Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Germ Layers”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources
PubMed2005

Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines.

BACKGROUND: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos. METHODS AND RESULTS: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice. CONCLUSION: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.

Animals↗
PubMedDate not supplied

[Neurogenesis in the adult mammalian brain].

The concept of the CNS cell composition stability has recently undergone significant changes. It was earlier believed that neurogenesis in the mammalian CNS took place only during embryonic and early postnatal development. New approaches make it possible to obtain new results overriding the dogma that neurogenesis is impossible in the adult brain. The present review summarizes the information about the neural stem cell. It has been demonstrated that new neurons are constantly formed in adult mammals, including man. In two brain zones, subventricular zone and denate gyrus, neurogenesis appears proceed throughout the entire life of mammals, including man. The newly arising neurons are essential for some important processes, such as memory and learning. Stem cells were found in the subependymal and/or ependymal layer. They express nestin, and have a low mitotic activity. During embryogenesis, the stem cell divides asymmetrically: one daughter cell resides as the stem cell in the ependymal layer and another migrates to the subventricular zone. There it gives rise very fast to a pool of dividing precursors, from which neural and glial cells differentiate and migrate to the sites of final localization. The epidermal and fibroblast growth factors act as mitogens for the neural stem cell. The neural stem cell gives rise to the cells of all germ layers in vitro and has a wide potential for differentiation in the adult organism. Hence, it can be used as a source of various cell types of the nervous tissue necessary for cellular transplantation therapy.

Animals↗
PubMed2001

Allocation and early differentiation of cardiovascular progenitors in the mouse embryo.

During gastrulation of the mouse embryo, progenitor cells of the endothelium of blood vessels are allocated to different compartments of the extraembryonic and embryonic tissues in accordance to the timing and the site of recruitment to the mesodermal layer. In the yolk sac, the endothelium and the erythropoietic progenitors are populated by different groups of mesodermal cells, suggesting that they may not be derived from a common pool of progenitors. An orderly pattern of movement of mesodermal cells and the provision of proper intercellular transforming growth factor beta (TGF beta) and vascular endothelial growth factor (VEGF) signaling by neighboring germ layer tissues are essential for normal morphogenesis of the vasculature.

Animals↗
PubMed2005

Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3.

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.

Cell Differentiation↗
PubMed2003

Feeder layer- and serum-free culture of human embryonic stem cells.

In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.

Animals↗
PubMed1999

Establishment of pluripotent cell lines from vertebrate species--present status and future prospects.

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.

Animals↗
PubMed1996

Establishment of an embryonic stem cell line from 8-cell stage mouse embryos.

The aim of this study was to try establishing mouse ES cell lines from the early developmental stage. Fifty-two uncompacted 8-cell stage embryos were dissociated and single blastomeres were seeded on primary embryonic fibroblasts in DMEM/F12 completed with 10% foetal calf serum, 10% new born calf serum. 10(-4) M beta-mercaptoethanol. After approximately 5 days of culture, multiple cell clones exhibiting stem cell morphology grew out and were dissociated. One cell line was established (MSB1) and characterised. The karyotype and the G-banding revealed a male diploid cell line. MSB1 cells were injected into syngenic mice and produced teratocarcinomas. Detailed histological examination of the tumours showed a great variety of cell types including representatives of all three primary germ layers. Several nests of undifferentiated stem cells were also present. Microinjections of MSB1 cells into 52 blastocysts produced 2 chimeras, 1 male and 1 female. These results demonstrate that a highly pluripotents ES cell line can be derived from 8-cell stage mouse embryos. However, the male chimera appeared sterile. More experiments would thus be necessary to prove that the cell line obtained is capable to colonise the germ line.

Animals↗
PubMed2000

Visceral endoderm mediates forebrain development by suppressing posteriorizing signals.

