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PubMed1999

Dual role of the basic helix-loop-helix transcription factor scleraxis in mesoderm formation and chondrogenesis during mouse embryogenesis.

Scleraxis is a basic helix-loop-helix (bHLH) transcription factor shown previously to be expressed in developing chondrogenic cell lineages during embryogenesis. To investigate its function in embryonic development, we produced scleraxis-null mice by gene targeting. Homozygous mutant embryos developed normally until the early egg cylinder stage (embryonic day 6.0), when they became growth-arrested and failed to gastrulate. Consistent with this early embryonic phenotype, scleraxis was found to be expressed throughout the embryo at the time of gastrulation before becoming restricted to chondrogenic precursor cells at embryonic day 9.5. At the time of developmental arrest, scleraxis-null embryos consisted of ectodermal and primitive endodermal cell layers, but lacked a primitive streak or recognizable mesoderm. Analysis of molecular markers of the three embryonic germ layers confirmed that scleraxis mutant embryos were unable to form mesoderm. By generating chimeric embryos, using lacZ-marked scleraxis-null and wild-type embryonic stem cells, we examined the ability of mutant cells to contribute to regions of the embryo beyond the time of lethality of homozygous mutants. Scleraxis-null cells were specifically excluded from the sclerotomal compartment of somites, which gives rise to the axial skeleton, and from developing ribs, but were able to contribute to most other regions of the embryo, including mesoderm-derived tissues. These results reveal an essential early role for scleraxis in mesoderm formation, as well as a later role in formation of somite-derived chondrogenic lineages, and suggest that scleraxis target genes mediate these processes.

Animals↗
PubMed1993

Visceral yolk sac-derived tumors.

Externalization of the visceral yolk sac, after fetectomy, induces the development of extra-embryonal fetal tumors in rodents. These tumors are either benign teratomas that appear 3 to 4 weeks after the displacement of the yolk sac or malignant tumors, i.e. yolk sac carcinomas. The latter appear 4 to 8 months after the surgery. If however, Mouse Sarcoma Virus (MSV) is injected in the placentas at the time of fetectomy (day 12 of pregnancy) the malignant tumors develop much earlier (2 to 3 months after surgery) and some display characteristics of embryonal carcinoma. Whether virus induced or not, the yolk sac carcinomas that develop from the displaced visceral yolk sac possess the same morphological and biological characteristics. They are composed of both parietal and visceral yolk sac structures and sometimes trophoblast. The tumors metastasize, grow in ascites form and kill their host. They are readily transplantable in syngeneic rats and grow in tissue culture as an epithelial-like sheet of cells. On the other hand, the benign teratomas are composed of various well differentiated adult tissues. In these tissues, derivatives of all three germ layers are observed. Numerous experiments prove that the stem cells for these various adult tissues are not germ cells. Instead the stem cells are multipotential cells that arise in the displaced yolk sac by a process of dedifferentiation. These poorly differentiated cells originate from the endoderm of the displaced visceral yolk sac. By redifferentiation they give rise to the various adult tissues characteristic for benign teratomas. The multipotential poorly differentiated cells are also likely to be the target cells for malignant transformation. Malignant transformation of these cells, whether induced by a virus or spontaneously occurring in the displaced yolk sac, leads not only to the development of yolk sac carcinomas and eventually embryonal carcinoma but also, although rarely, to choriocarcinoma. The latter tumor is transplantable in allogeneic hosts. It is hormonally active since it secretes lactogen and progesterone. The extra-embryonal fetal tumors and in particular the rat yolk sac carcinomas and choriocarcinoma proved to be a good source for the detection of oncofetal antigens. At least two different oncofetal endodermal antigens were detected with monoclonal antibodies (mab) made after immunization with yolk sac carcinoma. Another mab, made against choriocarcinoma, was found to react specifically with the cytotrophoblast both in the normal placenta and in the tumor. No other placental cells showed a positive reaction.

Animals↗
PubMed1975

An ultrastructural study of implanting mouse blastocysts: coated vesicles and epithelium formation.

