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)

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.
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PMID:Phenotypic correction of murine hemophilia A using an iPS cell-based therapy. 1913 14

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.
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PMID:Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. 1922 44

Authentic bovine embryonic stem (ES) cell lines have not been established despite progress made for more than two decades. Isolation and culture of primary ES cell colonies are the first critical step towards establishment of stable ES cell lines. Here we report a novel method designated as "Separate and Seed" that contributes remarkably to efficient derivation of bovine primary ES-like cell colonies from blastocysts. These primary cultured bovine ES-like cells exhibit morphology typical of ES cells and express pluripotent molecular markers including Oct4, Nanog and alkaline phosphatase. Interestingly, bovine primary ES-like cell colonies distinctively express both stage-specific embryonic antigens 1 and 4 (SSEA1 and SSEA4), unlike mouse and human ES cells. These pluripotent markers may be used for characterization of authentic bovine ES cell lines in later studies. In contrast, whole embryos or inner cell mass (ICM) used for primary culture by conventional methods fails to produce primary bovine ES cell colonies that express all pluripotent stem cell markers shown above. Furthermore, bFGF improves growth and maintained undifferentiated state of bovine ES-like cells for several passages, whereas LIF and ERK inhibitor PD98059 known to promote pluripotency of mouse ES cells are unable to sustain bovine ES-like cells. Although continued efforts are required for improving long-term culture of bovine ES cells, this novel "Separate and Seed" method provides an initial effective step that may eventually lead to derivation of authentic bovine ES cell lines.
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PMID:Isolation and culture of primary bovine embryonic stem cell colonies by a novel method. 1934 Aug 39

Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 +/- 5.1% vs MEF 84.1 +/- 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-a, integrinb1 and a6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare.
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PMID:Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation. 1938 38

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.
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PMID:Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. 1941 70

In this study we examine whether a somatic cell, once returned to a pluripotent state, gains the ability to reprogram other somatic cells. We reprogrammed mouse embryonic fibroblasts by viral induction of oct4, sox2, c-myc, and klf-4 genes. Upon fusion of the resulting iPS cells with somatic cells harboring an Oct4-GFP transgene we observed, GFP expression along with activation of Oct4 from the somatic genome, expression of key pluripotency genes, and positive immunostaining for Oct4, SSEA-1, and alkaline phosphatase. The iPS-somatic hybrids had the ability to differentiate into cell types indicative of the three germ layers and were able to localize to the inner cell mass of aggregated embryos. Furthermore, ntES cells were used as fusion partners to generate hybrids, which were also confirmed to be reprogrammed to a pluripotent state. These results demonstrate that once a somatic cell nucleus is reprogrammed, it acquires the capacity and potency to reprogram other somatic cells by cell fusion and shares this functional property with normal embryonic stem (ES) cells.
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PMID:Reprogramming of somatic cells after fusion with induced pluripotent stem cells and nuclear transfer embryonic stem cells. 1963 40

Studies revealed that PI3K/AKT/mTOR signaling is important in the regulation of human embryonic stem cell (hESC) self-renewal and differentiation. However, its action on osteogenic differentiation of hESCs is poorly understood. We tested the effects of pharmacological PI3K/AKT/mTOR inhibitors on their potential to induce osteogenic differentiation of hESCs. Under feeder-free culture conditions, rapamycin (an mTOR inhibitor) potently inhibited the activities of mTOR and p70S6K in undifferentiated hESCs; however, LY294002 (a PI3K inhibitor) and an AKT inhibitor had no effects. Treatment with any of these inhibitors down-regulated the hESC markers Oct4 and Nanog, but only rapamycin induced the up-regulation of the early osteogenic markers BMP2 and Runx2. We also observed that hESCs differentiated when treated with FK506, a structural analog of rapamycin, but did not exhibit an osteogenic phenotype. Increases in Smad1/5/8 phosphorylation and Id1-4 mRNA expression indicated that rapamycin significantly stimulated BMP/Smad signaling. After inducing both hESCs and human embryoid bodies (hEBs) for 2-3 weeks with rapamycin, osteoblastic differentiation was further characterized by the expression of osteoblastic marker mRNAs and/or proteins (osterix, osteocalcin, osteoprotegerin, osteonectin, and bone sialoprotein), alkaline phosphatase activity, and alizarin red S staining for mineralized bone nodule formation. No significant differences in the osteogenic phenotypes of rapamycin-differentiated hESCs and hEBs were detected. Our results suggest that, among these 3 inhibitors, only rapamycin functions as a potent stimulator of osteoblastic differentiation of hESCs, and it does so by modulating rapamycin-sensitive mTOR and BMP/Smad signaling.
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PMID:Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway. 1964 65

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.
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PMID:p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells. 1972 59

Mouse embryonic stem cells (mESCs) have played a key role in the newly emerging fields of stem cell research. The traditional derivation and culture of mESCs have been based on the use of mouse embryonic fibroblasts (MEFs) treated with exogenous leukemia inhibitory factor (LIF). However, the rapid senescence of MEFs, coupled with the high cost of LIF, has significantly hampered the widespread use of mESCs in stem cell research. Thus, we present a novel exogenous LIF-free culture system for general mESCs applications, comprising fibroblast-like cells derived from the rabbit spleen (RSFs). We demonstrated that mESCs cultured on RSFs (mESCs-RSFs) maintained all mESC features after prolonged LIF-free culture, including alkaline phosphatase, cell surface markers (SSEA-1), molecular markers (OCT-4, NANOG, TERT, REX-1), karyotype, and pluripotency. The high expression level of both LIF and WNT3A in the RSFs may account for their ability to maintain mESCs without exogenous LIF. Moreover, this exogenous LIF-free culture system was verified to be of microbiological quality through analysis with electron transmission microscopy.
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PMID:Establishment of an exogenous LIF-free culture system for mouse embryonic stem cells. 1975 Nov 13

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.
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PMID:[The characters and specific features of new human embryonic stem cells lines]. 1976 48


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