The anterior visceral endoderm (AVE) has attracted recent attention as a critical player in mouse forebrain development and has been proposed to act as "head organizer" in mammals. However, the precise role of the AVE in induction and patterning of the anterior neuroectoderm is not yet known. Here we identified a 5'-flanking region of the mouse Otx2 gene (VEcis) that governs the transgene expression in the visceral endoderm. In transgenic embryos, VEcis-active cells were found in the distal visceral endoderm at 5.5 days postcoitus (dpc), had begun to move anteriorly at 5.75 dpc, and then became restricted to the AVE prior to gastrulation. The VEcis-active visceral endoderm cells exhibited ectodermal morphology distinct from that of the other endoderm cells and consisted of two cell layers at 5.75 dpc. In the Otx2(-/-) background, the VEcis-active endoderm cells remained distal even at 6.5 dpc when a primitive streak was formed; anterior definitive endoderm was not formed nor were any markers of anterior neuroectoderm ever induced. The Otx2 cDNA transgene under the control of the VEcis restored these Otx2(-/-) defects, demonstrating that Otx2 is essential to the anterior movement of distal visceral endoderm cells. In germ-layer explant assays between ectoderm and visceral endoderm, the AVE did not induce anterior neuroectoderm markers, but instead suppressed posterior markers in the ectoderm; Otx2(-/-) visceral endoderm lacked this activity. Thus Otx2 is also essential for the AVE to repress the posterior character. These results suggest that distal visceral endoderm cells move to the future anterior side to generate a prospective forebrain territory indirectly, by preventing posteriorizing signals.

Animals↗
PubMed2000

The embryonic development of the rhabdocoel flatworm Mesostoma lingua (Abildgaard, 1789).

The embryonic development of the flatworm Mesostoma lingua was studied using a combination of life observation and histological analysis of wholemount preparations and sections (viewed by both light and electron microscopy.) We introduce a series of stages defined by easily recognizable morphological criteria. These stages are also applicable to other platyhelminth taxa that are currently under investigation in our laboratory. During cleavage (stages 1 and 2), the embryo is located in the center of the egg, surrounded by a layer of yolk cells. After cleavage, the embryo forms a solid, disc-shaped cell cluster. During stage 3, the embryo migrates to the periphery of the egg and acquires bilateral symmetry. The side where it contacts the egg surface corresponds to the future ventral surface of the embryo. Stage 4 is the emergence of the first organ primordia, the brain and pharynx. Gastrulation, as usually defined by the appearance of germ layers, does not exist in Mesos-toma; instead, organ primordia emerge "in situ" from a mesenchymal mass of cells. Organogenesis takes place during stages 5 and 6. Cells at the ventral surface form the epidermal epithelium; inner cells differentiate into neurons, somatic and pharyngeal muscle cells, as well as the pharyngeal and protonephridial (excretory) epithelium. A junctional complex, consisting initially of small septate junctions, followed later by a more apically located zonula adherens, is formed in all epithelial tissues at stage 6. Beginning towards the end of stage 6 and continuing throughout stages 7 and 8, cytodifferentiation of the different organ systems takes place. Stage 7 is characterized by the appearance of eye pigmentation, brain condensation and spindle-shaped myocytes. Stage 8 describes the fully dorsally closed and differentiated embryo. Muscular contraction moves the body in the egg shell. We discuss Mesostoma embryogenesis in comparison to other animal phyla. Particular attention is given to the apparent absence of gastrulation and the formation of the epithelial junctional complex.

Animals↗
PubMed1984

DNA transformation of a pluripotent mouse embryonal stem cell line with a dominant selective marker.

The mouse embryonal stem cell line BLC 1 growing on feeder layer was treated with a calcium phosphate/DNA precipitate prepared with DNA of plasmid pAG60 which harbors the Tn5-derived neo gene, thus encoding resistance to G418, an aminoglycoside antibiotic. Transfection performed on feeder layer resulted in the formation of G418-resistant clones T1 and T2/K26. The stable integration of the transformed neo gene was confirmed by dot hybridization in all descendant cultures of clones T1 and T2/K26 as well as in the tumors derived from them. In vivo and in vitro differentiation revealed the pluripotent status of the transformants. Tumors derived from T1 and T2/K26 contained various tissues with derivatives of all three primary germ layers.

