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Effects of type IV collagen and laminin on the cryopreservation of human embryonic stem cells.

Previous reports have indicated that extracellular matrices (ECMs) affect the developmental fate of human embryonic stem cells (hESCs). Specially, type IV collagen and laminin, which belong to a group of macromolecular proteins with a substantial proportion of ECMs, are known to influence the proliferation and differentiation of hES cells. In this study, we evaluated the effects of type IV collagen and laminin in freezing medium on the survival and differentiation rates of hES cells after slow freezing and rapid thawing. The addition of type IV collagen (1 microg/ml) to the freezing medium significantly increased the survival rate of hES cells after thawing compared with that of a control group. The spontaneous differentiation rates of groups treated with type IV collagen (1 microg/ml) or laminin (1 microg/ml) were significantly lower than those of the control group. Frozen-thawed hES cells have currently been cultured for more than 70 passages and retain key properties of hES cells such as morphological characteristics, normal karyotype, marker expression (alkaline phosphatase, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Rex-1, and Oct-4), basement membrane-related gene expression, and the potential to differentiate into derivatives of all three germ layers. This new slow freezing method by ECM treatment is a reliable and effective cryopreservation method for pluripotent hES cells.

Alkaline Phosphatase↗

Craniofacial development and the evolution of the vertebrates: the old problems on a new background.

Based on recent advances in experimental embryology and molecular genetics, the morphogenetic program for the vertebrate cranium is summarized and several unanswered classical problems are reviewed. In particular, the presence of mesodermal segmentation in the head, the homology of the trabecular cartilage, and the origin of the dermal skull roof are discussed. The discovery of the neural-crest-derived ectomesenchyme and the roles of the homeobox genes have allowed the classical concept of head segmentation unchanged since Goethe to be re-interpreted in terms of developmental mechanisms at the molecular and cellular levels. In the context of evolutionary developmental biology, the importance of generative constraints is stressed as the developmental factor that generates the homologous morphological patterns apparent in various groups of vertebrates. Furthermore, a modern version of the germ-layer theory is defined in terms of the conserved differentiation of cell lineages, which is again questioned from the vantage of evolutionary developmental biology.

Anatomy, Comparative↗

Heterotopic intestinal membrane in a retroperitoneal tumor.

Plain abdominal X-rays of a 13-year-old girl with chief complaints of back pain revealed calcification in the upper left abdomen. A calcified tumor was confirmed at the dorsal side of the pancreatic tail upon admission. A completely formed colic membrane free of all other germ layers was discovered within the tumor, leading to a diagnosis of heterotopic colonic membrane. To our knowledge, there have been no other cases of heterotopic intestinal tissue of this type, so we consider this an extremely rare case worth reporting.

Adolescent↗

No evidence for significant transdifferentiation of bone marrow into pancreatic beta-cells in vivo.

Several recent studies have suggested that the adult bone marrow harbors cells that can differentiate into tissues from all three germ layers. Other reports have contradicted these findings or attributed them to cell fusion. In this study, we investigated whether bone marrow-derived cells contribute to the renewal of adult pancreatic endocrine cells, in particular insulin-producing beta-cells, in vivo. To address this issue, we studied mice transplanted with green fluorescent protein (GFP)-positive, sex-mismatched bone marrow. We also extended our studies to pancreatic injury models (partial pancreatectomy and streptozotocin administration). All animals showed stable full donor chimerism in the peripheral blood and microscopic analysis at 4-6 weeks and 3 months after transplantation, indicating that the GFP(+) and Y chromosome-positive donor bone marrow contributed substantially to blood, lymphatic, and interstitial cells in the pancreas. However, after examining >100,000 beta-cells, we found only 2 beta-cells positive for GFP, both of which were in control animals without pancreatic injury. Thus our study results did not support the concept that bone marrow contributes significantly to adult pancreatic beta-cell renewal.

Animals↗

Lung growth and development.

