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PubMed2003

Exploitation of stem cell plasticity.

For many years, adult haemopoietic stem cells (HSCs) have been considered 'plastic' in their proliferative and differentiation capacities. Recently, evidence that supports newer concepts of adult stem cell plasticity has been reported. In particular, stem cells from haemopoietic tissues seem to have 'extraordinary' abilities to generate or switch between haemopoietic and nonhaemopoietic lineages, exhibiting an unexpected degree of developmental or differentiation potential. The mechanisms by which cell fate reprogramming occurs are still poorly understood. Nevertheless, an increasing number of studies is challenging one of the main dogmas in biology, namely that mammalian cell differentiation follows established programmes in a hierarchical fashion, and once committed to a particular somatic cell lineage, cells do not change into another somatic lineage. The 'nonhierarchical', 'reversible' phenotype of stem cells in haemopoietic tissues, if it exists, would be an advantage that could be exploited in regenerative medicine. Here, we review the recent advances in HSC biology and discuss the general concepts of adult stem cell plasticity with respect to these cells and how these might be exploited clinically.

Adult↗
PubMed2005

Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion.

Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell-cell fusion. Using an OP9 coculture system, we produced CD45+ CD33+ myeloperoxidase+ myeloid precursors from an Oct4-enhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.

Animals↗
PubMed2002

Amelogenin gene expression in porcine odontoblasts.

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.

Ameloblasts↗
PubMed2004

Cryopreservation of human embryonic stem cells by vitrification.

BACKGROUND: The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method. METHODS: Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified. RESULTS: Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers. CONCLUSIONS: Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

Cell Differentiation↗
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

Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors.

The primitive ectoderm of the mouse embryo arises from the inner cell mass between 4.75 and 5.25 days post coitum, around the time of implantation. Positioned at a pivotal time in development, just prior to formation of the three germ layers of the embryo proper, the primitive ectoderm responds directly to the signals generated during gastrulation. We have identified a conditioned medium, MEDII, which caused the homogeneous conversion of ES cells to a morphologically distinct cell population, termed early primitive ectoderm-like (EPL) cells. EPL cells expressed the pluripotent cell markers Oct4, SSEA1 and alkaline phosphatase. However, the formation of EPL cells was accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chimaera forming ability, changes in factor responsiveness and modified differentiation capabilities, all consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post coitum embryo. EPL cell formation could be reversed in the presence of LIF and withdrawal of MEDII, which suggested that EPL cell formation was not a terminal differentiation event but reflected the ability of pluripotent cells to adopt distinct cell states in response to specific factors. Partial purification of MEDII revealed the presence of two separable biological activities, both of which were required for the induction and maintenance of EPL cells. We show here the first demonstration of uniform differentiation of ES cells in response to biological factors. The formation of primitive ectoderm, both in vivo and in vitro, appears to be an obligatory step in the differentiation of the inner cell mass or ES cells into cell lineages of the embryonic germ layers. EPL cells potentially represent a model for the development of lineage specific differentiation protocols and analysis of gastrulation at a molecular level. An understanding of the active components of MEDII may provide a route for the identification of factors which induce primitive ectoderm formation in vivo.

Animals↗
PubMed1991

Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat.

Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13-17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149-173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1-148 of the antigen. Immunoelectro-transfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13-17 kDa nonamelogenins. The antiserum against the 13-17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13-17 kDa nonamelogenins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amelogenin↗
PubMed2003

Origin of feathers: Feather beta (beta) keratins are expressed in discrete epidermal cell populations of embryonic scutate scales.

The feathers of birds develop from embryonic epidermal lineages that differentiate during outgrowth of the feather germ. Independent cell populations also form an embryonic epidermis on scutate scales, which consists of peridermal layers, a subperiderm, and an alpha stratum. Using an antiserum (anti-FbetaK) developed to react specifically with the beta (beta) keratins of feathers, we find that the feather-type beta keratins are expressed in the subperiderm cells of embryonic scutate scales, as well as the barb ridge lineages of the feather. However, unlike the subperiderm of scales, which is lost at hatching, the cells of barb ridges, in conjunction with adjacent cell populations, give rise to the structural elements of the feather. The observation that an embryonic epidermis, consisting of peridermal and subperidermal layers, also characterizes alligator scales (Thompson, 2001. J Anat 198:265-282) suggests that the epidermal populations of the scales and feathers of avian embryos are homologous with those forming the embryonic epidermis of alligators. While the embryonic epidermal populations of archosaurian scales are discarded at hatching, those of the feather germ differentiate into the periderm, sheath, barb ridges, axial plates, barbules, and marginal plates of the embryonic feather filament. We propose that the development of the embryonic feather filament provides a model for the evolution of the first protofeather. Furthermore, we hypothesize that invagination of the epidermal lineages of the feather filament, namely the barb ridges, initiated the formation of the follicle, which then allowed continuous renewal of the feather epidermal lineages, and the evolution of diverse feather forms.

Amino Acid Sequence↗
PubMed2004

Repression of nodal expression by maternal B1-type SOXs regulates germ layer formation in Xenopus and zebrafish.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

Amino Acid Sequence↗
PubMed2005

Differentiation pathways in human embryonic stem cell-derived cardiomyocytes.

