Blood–brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug devel- opment for the human brain. Yet providing stem cell–derived... more
Blood–brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug devel- opment for the human brain. Yet providing stem cell–derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vas- cular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/β-catenin signaling and inhibition of the TGF-β pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/β-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell–derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.
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We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central nervous system development in mice and that Rybp null mutant (Rybp−/−) mouse embryonic stem (ES) cells form more progenitors and less... more
We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central nervous system development in mice and that Rybp null mutant (Rybp−/−) mouse embryonic stem (ES) cells form more progenitors and less terminally differentiated neural cells than the wild type cells in vitro. Accelerated progenitor formation coincided with a high level of Pax6 expression in the Rybp−/− neural cultures. Since Pax6 is a retinoic acid (RA) inducible gene, we have analyzed whether altered RA signaling contributes to the accelerated progenitor formation and impaired differentiation ability of the Rybp−/− cells. Results suggested that elevated Pax6 expression was driven by the increased activity of the RA signaling pathway in the Rybp−/− neural cultures. RYBP was able to repress Pax6 through its P1 promoter. The repression was further attenuated when RING1, a core member of ncPRC1s was also present. According to this, RYBP and PAX6 were rarely localized in the same wild type cell...
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The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse... more
The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells. Consistent with this result, rybp homozygous null embryos exhibited lethality at the early postimplantation stage. At this time, Rybp was essential for survival of the embryo, for the establishment of functional extraembryonic structures, and for the execution of full decidualization. Through the use of a chimeric approach, the embryonic lethal phenotype was circumvented and a role for Rybp in central nervous system development was uncovered. Specifically, the presence of Rybp-defi...
Research Interests: Stem Cells, Biology, Immunohistochemistry, Medicine, Transgenic Mice, and 15 moreBiological Sciences, Brain, Molecular and cellular biology, Mutation, Mice, Animals, Cell Death, Central Nervous System, Blastocyst, Neurons, Phenotype, Genotype, Cell Survival, embryo implantation, and Medical and Health Sciences
Research Interests: Engineering, Biology, Cell Adhesion, Cell Biology, Medicine, and 15 moreBiological Sciences, Cell Differentiation, Mice, Animals, Embryonic Stem Cell, Embryonic Stem Cells, Bioreactors, Large Scale, Genetic Markers, ES cell, Gene expression profiling, Cell Size, reverse transcriptase polymerase chain reaction, embryoid bodies, and Cell culture techniques
One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver... more
One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.
Research Interests: Flow Cytometry, Cellular Reprogramming, Cell Differentiation, Humans, Mice, and 15 moreElectroporation, Animals, Heart, Lentivirus, Embryonic Stem Cells, Neurons, Alkaline phosphatase, Clinical Sciences, Genetic Recombination, Cell Proliferation, Induced Pluripotent Stem Cells, Human Fibroblasts, Academic, Biochemistry and cell biology, and fibroblasts
Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for... more
Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in ...
Research Interests: Biology, Induced Pluripotent Stem Cells (I Psc), Immunohistochemistry, Transcription Factors, Theriogenology, and 12 moreBiological Sciences, Cell line, Cell Differentiation, Animals, Embryonic Stem Cells, Behavioral Animal Models, microRNAs, Chimera, Rabbits, Induced Pluripotent Stem Cells, Pluripotent Stem Cells, and Medical and Health Sciences
Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of... more
Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR-outbred, (2) C57BL/6-inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers-e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells.
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Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The... more
Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. Results The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1...
Research Interests: Comparative sequence analysis, Biology, Medicine, Gene expression, Humans, and 15 moreFemale, Animals, Embryonic Stem Cell, Blastocyst, Promoter Analysis, Embryonic Development, Optometry and Ophthalmology, Molecular Marker, Base Sequence, Embryos, Biochemistry and cell biology, Molecular Sequence Data, Inner Cell mass, Medical biochemistry and metabolomics, and Expression pattern
We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In... more
We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In order to investigate the underlying molecular events of this phenotype, we compared the transcriptomic profile of the wild type and Rybp null mutant ES cells and CMCs differentiated from these cell lines. We identified genes related to ion homeostasis, cell adhesion and sarcomeric organization affected in the Rybp null mutant CMCs, by using hierarchical gene clustering and Gene Ontology analysis. We have also demonstrated that the amount of RYBP is drastically reduced in the terminally differentiated wild type CMCs whilst it is broadly expressed in the early phase of differentiation when progenitors form. We also describe that RYBP is important for the proper expression of key cardiac transcription factors including Mesp1, Shh and Mef2c. These findi...
