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Anna M Wobus

Publications and source records attributed to Anna M Wobus.

At least 19 recordsLinked to original sources
PubMed2006

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro.

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.

Animals↗
PubMed2006

Insulin-producing cells.

Embryonic stem (ES) cells offer great potential for cell replacement and tissue engineering therapies because of their almost unlimited proliferation capacity and the potential to differentiate into cellular derivatives of all three primary germ layers. This chapter describes a strategy for the in vitro differentiation of mouse ES cells into insulin-producing cells. The three-step protocol does not select for nestin-expressing cells as performed in previous differentiation systems. It includes (1) the spontaneous differentiation of ES cells via embryoid bodies and (2) the formation of progenitor cells of all three primary germ layers (multilineage progenitors) followed by (3) directed differentiation into the pancreatic lineage. The application of growth and extracellular matrix factors, including laminin, nicotinamide, and insulin, leads to the development of committed pancreatic progenitors, which subsequently differentiate into islet-like clusters that release insulin in response to glucose. During differentiation, transcript levels of pancreas-specific transcription factors (i.e., Pdx1, Pax4) and of genes specific for early and mature beta cells, including insulin, islet amyloid pancreatic peptide, somatostatin, and glucagon, are upregulated. C-peptide/insulin-positive islet-like clusters are formed, which release insulin in response to high glucose concentrations at terminal stages. The differentiated cells reveal functional properties with respect to voltage-activated Na+ and ATP-modulated K+ channels and normalize blood glucose levels in streptozotocin-treated diabetic mice. In conclusion, we demonstrate the efficient differentiation of murine ES cells into insulin-producing cells, which may help in the future to establish ES cell-based therapies in diabetes mellitus.

Animals↗
PubMed2006

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation.

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.

Animals↗
PubMed2006

Pluripotency: capacity for in vitro differentiation of undifferentiated embryonic stem cells.

Embryonic stem (ES) cells, the pluripotent cells of early embryos have been successfully cultured as undifferentiated cells. The cells are characterized by two unique properties, unlimited self-renewal capacity and the ability to differentiate into all cells of the body. Because of the high in vitro differentiation potential, ES cells have been used as model system in cell and developmental biology. Here we present methods that use mouse embryonic stem cells for the in vitro differentiation and characterization of neuronal, cardiac, pancreatic and hepatic cells, derivatives of the ectoderm, mesoderm and endoderm, respectively. In the future, differentiated cells may be also generated from human ES cells by cultivation of early embryos or from reprogrammed cells derived by nuclear transfer. Such cells could represent potential sources for tissue repair of serious human diseases.

Animals↗
PubMed2006

In vitro differentiation of embryonic stem cells into the pancreatic lineage.

Embryonic stem (ES) cells represent a potential source of transplantable cells for the treatment of diabetes because of their almost unlimited proliferation capacity and the potential to differentiate into insulin-producing cells. The differentiation conditions significantly affect the development of pancreatic cells from ES cells in vitro. Here, we describe an efficient strategy for the in vitro generation of insulin-producing cells. The protocol includes the spontaneous generation of multilineage progenitor cells and their differentiation induction by growth and extracellular matrix factors into C-peptide/insulin-positive islet-like clusters. The differentiated cells release insulin in response to high glucose concentrations. No specific selection for nestin-positive cells is performed at any time of ES cell differentiation.

Animals↗
PubMed2005

Electromagnetic fields affect transcript levels of apoptosis-related genes in embryonic stem cell-derived neural progenitor cells.

