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)

The success of ex vivo viral gene therapy systems for promoting bone formation could be improved through the development of systems to spatially localize gene expression. Towards this goal, we have encapsulated adenovirus-transduced human diploid fetal lung fibroblasts (MRC-5) expressing bone morphogenetic protein-type 2 (BMP-2) within non-degradable poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels and implanted these intramuscularly to promote endochondral bone formation. To optimize BMP-2 secretion, the molecular weight of the polymers and cell densities were varied. Polymers with molecular weights of 6, 10, and 20 kDa were used to prepare hydrogels containing 1, 5, or 10 million transduced cells. The results showed that 10 million transduced fibroblasts that was the maximum number of cells feasible for encapsulation within PEG-DA 10 and 20 kDa hydrogels produced the highest amount of secreted BMP-2 protein. Encapsulation of MRC-5 and transduced fibroblasts resulted in 71 and 58% cell viability, respectively. The bioactivity of secreted BMP-2 protein from the hydrogels was confirmed with an alkaline phosphatase assay. Micro-CT of the lower limb muscles of NOD/SCID mice following implantation with hydrogels showed 39.5 +/- 25.0 mm3 mineralized tissue and 31.8 +/- 7.8 mm3 for the cell-injected mice, and the bone was localized to the hydrogel surfaces. Histology revealed bone as well as cartilage for both hydrogel implanted and cell-injected animals.
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PMID:Endochondral bone formation from hydrogel carriers loaded with BMP2-transduced cells. 1734 Jan 96

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

Strategies to expand human bone marrow stromal cells (HBMSC) for bone tissue engineering are a key to revolutionising the processes involved in three-dimensional skeletal tissue reconstruction. To facilitate this process we believe the use of biodegradable porous poly(DL-lactide-co-glycolide) (PDL LGA) hollow fibres as a scaffold used in combination with HBMSC to initiate natural bone repair and regeneration offers a potential solution. In this study, the biocompatibility of 75:25 PDL LGA fibres with HBMSC and the capacity of a PDL LGA fibre-associated HBMSC-monolayer to establish an osteogenic phenotype in vivo was examined. A high proportion of HBMSC survived when expanded on PDL LGA fibres for 6 days, with only 10% of the propidium iodide (pI)-labelled population represented in the sub-G1 DNA peak on analysis by flow cytometry. Tracking carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labelled HBMSC by flow cytometry indicated that HBMSC attachment to the P(DL)LGA fibres does not interfere with their rate of proliferation. Furthermore, in response to osteogenic stimuli, HBMSC expanded on PDL LGA fibres can differentiate, as expected, along the osteogenic lineage with associated alkaline phosphatase activity. Following implantation into SCID mice, osteogenic-conditioned PDL LGA fibre-HBMSC graft resulted in type I collagen deposition and associated bone mineralisation and osteoid formation, as evidenced by immunohistochemistry and histology. These studies provide evidence that porous PDL LGA hollow fibre-HBMSC graft is an innovative biomaterial that offers new approaches to mesenchymal cell expansion, which could be utilised as a scaffold for skeletal tissue generation.
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PMID:Expansion of human bone marrow stromal cells on poly-(DL-lactide-co-glycolide) (PDL LGA) hollow fibres designed for use in skeletal tissue engineering. 1782 56

Human embryonic stem cells (hESC) face ethical sensitivities and the problem of teratoma formation. Although Wharton's jelly stem cells (WJSC), also of embryonic origin, may not face such ethical concerns, it is not definitely known whether under hESC culture conditions they would be as pluripotent as hESC. WJSC grown on plastic showed two types of morphology (epithelioid and short fibroblastic) in primary culture depending on the culture medium used, and only fibroblastic morphology when passaged. When grown in the presence of hESC medium on mouse feeder cells, they produced atypical colonies containing hESC-like cells with high-nuclear cytoplasmic ratios and prominent nucleoli. They were positive for the hESC markers Tra-1-60, Tra-1-81, SSEA-1, SSEA-4, Oct-4 and alkaline phosphatase, negative for SSEA-3, showed normal karyotypes, developed embryoid body (EB)-like structures, did not produce teratomas in SCID mice and differentiated into neuronal derivatives. They were also positive for the mesenchymal CD markers (CD105, CD90, CD44), negative for CD34 and HLA, and although nine out of 10 embryonic stem cell genomic markers were detectable, these were expressed at low levels. WJSC are thus not as pluripotent as hESC but widely multipotent, and have the advantages of being able to be scaled up easily and not inducing teratomas.
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PMID:Comparative growth behaviour and characterization of stem cells from human Wharton's jelly. 1806 71

This study was aimed to evaluate the role of commensal Gram-negative bacterium Bacteroides ovatus in murine model of chronic intestinal inflammation. The attempt to induce chronic colitis was done in Bacteroides ovatus-monoassociated, germ-free and conventional mice either in immunocompetent (BALB/c) mice or in mice with severe combined immunodeficiency (SCID), using 2.5 % dextran-sodium sulfate (DSS) in drinking water (7 days DSS, 7 days water, 7 days DSS). Conventional mice developed chronic colitis. Some of germ-free BALB/c and the majority of germ-free SCID mice did not survive the long-term treatment with DSS due to massive bleeding into the intestinal lumen. However, monocolonization of germ-free mice of both strains with Bacteroides ovatus prior to long-term treatment with DSS protected mice from bleeding, development of intestinal inflammation and precocious death. We observed that though DSS-treated Bacteroides ovatus-colonized SCID mice showed minor morphological changes in colon tissue, jejunal brush-border enzyme activities such as gamma-glutamyltranspeptidase, lactase and alkaline phosphatase were significantly reduced in comparison with DSS-untreated Bacteroides ovatus-colonized mice. This modulation of the enterocyte gamma-glutamyltranspeptidase localized to the brush border membrane has been described for the first time. This enzyme is known to reflect an imbalance between pro-oxidant and anti-oxidant mechanisms, which could be involved in protective effects of colonization of germ-free mice with Bacteroides ovatus against DSS injury.
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PMID:Monocolonization with Bacteroides ovatus protects immunodeficient SCID mice from mortality in chronic intestinal inflammation caused by long-lasting dextran sodium sulfate treatment. 1819 84