Animals↗
PubMed1995

Expression of the small proteoglycans biglycan and decorin in the adult human testis.

The genes coding for the core proteins of the small chondroitin sulfate/dermatan sulfate proteoglycans (PGs) biglycan and decorin are both expressed in the adult human testis. Northern hybridization of human testicular mRNA indicated the presence of one specific transcript for biglycan at 2.6 kb and two specific transcripts for decorin at 1.6 kb and 1.9 kb. In situ hybridization localized the mRNA for biglycan to the peritubular tissue as well as to the tunica muscularis and adventitia of arteries. Leydig cells and cells of the seminiferous epithelium both proved to be negative. For decorin mRNA, strong signals were found over single, dispersed interstitial cells (possibly fibroblasts), the adventitious layer of large arteries, and the perivascular tissue cells of small arteries, arterioles, and capillaries. In the peritubular cell layers, decorin gene expression was comparably lower and was restricted to the outer fibroblast layers. Germ cells and Sertoli cells were devoid of any positive signal. Immunoblot analysis using specific antisera directed against the core proteins of biglycan and decorin confirmed their presence in chondroitin ABC lyase-treated proteinaceous extracts of the human testis. Immunohistochemistry for biglycan showed a good spatial correlation with the in situ hybridization data and revealed in addition a nonuniform distribution of the antigenic material, which was located predominantly over peritubular myoid cells and the adventitious layer of arteries. Together these results indicate that the expression of both genes in the human testis is differentially regulated, despite the overall similarity of their protein products, and suggest distinct roles for these PGs in testicular function.

Base Sequence↗
PubMed2004

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers.

BACKGROUND: Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. METHODS: Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. RESULTS: Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. CONCLUSIONS: This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.

Alkaline Phosphatase↗
PubMed1999

Establishment of pluripotent cell lines from porcine preimplantation embryos.

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.

Alkaline Phosphatase↗
PubMed1999

Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation.

The embryonic gastrointestinal tract develops from a simple tube into a coiled, flexed, and regionalized structure. The changes in gut morphology coincide with the differentiation of multiple cell types in concentric layers, and include colonization by migratory neuron precursors, and the development of gastrointestinal motility. We describe a reliable method for growing embryonic mouse intestine in vitro by the attachment of segments of intestinal tract by their cut ends, with the intervening region suspended in the culture medium. These are termed "catenary cultures." E11-E11.5 mouse midgut, hindgut, or mid- plus hindgut segments were grown in catenary culture for up to 10 days and their growth, morphology, cell differentiation, ability to support neural precursor migration, and contractile activity were assessed. The increase in size of the cultured explants was not large, but morphogenesis proceeded, best exemplified by elongation of the caecum. Cell differentiation also proceeded. In the mucosa, goblet cells differentiated. Muscle layers, characterized by desmin expression, and kit-positive interstitial cells of Cajal differentiated in the correct positions. Where segments initially included neural precursors in a small sub-region, these migrated and proliferated to form uniform neuronal networks throughout the entire explant, and the cells expressed the neuron markers nitric oxide synthase and neuron specific enolase. Gut motility was attained 5-6 days into the culture period, and both contractile- and mixing-type movements were observed. Thus, cell types representative of all three germ layer contributions developed, and in addition, the gut, being mainly free, was able to elongate and bend (unlike on solid support cultures), while retaining its rostrocaudal identity.

Animals↗
PubMed1999

Can exogenous stem cells be used in transplantation?