Mouse embryonic stem (ES) cells were used as an experimental model to study the effects of electromagnetic fields (EMF). ES-derived nestin-positive neural progenitor cells were exposed to extremely low frequency EMF simulating power line magnetic fields at 50 Hz (ELF-EMF) and to radiofrequency EMF simulating the Global System for Mobile Communication (GSM) signals at 1.71 GHz (RF-EMF). Following EMF exposure, cells were analyzed for transcript levels of cell cycle regulatory, apoptosis-related, and neural-specific genes and proteins; changes in proliferation; apoptosis; and cytogenetic effects. Quantitative RT-PCR analysis revealed that ELF-EMF exposure to ES-derived neural cells significantly affected transcript levels of the apoptosis-related bcl-2, bax, and cell cycle regulatory "growth arrest DNA damage inducible" GADD45 genes, whereas mRNA levels of neural-specific genes were not affected. RF-EMF exposure of neural progenitor cells resulted in down-regulation of neural-specific Nurr1 and in up-regulation of bax and GADD45 mRNA levels. Short-term RF-EMF exposure for 6 h, but not for 48 h, resulted in a low and transient increase of DNA double-strand breaks. No effects of ELF- and RF-EMF on mitochondrial function, nuclear apoptosis, cell proliferation, and chromosomal alterations were observed. We may conclude that EMF exposure of ES-derived neural progenitor cells transiently affects the transcript level of genes related to apoptosis and cell cycle control. However, these responses are not associated with detectable changes of cell physiology, suggesting compensatory mechanisms at the translational and posttranslational level.

Apoptosis↗
PubMed2005

Embryonic stem cells: prospects for developmental biology and cell therapy.

Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.

Animals↗
PubMed2005

Loss of beta 1 integrin function results in upregulation of connexin expression in embryonic stem cell-derived cardiomyocytes.

We show that loss of beta1 integrin function affects connexin (Cx) expression in embryonic stem (ES) cell-derived cardiomyocytes. Both loss of beta1 integrin function and inhibition of integrin clustering by RGD peptides in wild type ES cells correlated with upregulated expression of gap junctional proteins in ES-derived cardiomyocytes. The upregulation of connexin transcript levels in beta1 integrin-deficient cells is paralleled by a higher fraction of cells co-expressing Cx40 and Cx43. These observations demonstrate that the expression of connexins in developing cardiomyocytes is correlated to integrin-dependent mechanisms. Further, we found that upregulated cardiac connexin expression in beta1 integrin-deficient cells is related to Rho- and Wnt-dependent pathways. beta1 integrin-deficient cardiac cells displayed high levels of cytoplasmic gamma- and beta-catenins throughout the differentiation period. The administration of lithium, an activator of beta-catenin-dependent pathways, resulted in up-regulated connexin mRNA levels in wild type cardiomyocytes at intermediate, but not at early stages, indicating that the effects are restricted to advanced stages of cardiac differentiation. On the other hand, inhibition of Rho-dependent integrin signaling by treatment of cardiac cells with exoenzyme C3 resulted in immediate up-regulation of cardiac connexin transcript levels at early differentiation stages. Our data indicate that integrin function affects connexin expression in cardiomyocytes via direct effects on RhoA-dependent signaling mechanisms at early differentiation stages and of beta-catenin/Wnt-dependent pathways at advanced stages.

Animals↗
PubMed2004

Stem cells and pancreatic differentiation in vitro.

Cell therapy using pancreatic islets would be a promising therapy to treat diabetes. But, because of the limited supply of human donor islets, other cellular sources have to be considered. Stem cells characterized by extensive proliferation and differentiation capacity may be a valuable source for the in vitro generation of islets. Insulin-producing cells derived from embryonic stem (ES) cells have been shown to reverse experimentally induced diabetes in animal models. However, the oncogenic properties of ES cells are critical in the context of clinical applications and efficient cell-lineage selection systems need to be established. Future studies have to demonstrate whether somatic stem cells residing in adult tissues, such as bone marrow, pancreatic ducts, intestine or liver may provide alternatives to generate functional pancreatic endocrine cells.

Animals↗
PubMed2004

Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects.

Primary cultures or established cell lines of vertebrates are commonly used to analyse the cytotoxic potential of chemical factors, drugs and xenobiotics in vitro. An alternative approach will be provided by permanent lines of pluripotent embryonic stem (ES) cells, which are able to differentiate into specialised somatic cell types in vitro. Here, we demonstrate the capacity of ES cells to generate functional cardiac, neuronal and pancreatic cells. We show that during ES cell differentiation, tissue-specific genes, proteins as well as functional properties are expressed in a developmentally regulated manner recapitulating processes of early embryonic development. We present data that show the use of ES-derived cardiomyocytes and dopaminergic neurons in toxicological studies and the potential of ES-derived pancreatic beta-like cells in future in vitro assays. The application of these differentiation systems to human ES cells opens up new perspectives in basic and applied toxicology.

