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)

While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal fibroblasts (STO) as feeder-cells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epithelial-like polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-1 (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCC1), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA-1 and AP. In conclusion, SSEA-1 and EMA-1 can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures.
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PMID:Long-term culture and characterization of goat primordial germ cells. 1079 85

Human embryonic stem (hES) cells have been traditionally cultured on primary mouse embryonic fibroblasts (PMEFs). However, though STO cells have some advantages over PMEFs and human embryonic fibroblasts (hEFs) as feeder cells, they have never been used as feeder cells to establish hES cell lines. In this study, three hES cell lines (Miz-hES1, Miz-hES2, and Miz-hES3) were established from inner cell masses (ICM), using STO as feeder cells. The three hES cell lines had normal karyotypes and expressed high levels of alkaline phosphatase (AP), cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and transcription factor Oct-4. After culture on STO cells for 2 yr, hES cells maintained the potential to form derivatives of all three embryonic germ layers. Our results show that STO feeder cells have the potential to support the establishment and maintenance of hES cell lines. In addition, our results suggest that laminin may play an important role in maintaining the undifferentiated proliferation of hES cells.
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PMID:Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. 1293 Jul 26

The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.
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PMID:Long-term culture and transplantation of murine testicular germ cells. 1295 55

A long-term cultivation (5-8 months) of human blastocyst-derived embryonic cells (hES) was performed. Several properties of hESs were examined to prove the state of continuous cell lines. These cells have passed through 100-175 population doublings with the average population doubling time equal to 37.0 +/- 1.5 h. Isolated hESs, referred to as HESC-1, HESC-2, HESC-3, HESC-4, cultivated on mitotically inactivated mouse embryonic fibroblasts (STO continuous cell line), formed multilayer colonies of various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription factor Oct4, and all this characterizes embryonic stem cells of different origin. Expression of hES specific cell surface antigens (SSEA-3, SSEA-4, TRA-1-81 and TRA-11-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal antibodies. An additional prove for species specificity of HESC lines is the lack of expression of mouse specific surface antigen SSEA1. The cell cycle of HESC-1 undifferentiated cells and embryoid bodies was analysed cytofluorimetrically.
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PMID:[Isolation and characterisation of continuous human embryonic stem cell lines]. 1502 49

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.
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PMID:Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells. 1651 61

To establish human pluripotent embryonic germ (EG) cell lines, human primordial germ cells (PGCs) of embryos aborted in 5-9 week were cultured on inactive mouse STO fibroblast feeder. The medium contained human leukemia inhibitory factor (hLIF), human basic fibroblast growth factor (hbFGF) and forskolin. The EG cells could be passaged continuously until 12 generations. Most cells were positive in alkaline phosphatase staining and expressed cell surface antigen SSEA-3 and pluripotent marker Oct-4. These EG cell populations that retained normal karyotype could form embryoid body in culture and differentiate further into neuron-like cells, mucous epithelial cells, epithelial cells and other types of the cells spontaneously. These results indicated the cell clones derived from human PGCs resemble pluripotent EG cells from mouse PGCs in appearance or nature.
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PMID:[Isolation and culture of human pluripotent embryonic germ cells]. 1534 33

Here we report the derivation and characterization of new human embryonic stem cell (hESC) lines, SNUhES1, SNUhES2, and SNUhES3. These cells, established from the inner cell mass using an STO feeder layer, satisfy the criteria that characterize pluripotent hESCs: The cell lines express high levels of alkaline phosphatase, cell surface markers (such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), transcription factor Oct-4, and telomerase. When grafted into severe combined immunodeficient mice after prolonged proliferation, these cells maintained the developmental potentials to form derivatives of all three embryonic germ layers. The cell lines have normal karyotypes and distinct identities, revealed from DNA fingerprinting. Interestingly, analysis by electron microscopy clearly shows the morphological difference between undifferentiated and differentiated hESCs. Undifferentiated hESCs have a high ratio of nucleus to cytoplasm, prominent nucleoli, indistinct cell membranes, free ribosomes, and small mitochondria with a few crista, whereas differentiated cells retain irregular nuclear morphology, desmosomes, extensive cytoplasmic membranes, tonofilaments, and highly developed cellular organelles such as Golgi complex with secretory vesicles, endoplasmic reticulum studded with ribosomes, and large mitochondria. Existence of desmosomes and tonofilaments indicates that these cells differentiated into epithelial cells. When in vitro differentiation potentials of these cell lines into cardiomyocytes were examined, SNUhES3 was found to differentiate into cardiomyocytes most effectively.
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PMID:Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. 1567 Nov 44

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.
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PMID:Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts. 1620 32

To develop an alternative source for chicken pluripotent cells, we examined (1) whether undifferentiated preblastodermal cells could be subcultured in vitro for an extended period and (2) how subculturing affected the physiological properties of preblastodermal cells. The average number of preblastodermal cells was 2,397 in stage V embryos and 36,345 in stage VII embryos; stage X embryos had an average of 53,857 blastodermal cells. The average cell size decreased significantly (70.63-18.83 microm in diameter; P < 0.0001) as the embryo grew; this was closely related to a reduction in the size and number of lipid vesicles in the cell cytoplasm. The culture conditions were optimized for the stage V preblastodermal cells and the control stage X blastodermal cells. On STO feeder cells, the preblastodermal cells achieved stable growth in vitro only in HES medium or a mixed medium of the Knockout DMEM and HES media. However, more than 10 passages of preblastodermal cells at intervals of 3-4 days was possible only by using the Knockout/HES mixed medium and BRL cell-conditioned HES medium for the primary cultures and subcultures, respectively. Colony-forming preblastodermal cells had well-delineated cytoplasm, which was positively stained for stem cell-specific markers by anti-stage-specific embryo antigen-1 antibody, periodic acid-Schiff's solution, and alkaline phosphatase. When preblastodermal cells with or without culturing were transferred into the blastodermal cavity of stage X embryos, only in vitro-cultured preblastodermal cells at stage V (4/5 = 80%) and stage VII (2/8 = 25%) induced somatic chimerism in recipient chickens. In conclusion, undifferentiated preblastodermal cells could be subcultured, and only the colony-forming preblastodermal cells that stained positively for stem cell markers could induce somatic chimerism.
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PMID:Establishment of an in vitro culture system for chicken preblastodermal cells. 1642 35

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.
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PMID:Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts. 1739 46


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