The organogenesis of lung involves several complex mechanisms, including interactions between cells originating from two germ layers--endoderm and mesoderm. Regulation of lung branching morphogenesis with reference to its architecture, growth pattern, differentiation, interactions between epithelium and mesenchyme and / or endothelium, as well as genes regulating these processes have been addressed by the pulmonary biologists through careful molecular biology and genetic experimental approaches. The mammalian lung develops by outpouching from the foregut endoderm as two lung buds into the surrounding splanchnic mesenchyme. Several different regions of the foregut are specified to develop into different thoracic and visceral organs. The lung-buds further elongate and branch, and the foregut longitudinally gets separated into esophagus and trachea. In rodents (mice and rats), this occurs around embryonic day 11, where the right lung bud develops into four different lobes and left lung develops as a single lobe. In humans, these processes occur by 3-4 weeks of embryonic development, where the right lung is a trilobar lung and the left lung is a bilobar lung. Several generations of dichotomous branching occur during embryonic development, followed by secularization and alveolarization pre- and post-natally, which transform a fluid-filled lung into an air-breathing lung able to sustain the newborn. During these different developmental stages from embryonic to newborn stage, the lung architecture undergoes profound changes, which are marked by a series of programmed events regulated by master genes (e.g., homeobox genes), nuclear transcription factors, hormones, growth factors and other factors. These programmed events can be altered by undesirable exposure to overdoses of hormones/vitamins/growth factors, synthetic drugs, environmental toxins, radiation and other agents. In the recent years molecular techniques have opened avenues to study specific functions of genes or their products (proteins) in vivo or in vitro at a cellular or an organelle level, some of these include targeted disruption, knock-in / knock-out genes, in vitro mutagenesis, use of sense and anti-sense oligonucleotides. Some of these aspects with reference to regulation of normal lung development and growth and a specific example of pulmonary hypoplasia as an abnormal lung formation are discussed in this review.

Animals↗

Composition, regulation, and function of the cytoskeleton in mammalian eggs and embryos.

The cytoskeleton of the mammalian egg and early embryo is highly unique when compared to the cytoskeleton of their somatic cell brethren. Although all three cytoskeletal systems, actin filaments, microtubules, and intermediate filaments, are present as early as the unfertilized egg; each system has adapted features that allow the egg and early embryo to meet the strict demands of the developmental process. The major demands placed upon eggs and embryos are developmental transitions (i.e., fertilization, compaction, blastocyst formation, germ layer formation and gastrulation), each of which must be traversed in order for the embryo to form a new individual. To successfully complete all of the necessary processes during early development, eggs and embryos must call upon many signal transduction mechanisms, cytoskeletal components, and genes that are both unique to embryogenesis and ubiquitous among many types of somatic cells. It is the goal of this review to provide some current details into the mechanisms that drive early development primarily focusing on the cytoskeletal components eggs and embryos have adapted to promote embryogenesis.

Actins↗

Genetic interactions in early neurogenesis of Drosophila melanogaster.

A number of genetic loci, called neurogenic, have been found to be involved in directing the segregation of neural and epidermal lineages within the ectodermal germ layer of Drosophila melanogaster. With the aim of understanding the regulation of this developmental function we have studied interactions of the loci N, Dl and E(spl) among each other and with another locus (H), by means of increasing and decreasing the number of wild-type copies of one of these genes in the presence of mutations in another one. The results reveal functional community which exists among these neurogenic loci. E(spl) overlaps functionally with both N and Dl because genotypes involving only one copy of E(spl)+ and either an N or Dl mutation are lethal. Furthermore the normal H+ allele behaves as if it represses the activity of the 3 neurogenic loci; and, whereas E(spl) seems to be a close target of H repressing action, the influence of H upon the other two seems to be indirect.

Alleles↗

The murine amelogenin promoter: developmentally regulated expression in transgenic animals.