Cell Differentiation↗
PubMed2004

Non-thermal effects of power-line magnetic fields (50 Hz) on gene expression levels of pluripotent embryonic stem cells-the role of tumour suppressor p53.

The diffusion of extremely low-frequency (50 Hz) electromagnetic fields (ELF-EMF) in the human environment raises the question of the induction of biological effects of EMF on mammalian cells. We used the model of mouse pluripotent embryonic stem (ES) cells, which have the capacity to develop in vitro into cells of all lineages, to analyse non-thermal effects of ELF-EMF. Wild type (wt) and p53-deficient ES cells were exposed under controlled conditions to ELF-EMF signals simulating power-line (50 Hz) magnetic field (PL-MF) exposure. Different flux densities of 0.1 mT, 1.0 mT or 2.3 mT and intermittency schemes with various ON/OFF cycles were applied for 6 h or 48 h during the first stages of cell differentiation. Transcript levels of regulatory genes, such as egr-1, p21, c-jun, c-myc, hsp70 and bcl-2, were analysed by semi-quantitative RT-PCR immediately after exposure or after a recovery time of 18 h. Intermittent PL-MF exposure to 5 min ON/30 min OFF cycles at a flux density of 2.3 mT for 6 h resulted in a significant up-regulation of c-jun, p21 and egr-1 mRNA levels in p53-deficient, but not in wild-type cells. No significant effects were observed in both cell systems by PL-MF at lower flux densities, longer exposure time or after 18 h recovery time. Our data indicate that 5 min ON/30 min OFF intermittent PL-MF exposure is capable of evoking non-thermal responses in ES cells, dependent on the cellular p53 function. The nature of the biological responses triggered by PL-MF is discussed.

Animals↗
PubMed2004

High frequency electromagnetic fields (GSM signals) affect gene expression levels in tumor suppressor p53-deficient embryonic stem cells.

Effects of electromagnetic fields (EMF) simulating exposure to the Global System for Mobile Communications (GSM) signals were studied using pluripotent embryonic stem (ES) cells in vitro. Wild-type ES cells and ES cells deficient for the tumor suppressor p53 were exposed to pulse modulated EMF at 1.71 GHz, lower end of the uplink band of GSM 1800, under standardized and controlled conditions, and transcripts of regulatory genes were analyzed during in vitro differentiation. Two dominant GSM modulation schemes (GSM-217 and GSM-Talk), which generate temporal changes between GSM-Basic (active during talking phases) and GSM-DTX (active during listening phases thus simulating a typical conversation), were applied to the cells at and below the basic safety limits for local exposures as defined for the general public by the International Commission on Nonionizing Radiation Protection (ICNIRP). GSM-217 EMF induced a significant upregulation of mRNA levels of the heat shock protein, hsp70 of p53-deficient ES cells differentiating in vitro, paralleled by a low and transient increase of c-jun, c-myc, and p21 levels in p53-deficient, but not in wild-type cells. No responses were observed in either cell type after EMF exposure to GSM-Talk applied at similar slot-averaged specific absorption rates (SAR), but at lower time-averaged SAR values. Cardiac differentiation and cell cycle characteristics were not affected in embryonic stem and embryonic carcinoma cells after exposure to GSM-217 EMF signals. Our data indicate that the genetic background determines cellular responses to GSM modulated EMF. Bioelectromagnetics 25:296-307, 2004.

Animals↗
PubMed2004

Embryonic stem cells differentiate into insulin-producing cells without selection of nestin-expressing cells.

We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-producing cells via a multi-step process without selection and induction of nestin-positive cells. During ES cell differentiation, transcript levels of genes characteristic of early and mature beta cells including Pdx1, Pax4, insulin and islet amyloid pancreatic peptide are up regulated. Islet-like clusters are characterized by expression of C-peptide, insulin and partially cytokeratin 19 as well as by ion channel activity similar to that found in embryonic beta cells. Cells of islet-like clusters show glucose-dependent insulin release at terminal stage. At an intermediate stage, nestin is partially co-expressed with C-peptide and cytokeratin 19, whereas islet-like clusters at the terminal stage are nestin-negative. We conclude that expression of nestin and cytokeratin 19 is a normal property of ES cells preceding differentiation into C-peptide/insulin-producing cells without any selection for nestin-positive phenotypes.