Electron microscopial techniques were used to study mouse embryos at implantation stages (5 1/2 days post coitum). Evidence was obtained indicating that trophoblast cells remove uterine luminal epithelial cells by phagocytosis. Small coated vesicles very probably play an important role in the process of digestion of uterine cells by the trophoblast. Two types of endoderm cell were present: the embryonic endodermal cells contained organelles appropriate to a nutritive pathway; the extra-embryonic endodermal cells were relatively poorly developed compared with those of the embryonic endoderm, and remained as a discontinuous layer. The ectoderm contained few organelles. Epithelium formation is described. Junctions only develop between cells in the same germ layer except for occasional ones, including desmosomes, between trophoblast and uterine epithelial cells. The author wishes to thank Professors J.M.F. Landsmeer and W.T. Daems for their stimulating discussions during this research project and for their helpful comments during the preparation of the manuscript. The Laboratory for Electron Microscopy kindly provided full facilities. The assistance of Mr J. Tinkelenberg in the preparation of the drawing (Fig. 1) is acknowledged. Thanks are also due to Mrs 2. seeger for reading the English manuscript and to Miss Ria Stokman for typing it.

Animals↗
PubMed2004

Ectodermal dysplasias.

Ectodermal dysplasias are a large group of heritable conditions characterized by congenital defects of one or more ectodermal structures and their appendages: hair (hypotrichosis, partial, or total alopecia), nails (dystrophic, hypertrophic, abnormally keratinized), teeth (enamel defect or absent), and sweat glands (hypoplastic or aplastic). The ectodermal dysplasias, as a rule, are not pure "one-layer diseases." Mesodermal and, rarely, endodermal dysplasias coexist. Embryogenesis exhibits distinct tissue organizational fields and specific interactions among the germ layers that may lead to a wide range of ectodermal dysplasias when genes important for development are mutated or otherwise altered in expression. Of the approximately 200 different ectodermal dysplasias, about 30 have been studied at the molecular level with identification of the causative gene. Freire-Maia and Pinheiro used the clinical aspects for their classification, and Priolo integrated molecular genetic and clinical aspects for her scheme. Those two more historical classification schemes have the difficulty that, when applied strictly, several additional groups of diseases should be integrated within the term "ectodermal dysplasias," e.g. keratodermas with skin or hair alterations or the ichthyoses with associated features. Such consequent classification would lead to an endless list of diseases and would be useless for the practical work. Recent evidence implicates a genetic defect in different pathways orchestrating ectodermal organogenesis. Modern molecular genetics will increasingly elucidate the basic defects of the different syndromes and yield more insight into the regulatory mechanisms of embryology. In this way, a reclassification of ectodermal dysplasias will be possible according to the function of their involved mutated genes. Lamartine recently proposed a helpful classification according to the functions of the genes discovered in different types of ectodermal dysplasias. Accordingly, the present overview categorizes the various ectodermal dysplasias into four major functional subgroups: cell-cell communication and signaling, adhesion, transcription regulation, and development.

Cell Adhesion↗
PubMed2002

Hyaluronan is an abundant constituent of the extracellular matrix of Xenopus embryos.

The spatiotemporal distribution of hyaluronan (HA), a major constituent of the vertebrate extracellular matrix, was analyzed during early embryonic development of Xenopus laevis. This polysaccharide is abundantly present in ventricular structures such as the blastocoel, the archenteron as well as later on in the hepatic cavity, the brain ventricles and the developing heart. At the blastula stage, HA was detected in the extracellular matrix of the ecto- and mesodermal primordia. Shortly before gastrulation, it becomes enriched at the basal site of the superficial cell layer of the ectoderm. During gastrulation, enhanced synthesis of HA takes place in the involuting marginal zone, shortly before invagination starts, hence, resulting in a torus-like deposition in the deep layer of the equatorial mesodermal primordium. After gastrulation, HA appears to accumulate within the extracellular matrix demarcating the primary germ layers. During tailbud stages, it is found highly enriched in many mesodermal derivatives, e.g., in mesenchyme, the heart, precordal cartilage and the lung primordia. Furthermore, extracellular matrix of the ventral mesodermal cell layer in the trunk region and the immediate proximity of blood vessels contain high amounts of HA.