We are interested in understanding hierarchical regulation pathways that control gene expression in developing teeth. In pursuit of the molecular basis for the regulated expression of amelogenin by developing ameloblasts during tooth formation, we isolated the murine amelogenin promoter. Analysis of this promoter will provide additional details towards the identification of signals generated through instructive-, dissimilar-germ layer interactions that are for responsible for temporal- and spatial-regulation for amelogenin gene expression. Using transgenic mice we demonstrate that a 2263 nucleotide stretch of the murine amelogenin promoter conveys appropriate temporal- and spatial-regulation for amelogenin gene expression in response to instructive-signals. These transgenic animals are useful reagents to further dissect signaling pathways responsible for regulated gene expression by terminally differentiated ameloblasts.

3T3 Cells↗

Teratomas of the mediastinum.

Mediastinal teratomas are rare, constituting only 8-13% of tumours in this region. Teratomas often contain structures from all three germ layers, and 15-20% are malignant. A report is presented of eight patients operated on in 1978-1985 for mediastinal teratoma histologically classified as benign. Radical excision was performed in seven cases, but one tumour could not be radically removed because of infiltrative growth into vital structures, and the patient died 9 months postoperatively. In the other seven cases, including four with unusually rapid and dramatic tumour development, no signs of recurrence have appeared.

Adolescent↗

Expression of C-ETS1 in early chick embryo mesoderm: relationship to the hemangioblastic lineage.

In situ hybridization was used to detect the expression of the c-ets1 protooncogene during formation of the germ layers in the chick blastodisc. c-ets1 transcripts were present during the gastrulation process, i.e. when the mesodermal cells invaginated. The expression became down-regulated in lateral plate and the dorsal part of the somites while an intense signal was retained in the intermediate cell mass. When vasculogenesis started, c-ets1 transcripts labelled blood islands and endothelial cells. Before the mesoderm split, transcripts were present over the whole layer, more abundant however on its ventral side in contact with the endoderm. After the mesoderm split, silver grains became distributed asymmetrically: splanchnopleural mesoderm expressed c-ets1 messengers all over while expression in the somatopleural mesoderm was restricted to a few profiles corresponding to small endothelial cell groups. This asymmetrical distribution of c-ets1 transcripts is in agreement with our previous experimental findings establishing the different potentialities of the two mesodermal layers regarding hemopoiesis, vasculogenesis and angiogenesis processes.

Animals↗

Developmental expression of nicein adhesion protein (laminin-5) subunits suggests multiple morphogenic roles.

Nicein/kalinin (laminin-5) is a heterotrimeric laminin-like adhesion protein, which is secreted at the basement membrane of subsets of epithelia and is involved in the etiology of junctional epidermolysis bullosa, a severe human blistering disease characterized by disadhesion of epidermis from dermis. cDNA clones encoding the three chains of mouse nicein and antibodies specific to each polypeptide were used to examine the expression of the protein in the developing mouse embryo from 10.5 day post coitum to 7 days after birth. At various stages of development, co-expression of the three chains of nicein was observed in amnion, skin, and in epithelia of respiratory, urinary and digestive systems. High level expression of nicein was seen in enamel-secreting ameloblasts in developing teeth. Cell-specific distribution of nicein was also detected in other specialized tissues representative of the three primary embryonic germ layers with prominent secretory or protective functions. Differential and focal expression of nicein subunits was observed in the choroid plexus and the floor plate of the neural tube. Messenger for the heavy chain of nicein was detected in the floor plate, where mouse s-laminin messengers were also found. This suggests that nicein genes may play a role in the migration and polarization of motor neurons in the developing spinal cord.

Amino Acid Sequence↗

Hepatic teratomas in children.

This report describes a hepatic teratoma in a newborn child. Microscopic examination showed a predominantly immature teratoma containing tissue representative of all three germ layers. No evidence of malignant tissue was found in the tumor, and no clinical evidence of metastasis was found in the child. A markedly elevated serum alpha fetoprotein was documented. A review of the literature on hepatic teratomas in children is also presented.

Humans↗

Intracardiac teratoma in a child simulating an atrioventricular nodal tumor.