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Separation of germline cells from somatic lineages is one of the earliest decisions of embryogenesis. Genes expressed in germline cells include apoptotic and meiotic factors, which are not transcribed in the soma normally, but a number of... more
Separation of germline cells from somatic lineages is one of the earliest decisions of embryogenesis. Genes expressed in germline cells include apoptotic and meiotic factors, which are not transcribed in the soma normally, but a number of testis-specific genes are active in numerous cancer types. During germ cell development, germ-cell-specific genes can be regulated by specific transcription factors, retinoic acid signaling and multimeric protein complexes. Non-canonical polycomb repressive complexes, like ncPRC1.6, play a critical role in the regulation of the activity of germ-cell-specific genes. RING1 and YY1 binding protein (RYBP) is one of the core members of the ncPRC1.6. Surprisingly, the role of Rybp in germ cell differentiation has not been defined yet. This review is focusing on the possible role of Rybp in this process. By analyzing whole-genome transcriptome alterations of the Rybp-/- embryonic stem (ES) cells and correlating this data with experimentally identified bin...
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Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis... more
Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.
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Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp... more
Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp+/+) andrybp nullmutant (rybp-/-) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found thatrybp nullmutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack ofrybpcoincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes.
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POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting... more
POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting in spectacular segmental transformations due to the ectopic expression of homeotic genes. Homologs of Drosophila PcG genes were also identified in plants and in vertebrates and subsequent experiments revealed the general role of PCG proteins in the maintenance of the repressed state of chromatin through cell divisions. The past decades of gene targeting experiments have allowed us to make significant strides towards understanding how the network of PCG proteins influences multiple aspects of cellular fate determination during development. Being involved in the transmission of specific expression profiles of different cell lineages, PCG proteins were found to control wide spectra of unrelated epigenetic processes in vertebrates, such as stem cell pl...
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Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based... more
Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons. Undifferentiated SB-iPS and ES cells were aggregated into embryoid bodies (EBs) and cultured in neuronal differentiation medium supplemented with 5 μM all-trans retinoic acid. Thereafter, EBs were dissociated and plated to observe further neuronal differentiation. Samples were fixed on days 10 and 14 for immunocytochemistry staining using the NPC markers Pax6 and Nestin and the neuron marker βIII-tubulin/Tuj1. Nestin-labeled cells were analyzed further by flow cytometry. Our resul...
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Hyperthermia is used in the treatment of some human malignancies. Thermotolerance may interfere with the efficacy of hyperthermic treatment, and thermotolerant cells may also display an enhanced resistance to some anticancer drugs. We... more
Hyperthermia is used in the treatment of some human malignancies. Thermotolerance may interfere with the efficacy of hyperthermic treatment, and thermotolerant cells may also display an enhanced resistance to some anticancer drugs. We have earlier isolated stable heat-resistant rat hepatoma variants and examined whether heat resistance influenced the drug sensitivity of the cells. Heat-resistant variants were isolated by ten repeated cycles of heat exposure at 45 degrees C for 80 min. Highly multidrug-resistant variants were isolated by stepwise selection with colchicine. The heat-resistant variants became moderately multidrug resistant. This resistance was further increased by stepwise selection with colchicine (highly multidrug resistant variants). The levels of P-glycoprotein were elevated both in moderately and highly drug resistant variants. Decreased retention of antitumor drugs was observed in the multidrug resistant variants, verapamil increased doxorubicin retention signifi...
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Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis... more
Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.
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The past two decades of gene targeting experiments have allowed us to make significant strides towards understanding how the Myc/Max/Mad network influences multiple aspects of cellular behavior during development. Here we summarize the... more
The past two decades of gene targeting experiments have allowed us to make significant strides towards understanding how the Myc/Max/Mad network influences multiple aspects of cellular behavior during development. Here we summarize the findings obtained from the myc/max/mad knockout mice generated to date, namely those in which the N-myc, c-myc, L-myc, mad1, mxi1, mad3, mnt, or max genes have been targeted. A compilation of lessons we have learned from these myc/max/mad knockout mouse models, and suggestions as to where future efforts could be focused, are also presented.
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Human-induced pluripotent stem cells (iPSC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC would be highly useful for pre-clinical safety testing by autologous transplantation in a... more
Human-induced pluripotent stem cells (iPSC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. However, a major impediment is that currently, continuous expression of reprogramming factors is required to maintain the pluripotent state of porcine iPSC. In the mouse, neural progenitor cells (NPC) have proved to be highly amenable to reprogramming due to their partly stem-like epigenetic state and expression of pluripotency-related genes such as SOX2. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 mL of active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC and pKLF4 (pOSMK); all including 0.25 mL of transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and subcultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC and KLF4, as evaluated by immunocytochemistry and expression of NANOG, SSEA-1 and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The porcine iPSC-like lines could serve as an excellent platform for optimizing culture conditions, which may sustain the pluripotency network in the pig and could be applied for autologous stem cell transplantation in a porcine model for evaluation of safety and efficacy. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 (PartnErS, PIAP-GA-2008-218205; PluriSys, HEALTH-2007-B-223485).