Animals↗
PubMed1993

Analysis of the gooseberry locus in Drosophila embryos: gooseberry determines the cuticular pattern and activates gooseberry neuro.

The segment-polarity class of segmentation genes in Drosophila are primarily involved in the specification of sub-segmental units. In addition, some of the segment-polarity genes have been shown to specify cell fates within the central nervous system. One of these loci, gooseberry, consists of two divergently transcribed genes, gooseberry and gooseberry neuro, which share a paired box as well as a paired-type homebox. Here, the expression patterns of the two gooseberry gene products are described in detail. The gooseberry protein appears in a characteristic segment-polarity pattern of stripes at gastrulation and persists until head involution. It is initially restricted to the ectodermal and neuroectodermal germ layer, but is later detected in mesodermal and neuronal cells as well. The gooseberry neuro protein first appears during germ band extension in cells of the central nervous system and also, much later, in epidermal stripes and in a small number of muscle cells. P-element-mediated transformation with the gooseberry gene has been used to demonstrate that gooseberry transactivates gooseberry neuro and is sufficient to rescue the gooseberry cuticular phenotype in the absence of gooseberry neuro.

Animals↗
PubMed1992

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.

Animals↗
PubMed1985

Intracranial germ cell tumors: pathobiological and immunohistochemical aspects of 70 cases.

Seventy cases of histologically verified intracranial germ cell tumor were reviewed: 43 germinomas, 16 immature teratomas, seven mature teratomas, two embryonal carcinomas, one choriocarcinoma, and one yolk sac tumor. The male-to-female ratio was 2.6:1. The average age was 19 years in patients with germinoma, 11 years in patients with immature teratoma, and 17 years in patients with mature teratoma. Duration of symptoms averaged 19 months for germinoma, three months for immature teratoma, and 11 months for mature teratoma. Sixty-six lesions were located in the midline. Fifty-eight percent of the germinomas arose anterior to the pineal gland, whereas 29% of the immature and 14% of the mature teratomas were located anteriorly. The histologic appearance of the germinomas was indistinguishable from that of the usual testicular seminoma. The immature teratomas contained tissue from all three germ layers and exhibited morphologic features of fetal tissue. Of 14 immature teratomas, seven contained, in addition, foci of other malignant germ cell elements; thus, there were two teratocarcinomas, two lesions with germinoma and immature teratoma, two lesions with extensive rhabdomyoblastic differentiation in an immature teratoma, and one lesion with both germinoma and embryonal carcinoma in addition to immature teratoma. The seven mature teratomas consisted of fully differentiated epithelial and mesenchymal tissues. In 23 cases, immunoperoxidase stains for human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), and carcino-embryonic antigen (CEA) revealed patterns which, with minor exceptions, were essentially identical to those found in genital germ cell lesions. Survival was longest for patients with germinomas. In classifying germ cell tumors of the central nervous system, the World Health Organization's (WHO) classification of testicular germ cell tumors is preferable to its present classification of intracranial germ cell tumors.

Adolescent↗
PubMed1992

Platelet-derived growth factor-A and its receptor are expressed in separate, but adjacent cell layers of the mouse embryo.

The localized developmental expression of murine platelet-derived growth factor A (PDGF-A) was compared to that of its receptor (Pdgfra). Our in situ hybridization study included germ layers of primitive streak embryos, early axial structures (dermatome, myotome, sclerotome, floor plate), the skin and some of its derivatives (hair and mammary gland), the developing forelimb, the branchial arches and various sense organs (otic vesicle, olfactory epithelium and the eye). We report that PDGF-A and Pdgfra are expressed in separate, but adjacent cell layers in these structures and that in most, the ligand is expressed in the epithelium, whereas the receptor in the mesenchyme. This localization corresponds to classical experimental evidence for developmental interactions across cell layers. We suggest that the spatio-temporal regulation of PDGF-A and Pdgfra, and other related systems, represents one model for the spatial regulation of receptor-ligand interactions.

Animals↗
PubMed2004

Investigating the origins of triploblasty: 'mesodermal' gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa).

