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BackgroundHuman embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors.Methods and F... |
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be maintained in culture for an extended period of time without losing differentiation potential. The successful isolation of human ES cells (hESCs) has raised the hope that these cells may provide a universal ti... |
Cell Culture and FACSUndifferentiated hESCs, H1 (WA-01, XY, passages 40–65) and H9 (WA-09, XX, passages 35–45), were cultured on mitotically inactivated mouse embryonic fibroblasts (Specialty Media, Phillipsburg, New Jersey, United States) and maintained under growth conditions and passaging techniques described previo... |
Mesenchymal differentiation of hESCs (lines H1 [WA-01] and H9 [WA-09]) [9] was induced by plating undifferentiated hESCs on a monolayer of murine OP9 stromal cells [10], in the presence of 20% heat-inactivated FBS in alpha MEM medium. OP9 cells have been previously shown to induce blood cell differentiation from mouse ... |
Previous studies have demonstrated the derivation of neural cells [1–3], hematopoietic [17] and endothelial lineages [18], and cardiomyocytes [19] from hESCs. This study presents the induction of paraxial mesoderm with the generation of multipotent mesenchymal precursors. We calculate that under these conditions a sing... |
BackgroundUsing antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers. Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribut... |
Although the stem cell concept was introduced decades ago, to date, stem cells can only be defined functionally, not morphologically or phenotypically. Two functions define stem cells. Firstly, they are self-renewing, thus able to propagate to generate additional stem cells. Secondly they can differentiate into various... |
Characterization of undifferentiated human ES cells and differentiated EBs by antibodiesAll monoclonal antibodies were initially selected for their abilities to recognize recombinant proteins in direct ELISAs. A subset were also tested by Western Blot analysis using recombinant proteins and cell lysate to confirm bindi... |
The expression patterns detected using antibodies developed in our facility are consistent with data reported using reverse transcriptase-polymerase chain reaction or cDNA microarrays. Moreover several of the monoclonal antibodies have differing heavy chain subunits allowing double labeling using subtype specific marke... |
Cloning and expression of Brachyury, DPPA5, CD9, E-Cadherin, GATA1, GATA6, Nanog, Oct3/4, PDX-1, PODXL, SOX2 and SOX17Brachyury (aa. 1–202), DPPA5 (a.a. 1–116), GATA1 (a.a. 1–413), GATA6 (aa. 1–449), Nanog (aa. 153–305), Oct3/4 (aa. 1–265), PDX-1 (aa. 1–283), SOX2 (aa. 135–317) and SOX17 (aa. 177–414) were expressed in... |
BackgroundMany novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cel... |
Human embryonic stem cells (hES cells) show great promise for many novel cellular therapies due to their pluripotent nature [1]. These cells have the capacity to give rise to mature cells and tissues that arise from all three germ layers during embryonic development [2-4]. Several pluripotent hES cell lines have so far... |
Derivation of macrophages from hES cellsUndifferentiated hES cell colonies grown in media supplemented with 4 ng/ml bFGF displayed normal morphology of pluripotent human embryonic stem cells with tight and discreet borders on the MEF feeder layers (Fig 1A). Similarly, lentiviral vector transduced hES cell colonies, als... |
As a first step towards the use of hES cells for hematopoietic stem cell and HIV gene therapies, we have shown here that phenotypically and functionally normal macrophages could be derived from hES-CD34 cells. Both non transduced and lentiviral vector transduced hES cells were found to be capable of generating CD34 cel... |
Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV gen... |
Growth, propagation and lentiviral transduction of hES cellsThe NIH approved human ES H1 cell line was obtained from WiCell (Madison, Wisconsin). hES cell colonies were cultured on mouse embryonic fibroblasts (MEF) (Chemicon, Temecula, CA) in the presence of DMEM-F12 (Invitrogen, Carlsbad, CA) supplemented with 20% KNO... |
BackgroundIn order to compare the gene expression profiles of human embryonic stem cell (hESC) lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hESC lines and human fibroblast feeder cells using Illumina® bead arrays that contain probes for 24,131 tr... |
Embryonic stem cells (ESCs), derived from the inner cell mass of pre-implantation embryos, have been recognized as the most pluripotent stem cell population. Human ES cells (hESCs) can be maintained and propagated on mouse or human fibroblast feeders for extended periods in media containing basic fibroblast growth fact... |