Intracardiac teratoma is an extremely rare pediatric neoplasm. We studied the case of a 6-year-old girl with a right intraventricular cardiac mass. The tumor consisted of clusters of monotonous round epithelial cells scattered in a dense fibrotic stroma and was thought to represent an atrioventricular nodal tumor. Three years later the tumor recurred, with multiple mature elements derived from all three germ layers, and was diagnosed as mature cystic teratoma. Still present, however, were multiple areas that were histologically similar to the earlier lesion. Immunostaining revealed strong positivity for insulin, glucagon, somatostatin, and chromogranin consistent with overgrown pancreatic islets of Langerhans within a mature teratoma.

Atrioventricular Node↗

Maturation of intracranial immature teratomas. Report of two cases.

Immature teratomas arising within the central neuraxis are rare neoplasms. These tumors contain diverse cell lineages that retain an embryonal character and display phenotypic differentiation attributed to the three classic germ layers. The clinical management of these lesions is unclear, due in part to their low incidence and to an incomplete understanding of their natural history. Although the potential for phenotypic differentiation and cellular maturation within immature teratomas arising in the gonads is well documented, this has not been described in the intracranial tumors. In the present report, the authors describe two cases of intracranial immature teratomas, one involving the pineal region and the other involving the left frontotemporal lobes, which underwent cellular differentiation and maturation. At initial resection, the tumors from both cases were composed predominantly of primitive neuroepithelial tissue that was admixed with immature and differentiating mesenchymal and epithelial structures. No foci of germinoma, endodermal sinus, choriocarcinoma, or embryonal carcinoma tissue were present. Subsequent resections in both cases revealed an absence of immature tissue. The tumor in Case 1 contained only differentiated epithelial and mesenchymal tissue with no neuroepithelial component, whereas the tumor in Case 2 demonstrated abundant mature neuronal and glial tissue. These two cases from different intracranial sites suggest that spontaneous maturation may be a significant aspect of the natural history of intracranial immature teratomas.

Brain Neoplasms↗

Medulloblastoma arising from an immature teratoma of the posterior fossa. Case report.

Teratomas, a group of nongerminomatous germ cell tumors, account for 3% of all childhood tumors. These unusual lesions reproduce the cellular and structural phenotypic traits associated with the three classic germ layers in inappropriate places in the body and often retain an embryonal character. These immature cells can differentiate into more malignant neoplasms. An intracranial location for this lesion type is rare. The authors describe the case of a 2-year-old boy with a teratoma of the posterior fossa that had partially differentiated into a medulloblastoma.

Biopsy↗

Ovarian mature cystic teratoma containing homunculus: a case report.

We report a partial fetus-like structure (homunculus) in benign ovarian mature cystic teratoma removed from a 23-yr-old female. The cyst displayed various tissues derived from the three germ layers. The homunculus had a distinguished fetal cranial structure with one upper extremity. A partially developed osteocartilagenous skeleton was identified in the cranial structure of the homunculus. Intracranial structures such as cerebral cortex, retinal pigment, and leptomeninges were extruded from the partially disrupted calvarium.

Adult↗

In vitro development of secondary blastodiscs from dispersed blastoderm cells of Gallus domesticus.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

Animals↗

Coexistence of hepatocellular carcinoma and gastrointestinal stromal tumor: a case report.

Malignant gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors originating from the wall of the gastrointestinal tract. Their coexistence with other tumors originating from other germ layers is unique. We have reported a case of a 63-year-old GIST patient presenting as an epigastric mass associated with hepatic tumor. Histologically, the mesenteric tumor was composed of spindle cells showing both neural and smooth muscle differentiation. Immunohistochemical examination showed positive staining for CD117, vimentin, S-100, and SMA, while CD34 antigen was negative. The hepatic tumor was diagnosed as hepatocellular carcinoma (HCC). To the best of our knowledge, this is the first case of GIST and HCC coexistence. The rarity of the case, however, should not lead to ignoring such a possibility in differential diagnosis.

Carcinoma, Hepatocellular↗