23

One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.
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PMID:Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. 1621 Apr 10

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.
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PMID:Osteogenic differentiation of human dental papilla mesenchymal cells. 1651 58

The study was aimed to establish a protocol of isolating and culturing adult mesenchymal stem cells (MSC) from human bone marrow aspirate and identify them by surface antigen analysis and committed differentiation in order to provide an experimental foundation for achieving a therapeutic benefit in applying MSC in hematopoietic stem cell transplantation. MSCs were obtained from fresh human bone marrow aspirate by gradient centrifugation with Percoll (1.073 g/ml) and anchoring culture in L-DMEM with 10% fetal bovine serum by a full medium exchange every 3 days. The MSC surface antigens, including CD34, CD45, CD73, CD105, CD166, were analyzed on FACScan flow cytometer. Under culture in conditioned medium for osteogenesis (the hormone cocktail containing 0.1 micromol/L dexamethasone, 10 mmol/L glycerol-2-phosphate and 50 micromol/L ascorbic acid) and adipogenesis (the cocktail containing 1 micromol/L dexamethasone, 5 mg/L insulin, 0.5 mmol/L 1-methyl-3-isobutylxanthine and 60 micromol/L indomethacin), MSCs committedly differentiated into osteoblasts and adipocytes. The differentiated mesenchymal stem cells were identified by morphological observation and immunohistochemical staining. The results showed that by gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate. These cells were uniformly negative for CD34, CD45 and positive for CD73, CD105 and CD166. The osteogenic differentiated cells were positive for alkaline phosphatase (ALP) and the adipogenic differentiated cells displayed accumulation of lipid vacuoles, as detected by oil red O. It is concluded that MSC can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSC primary identification can be accomplished by flow cytometry and induced differentiation. The set of methods in current experiment shows somewhat practical value for basic research and clinical application.
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PMID:[A modified method to isolate and identify the adult mesenchymal stem cells from human bone marrow]. 1680 Sep 42

Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue engineering purposes, however, it would be advantageous to use the whole SVF, which can be transplanted without further in vitro selection or expansion steps. Because little is known about the freshly isolated ASCs in the SVF, we phenotypically characterized human freshly isolated ASCs, using flow cytometry. In addition, we investigated whether freshly isolated ASCs have functional properties comparable to cultured ASCs. For this, the differentiation potential of both freshly isolated ASCs and cultured ASCs into the osteogenic pathway was analyzed. Freshly isolated ASCs slightly differed in immunophenotype from cultured ASCs. Contrary to cultured ASCs, freshly isolated ASCs were shown to be highly positive for CD34, and positive for CD117 and HLA-DR. On the other hand, expression of CD105 and especially CD166 on the freshly isolated ASCs was relatively low. After osteogenic stimulation of freshly isolated ASCs, both Runx-2 and CollaI gene expression were significantly increased (p < 0.05). However, there was a difference in the kinetics of gene expression between freshly isolated and cultured ASCs and also between the different SVF isolates tested. There was no difference in alkaline phosphatase activity between freshly isolated ASCs and cultured ASCs. In addition, freshly isolated ASCs stained positive for osteonectin and showed matrix mineralization. We conclude that although there are minor differences in phenotype and kinetics of differentiation between freshly isolated ASCs and cultured ASCs, the use of freshly isolated ASCs for tissue engineering purposes involving bone repair is potentially applicable.
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PMID:Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. 1734 7

During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm2 had a significantly higher growth rate than at 5000 cell/cm2, which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm2 in an alpha-minimal essential medium-based medium containing dexamethasone.
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PMID:A rapid and efficient method for expansion of human mesenchymal stem cells. 1751 76

Stem cells or osteogenic precursor cells isolated from bone marrow, trabecular tissues in bone, cartilage, muscle, and fat are the most suitable source for bone tissue engineering. In this study, we investigated the osteogenic phenotypes and mineralization of cultured human periosteal-derived cells obtained from mandibular periosteums. These periosteal-derived cells were positive for CD44, CD90, and CD166 antigens. They are successfully differentiated into osteoblasts in the medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. We observed that alkaline phosphatase (ALP) was largely expressed in the earlier stage of osteoblastic differentiation according to histochemical staining and RT-PCR analysis, whereas osteocalcin was dominantly expressed and secreted into the medium at the later stage. In addition, mineralized nodule formation has been observed by von Kossa staining in a time-dependent manner. These results suggest that periosteal-derived cell has the potential osteogenic activity and could be a good candidate for tissue engineering to restore the bony defects of the maxillofacial region.
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PMID:Osteogenic phenotypes and mineralization of cultured human periosteal-derived cells. 1754 71

Mesenchymal stem-like cells identified in different tissues reside in a perivascular niche. In the present study, we investigated the putative niche of adipose-derived stromal/stem cells (ASCs) using markers, associated with mesenchymal and perivascular cells, including STRO-1, CD146, and 3G5. Immunofluorescence staining of human adipose tissue sections, revealed that STRO-1 and 3G5 co-localized with CD146 to the perivascular regions of blood vessels. FACS was used to determine the capacity of the CD146, 3G5, and STRO-1 specific monoclonal antibodies to isolate clonogenic ASCs from disassociated human adipose tissue. Clonogenic fibroblastic colonies (CFU-F) were found to be enriched in those cell fractions selected with either STRO-1, CD146, or 3G5. Flow cytometric analysis revealed that cultured ASCs exhibited similar phenotypic profiles in relation to their expression of cell surface markers associated with stromal cells (CD44, CD90, CD105, CD106, CD146, CD166, STRO-1, alkaline phosphatase), endothelial cells (CD31, CD105, CD106, CD146, CD166), haematopoietic cells (CD14, CD31, CD45), and perivascular cells (3G5, STRO-1, CD146). The immunoselected ASCs populations maintained their characteristic multipotential properties as shown by their capacity to form Alizarin Red positive mineralized deposits, Oil Red O positive lipid droplets, and Alcian Blue positive proteoglycan-rich matrix in vitro. Furthermore, ASCs cultures established from either STRO-1, 3G5, or CD146 selected cell populations, were all capable of forming ectopic bone when transplanted subcutaneously into NOD/SCID mice. The findings presented here, describe a multipotential stem cell population within adult human adipose tissue, which appear to be intimately associated with perivascular cells surrounding the blood vessels.
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PMID:Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. 1765 79

Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.
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PMID:Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells. 1860 Mar 96


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