Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We established a human embryonic stem cell line derived from frozen human embryos of Chinese origin. The cell line expressed the pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and alkaline phosphatase. The pluripotency of the cell line was also demonstrated in vivo by teratoma formation in severe combined immunodeficiency mice. The embryonic stem cells formed embryoid bodies after culturing in suspension for 7 days. The embryoid bodies were transferred to an adherent culture system in serum-free medium. The differentiating cells derived from the embryoid bodies expressed Nestin and Sox2, markers of neural progenitor cells. After the induction of cyclic AMP for 7 days, the neural progenitor cells had differentiated into neurons and glial cells.
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PMID:Derivation of embryonic stem cell line from frozen human embryos and neural differentiation. 1879 96

A current challenge in mesenchymal stem cell (MSC)-based cartilage repair is to solve donor and tissue-dependent variability of MSC cultures and to prevent chondrogenic cells from terminal differentiation like in the growth plate. The aim of this study was to select the best source for MSC which could promise stable cartilage formation in the absence of hypertrophy and ectopic in vivo mineralization. We hypothesized that MSC from synovium are superior to bone marrow- and adipose tissue-derived MSC since they are derived from a joint tissue. MSC were characterized by flow cytometry. MSC pellets were cultured under chondrogenic conditions and differentiation was evaluated by histology, gene expression analysis, and determination of alkaline phosphatase activity (ALP). After chondrogenic induction, pellets were transplanted subcutaneously into SCID mice. MSC from bone marrow, adipose tissue, and synovium revealed similar COL2A1/COL10A1 mRNA levels after chondrogenic induction and were positive for collagen-type-X. Bone marrow-derived and adipose tissue-derived MSC showed significantly higher ALP activity than MSC from synovium. Low ALP-activity before transplantation of pellets correlated with marginal calcification of explants. Surprisingly, non-mineralizing transplants specifically lost their collagen-type II, but not collagen-type I deposition in vivo, or were fully degraded. In conclusion, the lower donor-dependent ALP activation and reduced mineralization of synovium-derived heterotopic transplants did not lead to stable ectopic cartilage as known from articular chondrocytes, but correlated with fibrous dedifferentiation or complete degeneration of MSC pellets. This emphasizes that beside appropriate induction of differentiation, locking of MSC in the desired differentiation state is a major challenge for MSC-based repair strategies.
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PMID:Calcification or dedifferentiation: requirement to lock mesenchymal stem cells in a desired differentiation stage. 1910 42

Bone grafting is the current standard of care for treatment of fracture nonunions, while alternative strategies such as bone marrow-derived mesenchymal stem cells are also used. MSCs can be induced towards the osteogenic lineage by in vitro treatment with dexamethasone (dex). This study aimed to determine the optimal duration of dex treatment for osteogenic differentiation of MSCs in vitro and evaluate the effect of this dex pretreatment on bone formation in vivo. To determine the optimal dex treatment, MSCs were cultivated in osteogenic medium for 5 weeks with a varying dex withdrawal schedule, such that MSCs were exposed to dex for either 0, 1, 2, 3, 4, or 5 weeks. During this period, alkaline phosphatase, calcium, and DNA assays, as well as von Kossa staining and morphological observations were performed. One and 2 week dex-treated groups returned to control levels rapidly, whereas 3 and 4 week groups retained higher levels of differentiation markers, with the 4 week group being the highest. Based on these in vitro results, MSCs (with and without dex) and control fibroblasts were seeded into ceramic cubes, cultured for 4 weeks, and implanted into SCID mice, and harvested 6 weeks postimplantation for histologic evaluation. There was no bone formation in fibroblast-seeded controls, little bone formation in control (CS 1), and extensive bone formation (CS 3-4) in dex-treated MSCs. These results indicate that pretreatment of MSCs with dex results in greater bone formation than in untreated controls.
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PMID:In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo. 1913 80

There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.
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PMID:Osteoblastic and osteolytic human osteosarcomas can be studied with a new xenograft mouse model producing spontaneous metastases. 1921 26

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian-blue staining, qRT-PCR, and quantification of alkaline phosphatase (ALP) activity. Pre-differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)-beta was always required for chondrogenic differentiation and deposition of a collagen-type-II-positive extracellular matrix, while bone morphogenetic protein (BMP)-2, -4, -6, -7, aFGF, and IGF-I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF-beta, however, without preventing collagen type X expression. bFGF or parathyroid hormone-like peptide (PTHrP) inhibited the TGF-beta-responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up-regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor-independent pathway. While TGF-beta was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II(+)/collagen type X(-) cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes.
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PMID:Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells. 2004 52