Today's most urgent problem in transplantation is the lack of suitable donor organs and tissues and as the population ages, demands for organs and tissue therapies will only increase. One alternative to organ transplantation is cell therapy whose aim is to replace, repair or enhance the biological function of damaged tissue or diseased organs. One goal of cellular transplantation thus has been to find a renewable source of cells that could be used in humans. Embryonic stem (ES) cells have the potential to proliferate in vitro in an undifferentiated and pluripotent state. Theoretically, ES cells are capable of unlimited proliferation in vitro. ES cells spontaneously differentiate into derivatives of all three primary germ layers: endoderm, ectoderm and mesoderm, hence providing cells in vitro which can theoretically be isolated and used for transplantation. Furthermore, these pluripotent stem cells can potentially be used to produce large numbers of cells that can be genetically modified in vitro. Once available, this source of cells may obviate some of the critical needs for organ transplantation. Murine ES cells have been extensively studied and all available evidence indicates that all aforementioned expectations are indeed fulfilled by ES cells. ES cells as well as embryonic germ cells have recently been isolated and maintained in culture. The recent descriptions of human ES cells portend the eventual use of allogeneic in vitro differentiated cells for human therapy. This goal, however, is fraught with obstacles. Our aim is first to review the recent advances made with murine ES cells and then to point out potentials and difficulties associated with the use of human ES cells for transplantation.

Animals↗
PubMed1992

Origin of primordial germ cells, as characterized by the presence of nuage, in embryos of the teleost fish Barbus conchonius.

The presence, location and morphology of cells containing nuage, an ultrastructural characteristic of primordial germ cells (PGCs), is described from the moment of first morphological recognition of PGC (around 100% epiboly) in embryos of the teleost fish Barbus conchonius. Thus characterized cells were studied in relation to their cellular contacts with somatic germ layer cells, possibly involved in the determination of PGCs. The results show that from the very moment that cells, likely to be PGCs, can be light microscopically identified with morphological and positional criteria (from 10 h post fertilization (p.f.) onwards), they contain nuage near the nuclear envelope, which is a strong indication of their PGC-identity. During the studied period (9-12 h and 24 h p.f.) nuage-containing cells seem to translocate from the mesoderm towards the yolk syncytial layer (YSL). These PGCs usually appear not to be directly connected with the YSL but to remain separated from the YSL by one or more endodermal extensions, at least up to 12 h p.f. Also at 24 h p.f. somatic cells separate the PGCs from the YSL.

Animals↗
PubMedDate not supplied

[Prostatic tissue in a cystic teratoma of the ovary].

We report one example of mature cystic teratoma of the ovary containing prostatic tissue, in a 73 year-old female. The tumor is typical and it is composed of well-differentiated derivatives of the three germ layers. Prostatic tissue was an incidental finding in teratoma. This tissue contained ducts and acini in a simple branching pattern similar to that seen in the peripheral zone of the prostatic gland. The immunohistochemical stainings for PSA and PSAP throughout the glandular cells were similar to those present in normal prostate.

Aged↗
PubMed1988

Developmental potential of day 13 porcine embryonic disk under in vitro culture conditions.

Embryonic disks were microsurgically isolated from adjacent trophoblast tissue and cultured for varying periods in vitro. During the first 24 h of culture, vesicles (1 to 4/disk) composed of mesoderm and endoderm formed from the ventral surface. In the subsequent culture period, the vesicles continued to increase in size and by 96 h in vitro, most originally multivesiculated explants possessed a single vesicle formed by delamination and coalescence of smaller vesicles. Scanning electron microscopy revealed the formation of grooves and ridges in abnormal attempts at differentiation by the embryonic ectoderm. Endoderm comprising the outer tissue layer of the vesicle underwent a gradual alteration in surface morphology during in vitro culture. Initially flat, with a paucity of microvilli, these cells became dome-shaped with an abundance of microvilli. In addition, they became highly secretory as revealed by the presence of numerous secretory droplets at their surface. After culture for periods of up to 10 d, several explants displayed areas containing pulsating tissue, with contractions occurring at a rate of 20 to 30/minute, indicative of mesoderm differentiation. Culture of porcine isolated embryonic disk in vitro should enhance investigations into the regulation of germ layer formation and differentiation and assist in determining the tissue source of conceptus secretory products.

Animals↗