Mesoderm played a crucial role in the radiation of the triploblastic Bilateria, permitting the evolution of larger and more complex body plans than in the diploblastic, non-bilaterian animals. The sea anemone Nematostella is a non-bilaterian animal, a member of the phylum Cnidaria. The phylum Cnidaria (sea anemones, corals, hydras and jellyfish) is the likely sister group of the triploblastic Bilateria. Cnidarians are generally regarded as diploblastic animals, possessing endoderm and ectoderm, but lacking mesoderm. To investigate the origin of triploblasty, we studied the developmental expression of seven genes from Nematostella whose bilaterian homologs are implicated in mesodermal specification and the differentiation of mesodermal cell types (twist, snailA, snailB, forkhead, mef2, a GATA transcription factor and a LIM transcription factor). Except for mef2, the expression of these genes is largely restricted to the endodermal layer, the gastrodermis. mef2 is restricted to the ectoderm. The temporal and spatial expression of these 'mesoderm' genes suggests that they may play a role in germ layer specification. Furthermore, the predominantly endodermal expression of these genes reinforces the hypothesis that the mesoderm and endoderm of triploblastic animals could be derived from the endoderm of a diploblastic ancestor. Alternatively, we consider the possibility that the diploblastic condition of cnidarians is a secondary simplification, derived from an ancestral condition of triploblasty.

Amino Acid Sequence↗
PubMed1997

Patterns of cell death during gastrulation in chick and mouse embryos.

We have examined the distribution of cells at an early stage of the cell death process in gastrulating chick and mouse embryos, using a DNA nick end-labelling technique to label nuclei that are undergoing DNA fragmentation in situ. In the chick embryo, the incidence of nuclei showing DNA fragmentation was mapped by digitizing the occurrence of these nuclei from sections, and reconstructing the three separate layers of the entire embryo at several stages of gastrulation. In the chick, DNA fragmentation was found in nuclei throughout the embryo, in cells of all three germ layers, but most especially in the epiblast in the rostral germinal crescent and in the lateral marginal zones. This region of greatest cell death formed an arc rostrally and laterally in the epiblast, and was consistent through gastrulation and into the early neurulation stage. While the extensive cell death in the chick embryo may be due to cell redundancy, it is also possible that the pattern of death observed could be related to the compression of the embryo against the barrier of yolk at the periphery of the area pellucida during expansion. In a number of cases in the chick, local regions of elevated cell death were also observed in the primitive streak. This may be associated with the changing cell-cell and cell-matrix interactions experienced by cells traversing the primitive streak. In the gastrulating mouse embryo, by contrast, nuclei undergoing DNA fragmentation showed no consistent regions of elevated incidence, in any of the embryonic layers. DNA fragmentation in these embryos was, however, observed in nuclei of cells in the visceral endoderm and in the epiblast. The lack of any clear pattern of DNA fragmentation in the mouse embryo at this stage of development leaves the roles of the dying cells enigmatic. The death may, however, be lineage-related or be a reflection of a cellular redundancy necessary in a developing system that is undergoing extensive cell rearrangement and cellular adhesive change.

Animals↗
PubMed2001

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Animals↗
PubMed2005

The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations.

We present a detailed analysis of the cell lineage of the tardigrade Thulinia stephaniae with a 4D-microscopy system (3D time-lapse recording). The recording, of the entire development from embryogenesis until hatching, allowed us to analyze the fate of single descendants from early blastomeres up to germ layer formation and tissue development. The embryo undergoes an irregular indeterminate cleavage pattern without early fate restriction. During gastrulation, mesodermal and endodermal precursors, and a pair of primordial germ cells migrate through a blastopore at the prospective position of the mouth. Our results are not consistent with earlier descriptions of mesoderm formation by enterocoely in tardigrades. The mesoderm in Thulinia stephaniae originates from a variable number of blastomeres, which form mesodermal bands that later produce the serial somites. The nervous system is formed by neural progenitor cells, which delaminate from the neurogenic ectoderm. Early embryogenesis of Thulinia stephaniae is highly regulative, even after laser ablations of blastomeres at the two- and four-cell stages 'normal' juveniles are formed. This has never been observed before for a protostome. Germ cell specification occurs late during development between the sixth and seventh cell generation. Comparing the development of other protostomes with that of the Tardigrada, which occupy a basal position within the Arthropoda, suggests that an indeterminate cleavage and regulatory development is not only part of the ground pattern of the Arthropoda, but probably of the entire Ecdysozoa.

