Showing 49 - 60 of 78 results for "07923"
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- ReferenceTay FC et al. (OCT 2013) Journal of Gene Medicine 15 10 384--395
Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector-mediated zinc finger nuclease expression in human-induced pluripotent stem cells
Background The AAVS1 locus is viewed as a ‘safe harbor' for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright textcopyright 2013 John Wiley & Sons, Ltd. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceAkdemir KC et al. (JAN 2014) Nucleic Acids Research 42 1 205--223
Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells
How tumor suppressor p53 selectively responds to specific signals, especially in normal cells, is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation, induced by retinoic acid, versus DNA damage, caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and, in pluripotent hESCs, are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases, UTX and JMJD3, to chromatin. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceTrilck et al. ( 2013) Orphanet journal of rare diseases 8 144
Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks.
BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceZhu H et al. (OCT 2013) Nucleic Acids Research 41 19 e180
Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells
Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceSun N and Zhao H (MAY 2014) Biotechnology and Bioengineering 111 5 1048--53
Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs.
Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ, patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs), a recently emerged novel genome editing tool, to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon, TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD, which represents a significant advance toward hiPSC-based cell and gene therapies. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 72252 Thiazovivin 85850 mTeSR™1 07920 ACCUTASE™ Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 72252 Product Name: Thiazovivin Catalog #: 85850 Product Name: mTeSR™1 Catalog #: 07920 Product Name: ACCUTASE™ - ReferenceKim J et al. (NOV 2013) Stem Cell Research 11 3 978--989
Alginate microcapsule as a 3D platform for the efficient differentiation of human embryonic stem cells to dopamine neurons
Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction, cell proliferation and differentiation into specific lineages as well as tissue organization, it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers, particularly tyrosine hydroxylase (TH) by textgreater100 folds after 2weeks and at least 50% higher expression after 4weeks respectively, compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH+ cells co-expressed mdDA neuronal markers, forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/106 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages, particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies. ?? 2013 Elsevier B.V. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceLiberski AR et al. (JUL 2013) Journal of Proteome Research 12 7 3233--3245
Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture
Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceAmita M et al. (MAR 2013) Proceedings of the National Academy of Sciences of the United States of America 110 13 E1212--E1221
Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4
Human ES cells (hESC) exposed to bone morphogenic protein 4 (BMP4) in the absence of FGF2 have become widely used for studying trophoblast development, but the soundness of this model has been challenged by others, who concluded that differentiation was primarily toward mesoderm rather than trophoblast. Here we confirm that hESC grown under the standard conditions on a medium conditioned by mouse embryonic fibroblasts in the presence of BMP4 and absence of FGF2 on a Matrigel substratum rapidly convert to an epithelium that is largely KRT7+ within 48 h, with minimal expression of mesoderm markers, including T (Brachyury). Instead, they begin to express a series of trophoblast markers, including HLA-G, demonstrate invasive properties that are independent of the continued presence of BMP4 in the medium, and, over time, produce extensive amounts of human chorionic gonadotropin, progesterone, placental growth factor, and placental lactogen. This process of differentiation is not dependent on conditioning of the medium by mouse embryonic fibroblasts and is accelerated in the presence of inhibitors of Activin and FGF2 signaling, which at day 2 provide colonies that are entirely KRT7+ and in which the majority of cells are transiently CDX2+. Colonies grown on two chemically defined media, including the one in which BMP4 was reported to drive mesoderm formation, also differentiate at least partially to trophoblast in response to BMP4. The experiments demonstrate that the in vitro BMP4/hESC model is valid for studying the emergence and differentiation of trophoblasts. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceCherry ABC et al. (JUL 2013) Stem Cells 31 7 1287--1297
Induced pluripotent stem cells with a mitochondrial dna deletion
In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here, we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View PublicationCatalog #: Product Name: 04434 MethoCult™ H4434 Classic 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 04434 Product Name: MethoCult™ H4434 Classic Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceYang J-Y et al. (JUN 2013) Cell Transplantation 22 6 945--959
SSEA4-positive pig induced pluripotent stem cells are primed for differentiation into neural cells.
Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However, further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still, the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study, we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors, piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons, astrocytes, and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceEasley CA et al. (SEP 2012) Cell reports 2 3 440--6
Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1 - ReferenceBardy J et al. (SEP 2013) Tissue engineering. Part C, Methods 19 2 120904064742009
Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells.
Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10�?� cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10�?� cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin�?� neurons, GFAP�?� astrocytes, and O4�?� oligodendrocytes, showing that cells maintained their multilineage differentiation potential. View PublicationCatalog #: Product Name: 07923 Dispase (1 U/mL) 85850 mTeSR™1 Catalog #: 07923 Product Name: Dispase (1 U/mL) Catalog #: 85850 Product Name: mTeSR™1
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