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Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we... more
Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of ...
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Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is... more
Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is still low. To enable a large-scale culture of ES-derived cells, we have tested a scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, bioreactor/STLV (slow turning lateral vessel, Synthecon, Inc., Houston, TX, USA) following inoculation with a single cell suspension of mouse ES cells. Technical parameters for optimal cell expansion and efficient ES cell differentiation were compared, such as ES cell seeding density (3 × 105 and 5 × 105 cells mL-1) into the bioreactor and day of transfer and plating of EB on gelatinated petri dishes (Day 2, Day 3, Day 4, and Day 5). The quantity and quality of EB production including the yield and size of EB, as well as viability and apoptosis of cells, were analyzed. Furthermore,...
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Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety... more
Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycy...
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Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate... more
Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate knockout animal models. The goal of the current work was to close the gap in knowledge with regard to the molecular biological background for rabbit iPS work by isolating the putative pluripotency genes from the rabbit, based on the sequences published for other species. The sequence of known pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Nanog) were analysed and primers designed based on the similarity of sequences. Sequences of each individual rabbit pluripotency gene was compared to other vertebrates (e.g. human, mouse, bovine) phylogenetically. Rabbit ESCs and blastocyst stage embryos were collected from superovulated rabbits to isolate total RNA. Genes of interest were amplified using RT-PCR and electrophoretically separated for cDNA fragment is...
Research Interests: Endocrinology, Genetics, Andrology, Art, Fertility, and 15 moreCell Biology, Cryopreservation, Oogenesis, Embryo, Biological Sciences, Educational, Cloning, Fertilization, Embryonic Development, Folliculogenesis, Embryogenesis, Gametogenesis, Fertilisation, Embryo Development, and Medical and Health Sciences
Research Interests: Computational Biology, Cancer, Cell Cycle, Apoptosis, Cell line, and 15 moreHumans, Mice, Animals, HIstone Deacetylase, Oncogene, Plasmids, Immunoprecipitation, Clinical Sciences, HeLa cells, Amino Acid Sequence, DNA binding proteins, Gene Expression Regulation, Histone deacetylases, Molecular Sequence Data, and Expression pattern
Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation... more
Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte based screening platform is a useful tool for discovery of cytoprotective molecules.
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Background Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF... more
Background Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs. Results To examine the...
Research Interests: Bioinformatics, Genetics, Gene regulation, Life Sciences, Epigenetics, and 15 moreHeat Shock Transcription Factor, Neural stem cell, Cell Differentiation, Biomedical Research, Glial Cell, Chromatin remodeling, Enzyme, Chromatin, Chromatin structure, Embryonic Development, Model System, DNA binding, Gene Targeting, Retinal Development, and Electron Microscope
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We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68. Multidrug-resistant variants were... more
We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68. Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 microgram/ml colchicine (termed as highly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stable heat-resistant variants of clone 2 were also isolated, which showed increased drug resistance to several drugs, i.e. they became moderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heat-resistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against environmental stress such as heat.
Research Interests: Technology, Protein synthesis, Biological Sciences, Multidrug Resistance, Animals, and 14 moreDrug Resistance, P-glycoprotein, Doxorubicin, Colchicine, Rats, Physiological Stress Markers, Heat Shock Protein, Antineoplastic Agents, Glucocorticoids, Vinblastine, Cytotechnology, Verapamil, Stress Physiological, and Medical and Health Sciences
Research Interests: DNA repair, Recombinant DNA Technology, Biological Sciences, Pregnancy, Mutation, and 12 moreMice, Female, Animals, Microbial genetic and drug resistance, Phenotype, Spectrum, Gene Targeting, Base Sequence, Congenital abnormalities, alleles, Psychology and Cognitive Sciences, and Medical and Health Sciences
Background Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that... more
Background Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. Results Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased level...
Research Interests: Genetics, Electron Microscopy, Cornea, Gene expression, Biological Sciences, and 15 moreCell Differentiation, Pregnancy, Mice, Female, Animals, Retina, Transcription Factor, Physical Interaction, Eye, Mouse Model, Embryos, Knockout Mice, Tissue Specificity, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box... more
Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. Results To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses ne...