Animals↗
PubMed2006

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells.

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.

Amino Acid Sequence↗
PubMed2006

August Rauber (1841-1917): from the primitive streak to Cellularmechanik.

In the early 19th century Karl Ernst von Baer initiated a new research program searching for the mechanisms by which an egg transforms itself into an embryo. August Rauber (1841-1917) took up this challenge. He considered the phylogenetic principle as the right tool to explain the similitude of embryogenetic processes. In extending Baer's approach, he combined comparative embryology and histology in his studies of avian and mammalian embryos. His earlier work demonstrated that the two-layered chick embryo is a modified gastrula and not a "disc" as Wilhelm His had claimed. From the 1880s onwards, he concentrated on the issue of how the development of germ layers is related to tissue differentiation. To address this, he studied the blastopore, epiblast, primitive streak, teratology and the relative importance of nucleus and cytoplasm in heredity. This paper reconstructs some of Rauber's work and concludes that his observations and reflections constituted a new approach combining embryology and histology with "phylogenetic" reasoning.

Animals↗
PubMed1998

Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V.

This study describes the use of biotinylated annexin V for the histochemical detection of apoptotic cells in cultured chicken embryos during gastrulation. This method is based on the Ca2+-dependent binding of annexin V to phosphatidylserine, a negatively charged phospholipid, located at the inner leaflet of the cell membrane in living cells. However, in the early stages of apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane and can then be recognized by annexin V. Applying this method in cultured chicken embryos during gastrulation, we obtained labelling of apoptotic cells in the three germ layers. In the epiblast and mesoblast, labelling was predominantly present in the region lateral to the primitive streak. At the level of the germinal crescent, labelled cells were also found in the epiblast. Labelled cells in the deep layer, which is a heterogeneous tissue layer composed of endophyll, sickle endoblast and definitive endoblast, were rather scarce. The distribution of cells, as observed in this study after labelling with annexin V in light microscopy and confocal laser scanning microscopy, is consistent with distributions reported by other authors using other approaches and with our previous observations made with the TUNEL technique and by electron microscopy after fixation in a tannic acid-based fixative. The main advantages of this method over other more sophisticated methods is its easiness and rapidity of execution and the fact that both early and late stages of apoptosis are detected.

Animals↗
PubMed2005

In vitro neuronal differentiation of cultured human embryonic germ cells.

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.

Biomarkers↗
PubMed2004

Derivation of a diploid human embryonic stem cell line from a mononuclear zygote.

BACKGROUND: IVF occasionally produces aneuploid zygotes with one or three pronuclei (PN). Routinely, these zygotes are discarded. The aim of this work was to establish human embryonic stem cell (hESC) lines from blastocysts resulting from abnormal fertilization. METHODS: Abnormally fertilized zygotes were cultured to the blastocyst stage and, following zona pellucida digestion, zona-free blastocysts were placed on a mouse feeder layer. Culture of hESCs was carried out as described earlier. RESULTS: Six out of the nine developing blastocysts attached to the feeder layer. One hESC line, originating from a mononuclear zygote following ICSI, was successfully derived. This line displayed typical phenotype and embryonic surface markers, and exhibited the potential to develop into all three embryonic germ layers both in vitro (by embryoid body formation) and in vivo (teratoma generation). Genetic examination revealed normal diploid karyotype and heterozygotic appearance for metachromatic leukodystrophy (MLD). CONCLUSION: This method, which requires neither immuno nor mechanical removal of the trophectoderm, may facilitate the derivation of hESC lines in general, and those from abnormal embryos in particular. Furthermore, it is shown that aneuploid zygotes can be used as a source for normal hESC derivation and hold the potential to generate aneuploid hESC lines for research purposes.

Animals↗