Multiple hESC lines can be assessed by Illumina bead arrayForty-eight samples were selected from multiple laboratories and gene expression profiles were examined using a bead array containing 24,131 transcripts derived from the Human RefSeq database that included full length and splice variants. Each gene was represent... |
Undifferentiated hESCs have been analyzed by EST scan, MPSS, SAGE and microarray [5,10,16]. The goal of these experiments including our own is to develop a low cost reliable method to assess multiple samples to generate a global database of markers and to provide a method of identifying core measures of similarities an... |
In summary, the Illumina bead array has several key strengths including high throughput, low cost and high sensitivity. By using this array, we can readily detect contaminating feeders and spontaneous differentiation, differentiate male and female lines and distinguish between one undifferentiated population and anothe... |
hESC cultureThe hESC lines H1, H7 and H9 (WiCell, Madison, WI) were cultured on feeder layers derived from mitotically inactivated HS27 human fibroblast cells (HS27, ATCC), or mouse embryonic fibroblsts or under feeder-free conditions on Matrigel (BD, Franklin Lakes, NJ) coated plates for at least 10 passages. Culture ... |
BackgroundHuman stem cells are viewed as a possible source of neurons for a cell-based therapy of neurodegenerative disorders, such as Parkinson's disease. Several protocols that generate different types of neurons from human stem cells (hSCs) have been developed. Nevertheless, the cellular mechanisms that underlie the... |
Modern DNA microarrays permit a comprehensive analysis of quantitative and qualitative changes in RNA transcript abundance, outlining the cross-sections of gene expression and alterations of these in response to genetic or environmental stimuli. Genome-scale microarrays (cDNA- or oligonucleotide-based) are most valuabl... |
Stem cells have unique biological characteristics, but only a limited number of genes are currently recognized as established stem cell markers. Examples include POU domain, class 5, transcription factor 1 (Oct3/4), signal transducer and activator of transcription 3 (Stat3), teratocarcinoma-derived growth factor (Tdgf1... |
Recent technological advances have led to DNA microarrays which contain over hundred thousand of spots of DNA material, reaching a truly genomic scale. Highly specialized DNA microarrays of smaller scale (e.g. the NeuroStem Chip) still have an important role in the directed studies in particular fields. Since they are ... |
Human embryonic stem cell (hESC) culturesUndifferentiated hESCs of SA02 (Sahlgrenska 2) line (Cellartis AB, Göteborg, Sweden; see NIH Human Embryonic Stem Cell Registry at [46]) were maintained over a monolayer of human "feeder cells" (hFCs; human foreskin fibroblasts, ATCC; cell line CCD-1112Sk). Feeder cells were gro... |
This work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. Specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human emb... |
Embryonic stem cells (ESCs) are distinguished by their ability to self-renew and to differentiate into any other cell type via asymmetric cell divisions, in which one daughter cell maintains ‘stemness’ while the other daughter cell differentiates into a particular tissue type. ESCs, including those of human origin (hES... |
Regenerative responses of adult muscle stem cells and hESCs are dominantly inhibited by the aged systemic milieuPrevious work established that the upregulation of repair-specific molecular signaling mechanisms, such as Notch, and successful engagement of resident muscle stem cells in tissue repair are largely determine... |
The data presented here establish for the first time that both the local environment of old differentiated organ, e.g., skeletal muscle and the systemic milieu dramatically affect the regenerative potential of both hESCs and mouse post-natal myogenic progenitor cells. Not only are the factors promoting myogenic differe... |
Animal strainsYoung (2–3 months), C57-BL/6 male mice were obtained from pathogen-free breeding colonies at Jackson Laboratories (Bar Harbor, ME, USA). Aged 22–24 months C57-BL/6 male mice were obtained from the National Institute on Aging (NIH). Animals were maintained in the North-West Animal Facility of the Universit... |
Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recentl... |
Mapping the location of DNaseI-hypersensitive sites (DHSs) remains central to developing our understanding of transcriptional regulation. Elements with a range of transcriptional regulatory functions have been identified initially as DHSs. These include transcriptional enhancers (1,2) and repressors (3,4) as well as ch... |