In order to ensure that MSCs designed for in vivo cartilage repair do not untowardly differentiate into osteoblasts and mineralize in situ, we tested whether siRNA-induced suppression of cbfa1/Runx2 affected the osteogenic and chondrogenic differentiation potential of the murine cell line C3H10T1/2. Anti-cbfa1/Runx2 siRNA decreased the levels of cbfa1/Runx2 mRNA and protein by 65-80%, and also markedly reduced the expression of osteoblast-related genes such as Dlx5, osterix, collagen type I, alkaline phosphatase (AP), osteocalcin, SPARC/osteonectin and osteopontin, leading to a temporal expression of AP enzyme activity and mineralization potential delayed by at least some 7-9 days. Furthermore, siRNA-transfected cells, grown under chondrogenic conditions did not display biologically significant changes in the expression of aggrecan, collagen type II or type X, or histology when grown in micropellets or monolayer cultures. Finally, when cells were propagated in osteogenic medium and injected into the tibial muscles of SCID mice, no overtly mineralized bone tissue emerged. These experiments indicate that a major transient reduction of cbfa1/Runx2 expression in MSCs is sufficient to delay osteoblastic differentiation, both in vitro and in vivo, while chondrogenesis seemed to be sustained.
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PMID:Transient down-regulation of cbfa1/Runx2 by RNA interference in murine C3H10T1/2 mesenchymal stromal cells delays in vitro and in vivo osteogenesis, but does not overtly affect chondrogenesis. 1831 48

Human mesenchymal stem cells (hMSCs) from bone marrow were genetically marked by using a murine leukaemia virus construct encoding enhanced green fluorescent protein (eGFP). The marked cells were either directly implanted into the tibialis anterior muscle or introduced into a variety of other tissue sites in immunocompromised mice (NOD/SCID and C.B-17 SCID/beige) to investigate their fates and differentiation potentials. It was observed that the hMSCs survived for up to 12 weeks and showed site-specific morphological phenotypes. hMSCs delivered by intravenous injection were found mainly in the lungs and were detected rarely in other organs. Histomorphometry showed that, after implantation of hMSCs into the tibialis anterior muscle juxtaskeletally, the areas of reactive host callus formation at 1 and 2 weeks and of ectopic human bone formation at 1 week were significantly increased compared with the control group. Expression of eGFP and human RUNX2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type I mRNAs were detected in mice implanted with the labelled hMSCs but not in sham-treated samples. Active clearance of the reactive callus and ectopic calcified tissue by osteoclast-like tartrate-resistant acid phosphatase-positive cells was observed. We conclude that the eGFP-labelled hMSCs can survive and retain the potential to differentiate morphologically into a variety of apparent mesenchymal phenotypes in vivo. Absolute confirmation of differentiation capacity requires further study and is complicated by known possibilities of fusion of donor and host cells or limited transfer of genetic material. Nevertheless, the genetically marked hMSCs are shown to participate extensively in bone formation and turnover. Control of the host osteoclast/macrophage responses resulting in clearance of formed osteogenic tissue warrants further investigation to promote prolonged human osteogenesis in immunocompromised mice. Furthermore, any proposed general cytotherapeutic strategy for enhanced osteogenesis is likely to require supplementation of local bone-forming biological signals.
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PMID:Fates and osteogenic differentiation potential of human mesenchymal stem cells in immunocompromised mice. 1841 47

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.
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PMID:Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells. 1845 90

The interaction of stem cells and ceramics in bone regeneration is still poorly understood. The aim of this study was to examine the influence of the porosity (25%, 65% and 75%) of beta-tricalcium phosphate (TCP) ceramics on osteogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. For the in vitro portion of the study, TCP scaffolds loaded with MSC were kept in osteogenic induction medium for 21 days. For the in vivo portion of the study, scaffolds loaded with undifferentiated MSC were implanted subcutaneously into SCID mice for 8 weeks and compared with similarly implanted controls that were not loaded with MSC. Measurements of total protein as well as specific alkaline phosphatase (ALP) activity were taken as indicators of growth/matrix production and osteogenic differentiation. An increase in the total protein concentration was noted from day 1 to day 21 on the in vitro TCP 65% and TCP 75% scaffolds (p<0.05) with no such increase noted in the TCP 25% specimens. However, the specific alkaline phosphatase activity increased from day 1 to day 21 in all three in vitro specimens (p<0.02) and reached similar levels in each specimen by day 21. In vivo, ALP activity of cell-loaded TCP 65% ceramics was higher when compared with both the TCP 25% and TCP 75% specimens (p<0.046), and higher in the TCP 75% than TCP 25% specimens (p=0.008). Histology revealed mineralization by human cells in the pores of the TCP ceramic scaffolds with a trend toward greater calcification in TCP 65% and 75%. In summary, a higher porosity of TCP scaffolds does not necessarily mean a higher ALP activity in vivo. The distribution and size of the pores, as well as the surface structure, might play an important role for osteogenic differentiation in vivo.
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PMID:Porosity and pore size of beta-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: an in vitro and in vivo study. 1857 99

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.
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PMID:Isolation and culture of rabbit primordial germ cells. 1862 91


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