Human embryonic stem (hES) cells are pluripotent cell lines derived from the inner cell mass of the blastocyst-stage embryo. These unique cell lines can be propagated in the undifferentiated state in culture, while retaining the capacity to differentiate into derivatives of all three germ layers, including cardiomyocytes. The derivation of the hES cell lines presents a powerful tool to explore the early events of cardiac progenitor cell specification and differentiation, and it also provides a novel cell source for the emerging field of cardiovascular regenerative medicine. A spontaneous differentiation system of these stem cells to cardiomyocytes was established and the generated myocytes displayed molecular, structural, and functional properties of early-stage heart cells. In order to follow the in vitro differentiation process, the temporal expression of signaling molecules and transcription factors governing early cardiac differentiation was examined throughout the process. A characteristic pattern was noted recapitulating the normal in vivo cardiac differentiation scheme observed in other model systems. This review discusses the known pathways involved in cardiac specification and the possible factors that may be used to enhance cardiac differentiation of hES cells, as well as the steps required to fully harness the enormous potential of these unique cells.

Animals↗
PubMed1985

Regional specificity of glycoconjugates in Xenopus and axolotl embryos.

This paper reviews work on the presence, synthesis and developmental regulation of glycoconjugates (proteoglycans, glycoproteins and glycolipids) in the early amphibian embryo. In the axolotl there is little regional specificity of protein synthesis until the tailbud stage, but substantial regional specificity of glycoprotein synthesis from the end of gastrulation. Glycolipid synthesis is more uniform although a number of unique species are made in the epidermis. Isolated explants from axolotl early gastrulae show three types of behaviour in terms of glycoprotein synthesis, corresponding to the classical germ layers. Xenopus embryos at this stage show a higher degree of mosaicism. Changes of glycoprotein synthesis in response to mesodermal or neural induction follow the predicted course depending on the regional character of the induced tissue. The regional binding patterns of a number of lectins and monoclonal antibodies specific for particular carbohydrate determinants are presented and their significance discussed.

Ambystoma↗
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↗
PubMed2002

Microbial-host interactions specifically control the glycosylation pattern in intestinal mouse mucosa.

The glycosylation of the intestinal cell layer is thought to control several key functions of the gut such as vectorial transports, defence against microbial agents or immunological processes. It has been assumed that the gut microflora may modulate the glycosylation pattern of the intestinal cell layer. However, there is no direct evidence for this regulatory process. The first goal of this work was to establish the germ-free mice intestinal glycosylation baseline using a histochemical approach and a panel of ten lectins with defined glycan specificities to tissue sections prepared from various cellular compartments of the small and large intestine. Using this baseline, we have studied the contribution of the gut microflora on the carbohydrate composition of glycoconjugates of intestinal cells by comparing the germ-free and conventional mice glycosylation patterns. Analysis of the germ-free mice intestinal glycosylation baseline revealed that the expression of glycans depends on the proximodistal gradient (small to large intestine) and on the cell lineage (absorptive, goblet, crypt, and Paneth cells), indicating that mice are able to create and maintain a strict topological and cell lineage-specific regulation of glycosyltransferase expression. By comparing germ-free and conventional mice, we find that the gut microflora specifically modulates the gut glycosylation pattern, quantitatively as well as qualitatively by changing the cellular and subcellular distribution of glycans. This is the first report in mice to directly demonstrate the critical contribution of microflora to intestinal glycosylation, a key characteristic of the gut.

Animals↗
PubMed1988

Adherence of Candida albicans germ tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins.

Germ tubes of Candida albicans produced an additional fibrillar surface layer responsible for enhanced adherence to plastic. The correlation between germination of C. albicans and adherence of germ tubes to a plastic matrix led us to consider the existence of germ tube-specific adhesive components involved in the attachment process. Using concanavalin A-sensitized latex microspheres, we first detected extracellular molecules on the plastic surface after removal of the adherent germ tubes. Electron microscopy confirmed that fibrils of the germ tube involved in cell-substratum interconnections were retained on the plastic surface. Cytochemistry with concanavalin A-gold labeling demonstrated that these fibrillar structures contained mannoproteins. Dithiothreitol and iodoacetamide treatment of washed plastic allowed us to further characterize these fibrillar adhesins. Through analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two components with molecular weights (MWs) of 68,000 and 60,000 were detected on the plastic surface. The 68,000-MW component appeared to be one of the major constituents of the germ tube surface layers. Biosynthetic labeling experiments performed with L-[35S]methionine revealed two additional proteins: a high-MW component (greater than 200,000), and a 200,000-MW component. These four proteins, strongly labeled on the plastic surface and on the germ tube cell wall layers, were in contrast slightly labeled or even nonidentified in the culture supernatant, suggesting their involvement in germ tube adherence.

Adhesiveness↗
PubMed1978

A specific dimerization of rabbit beta-globin messenger ribonucleic acid.

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.

Animals↗
PubMed2004

Hematopoietic stem cells convert into liver cells within days without fusion.

Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2-7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.

Animals↗
PubMed1990

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.

Animals↗