This study investigated the hypothesis that cell sheets composed of multilayered rabbit bone marrow derived mesenchymal stem cells (MSC) could be assembled with two kinds of implants (surface-modified titanium and zirconia) for the construction of a MSC-implant. The MSC sheets were harvested from culture flasks, wrapped around implants to construct the complexes, and then cultured in osteogenic medium. The layered cell sheets integrated well with implants and remained viable, with small mineralized nodules visible on the implant surfaces for up to four weeks after culture. Cells on the implants underwent classical in vitro osteogenic differentiation with an associated elevation of alkaline phosphatase activity and bone- and vascular-related protein expression. In vivo, two kinds of cell sheet-implant complexes were transplanted under the skin of SCID mice and cultured for eight weeks. For the MSC sheet titanium implant complex, histological examination revealed that new bone tissue that formed around implants followed a predominantly endochondral pathway, exhibiting histological markers of native bone; for the MSC sheet zirconia implant complex, however, intramembranous ossification appeared to occur on the surface of the zirconia implant, as observed with typical osteocytes embedded in dense matrix and accompanied by both microvessels and marrow cavities. These findings demonstrate that MSC-implants possessing osteogenic and vascularization abilities can be produced using cell sheet engineering techniques in conjunction with routine implant materials, which provide a novel technology to modify the implant surface.
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PMID:The performance of bone marrow mesenchymal stem cell--implant complexes prepared by cell sheet engineering techniques. 2013 81

The pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for seeded cells to organize into a functioning tissue. In this report we have investigated the effects of different concentrations of silk fibroin protein on three-dimensional (3D) scaffold pore microstructure. Four pore size ranges of silk fibroin scaffolds were made by the freeze drying technique, with the pore sizes ranging from 50 to 300 microm. The pore sizes of the scaffolds decreased as the concentration of fibroin protein increased. Human bone marrow mesenchymal stromal cells (BMSC) transfected with the BMP7 gene were cultured in these scaffolds. A cell viability colorimetric assay, alkaline phosphatase assay and reverse transcription-polymerase chain reaction were performed to analyze the effect of pore size on cell growth, the secretion of extracellular matrix (ECM) and osteogenic differentiation. Cell migration in 3D scaffolds was confirmed by confocal microscopy. Calvarial defects in SCID mice were used to determine the bone forming ability of the silk fibroin scaffolds incorporating BMSC expressing BMP7. The results showed that BMSC expressing BMP7 preferred a pore size between 100 and 300 microm in silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production. Furthermore, in vivo transplantation of the silk fibroin scaffolds combined with BMSC expressing BMP7 induced new bone formation. This study has shown that an optimized pore architecture of silk fibroin scaffolds can modulate the bioactivity of BMP7-transfected BMSC in bone formation.
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PMID:The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7. 2018 72

Human embryonic stem cells (HESCs) are defined as self-renewing cells that retain their ability to differentiate into all cell types of the body. They have enormous potential in medical applications and as a model for early human development. There is a need for derivation of new HESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. Here, we describe a modified culture medium containing human recombinant leukemia inhibitory factor and human basic fibroblast growth factor that significantly increases the number of human blastocysts formed and their quality, as well as the efficiency of HESC derivation from poor-quality embryos. Culturing poor-quality embryos in modified medium resulted in a two-fold increase in the blastocyst formation rate and a seven-fold increase over the derivation efficiency in conventional medium. We derived 15 HESC lines from poor-quality embryos cultured in modified culture medium and two HESC lines from quality embryos cultured in conventional culture medium. All cell lines shared typical human pluripotent stem cell features including similar morphology, normal karyotypes, expression of alkaline phosphatase, pluripotency genes, such as Oct4, and cell surface markers (SSEA-4, TRA-1-60, TRA-1-81), the ability to form teratomas in SCID mice, and the ability to differentiate into cells of three embryonic germ layers in vitro. Our data suggest that poor-quality embryos that have reached the blastocyst stage in our modified culture medium are a robust source for normal HESC line derivation.
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PMID:A modified culture medium increases blastocyst formation and the efficiency of human embryonic stem cell derivation from poor-quality embryos. 2065 58

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.
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PMID:Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos. 2138 53

Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared not to induce ectopic bone formation, longer-term studies are required to determine whether it promotes the final stages of osteoblast formation and mineralization at gene and protein levels. While used in clinical applications, whether Emdogain and other commercial preparations of EMPs truly possess the capacity to induce the regeneration of bone or other components of the periodontium remains to be established.
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PMID:Effect of coating Straumann Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation. 2158 94


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