Generating a library of DHSsThe basic protocol is outlined in Figure 1. Nuclei are extracted from living cells, then digested on ice for 1 h with a range of DNaseI concentrations, as detailed in the ‘Materials and methods’ sections. Following RNaseA and proteinaseK treatment, the DNA is extracted and run on a 1% agaros... |
The development of techniques that permit the rapid comparative mapping of DHS between different cell types will greatly facilitate the study of transcriptional regulation in both normal and diseased cells. Recently published high-throughput techniques that map DHS sites using high-throughput sequencing (8,9) and genom... |
Cell cultureCell lines were maintained in culture as previously described (11). Care was taken to ensure maximum viability of cells when taken for experiments. The primary thymocytes used for Figure 2 were prepared by gentle physical disassociation of a whole thymus gland into PBS supplemented with 2% FCS. Cells were f... |
Human embryonic stem cells (hESCs) and neural progenitor (NP) cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disorders. While much effort had been undertaken to analyze transcriptional and epigenetic differences... |
The human central nervous system is composed of thousands of neuronal subtypes originating from neural stem cells (NSCs) that migrate from the developing neural tube. Such neuronal complexity is generated by a vast repertoire of molecular, genetic, and epigenetic mechanisms, such as the active retrotransposition of tra... |
Derivation of Neural Progenitors from Embryonic Stem CellsNPs were independently derived from two hESC lines, and RNA extracted from the cell lines was processed and hybridized onto Affymetrix Human 1.0 ST exon arrays. Immunohistochemical and reverse-transcriptase polymerase chain reaction (RT-PCR) analyses demonstrate... |
The ability of ESCs to generate all three embryonic germ layers has raised the exciting possibility that hESCs may become an unlimited source of cells for transplantation therapies involving organs or tissues such as the liver, pancreas, blood, and nervous system, and become tools to explore the molecular mechanisms of... |
Maintenance and differentiation of hESCs and hCNS-SCns.hESC line Cy203 (Cythera) was cultured as previously described [12]. To differentiate into neuroepithelial precursor cells, colonies were manually isolated from mouse embryonic fibroblasts (MEFs) and cut in small pieces. These pieces were transferred to a T75 flask... |
BackgroundMuch of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge ... |
Human embryonic stem cells (hESCs) are pluripotent cells isolated from the inner cell mass of the blastocyst [1]. They can be maintained for prolonged periods in culture and differentiate to representatives of the three germ layers as well as trophoblasts and germ cells. This differentiation potential may be used to mo... |
PowerBlot analysis of hESCsWe first employed a large-scale western blot screen, the PowerBlot system, to profile protein expression in undifferentiated hESCs. This system used 934 antibodies toward proteins representing 22 diverse classes of function, such as transcription factors, the MAP kinase (MAPK) pathway, and ap... |
Attempts to harness the potential of hESCs for models of human embryogenesis and cell therapy applications will be greatly enhanced by a detailed understanding of their molecular characteristics. This includes definition of the transcripts, splice variants, and protein isoforms expressed by these cells. Post-translatio... |
In summary, we generated a focused proteome of hESCs using large-scale western blotting and sorted the detected proteins according to function and signaling pathways. This characterization provides important basic information on expressed proteins, their isoforms and post-translational modifications, and tools for the ... |
Culture of human embryonic stem cellsFor the PowerBlot analysis, enzymatically passaged BG01 hESCs were grown as described previously [23]. These conditions were necessary to scale up the culture to generate the milligram amounts of protein lysate required for this analysis. These conditions maintain cell populations t... |
BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neura... |
To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associ... |
1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stim... |
Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and... |
hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µ... |
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