Animals↗
PubMed2004

The generation of insulin-producing cells from embryonic stem cells--a discussion of controversial findings.

The derivation of insulin-producing cells from embryonic stem (ES) cells has been controversially described. Whereas several authors showed successful differentiation of mouse ES cells into islet-like clusters, others could not confirm the results. Here, we present a detailed comparison of the various strategies used to generate pancreatic cells with respect to protocols and differentiation factors and give an explanation of the contradictory findings. It is suggested that the selection or enrichment of ES-derived nestin-positive cells should be avoided, since these cells are already committed to a neural fate before pancreatic differentiation is induced.

Animals↗
PubMed2004

Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes.

To use embryonic stem (ES) cells in future therapeutical applications, differentiated hepatic phenotypes with specific liver functions would be necessary. We analyzed albumin (ALB), alpha-fetoprotein (AFP) and hepatic transcription factor (TF) gene expression in tissues derived from embryonic, fetal and adult liver, and compared the gene expression profiles with those from mouse ES cells after hepatic differentiation and from cultured adult hepatocytes. The mRNA expression of hepatocyte nuclear factor (HNF)-1alpha,beta, -3alpha,beta, -4alpha, -6, CCAAT/enhancer binding protein (C/EBP) alpha,beta, ALB and AFP relative to glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) were studied by "real time" RT-PCR. ALBand AFP-expression was also determined by in situ hybridization (tissue) and immunofluorescence (ES-derived cells after hepatic differentiation, ES-HPC). Peak levels for HNF-1alpha, -3alpha, -4alpha and -6 were detected in early liver development at d9.5 and d11.5. C/EBPalpha and beta were most abundantly expressed in adult liver. ALB mRNA increased steadily from d10.5 on and was maximally present in adult liver. AFP was present at d9.5, peaked at d15.5 and dramatically declined in mature liver tissue. Based on immunofluorescence, ALB and AFP were expressed in approximately 20% of ES-HPC. While expression of HNF-3, 4 and 6 reached levels similar to adult hepatocytes, ALB and AFP expression was several orders of magnitude lowerthan in adult tissue or cells. Stages of liver organogenesis are characterized by specific expression patterns of developmentally regulated genes. With sophisticated differentiation protocols, hepatic gene expression can be induced in a proportion of ES cells with gene expression patterns similar to early fetal liver.

Albumins↗
PubMed2004

Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells.

We present a novel two-step protocol for the differentiation of embryonic stem (ES) cells into the hepatic lineage. Differentiated hepatocyte-like cells express genes and proteins characteristic for endodermal and hepatic cells and acquire a functional hepatic phenotype as demonstrated by albumin secretion and glycogen storage. During differentiation, alpha-fetoprotein, albumin, transthyretin, alpha-1-antitrypsin, cytochrome P450 subunits 2b9 and 2b13 and tyrosine aminotransferase transcripts are upregulated. Quantitative RT-PCR data revealed a fetal hepatic phenotype corresponding to day 13-14 of liver development. Terminally differentiated hepatocyte-like cells show a bi-nucleated, cuboidal morphology labeled by albumin, alpha-1-antitrypsin, liver amylase, dipeptidyl peptidase IV, c-met and cytokeratin 18. ES-derived intermediate cell types transiently and partially co-express nestin with albumin and alpha-fetoprotein, respectively, but not cytokeratin 19. This finding suggests an ES-derived potential hepatic progenitor cell type, which is partially nestin-, albumin- and alpha-fetoprotein-positive, but cytokeratin 19-negative.

Albumins↗
PubMed2003

Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells.

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4(+)), and to a lesser extent Pdx1 (Pdx1(+)), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult beta cells. In response to glucose, Pax4(+) and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

Animals↗