Ipsac track meet 2015

Keystone Symposia | Scientific Conferences on Biomedical and Life Science Topics

ipsac track meet 2015

MORE TEAMS: Men's Track & Field Sep 22, , Mountain Dew Gator Cross Country Invitational Aug 31, , Covered Bridge CC Open Meet. M.D. (–), a member of the Guidelines Update Task Force who passed away and mentor. © , INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH not meet its standards. . number of recipients, iPSC or other pluripotent- Careful adherence to regulations and tracking of. The track coach at Marshall, Christopher Fields. boy's meter run, at Arsenal Tech High School, Indianapolis, Friday, May 8, They're from the IPSAC meet the week before, four photos of Stoney's epic anchor lap.

The injury phenotype had three components: This model creates novel opportunities for drug discovery and exploration of the role of human genotype in TBI pathology.

Here we demonstrate that nanoscale substrate cues, designed to mimic the architecture of the extracellular matrix, drastically improve cardiomyocyte structural development. Moreover, we show that when evaluating the structural response of dystrophic cardiomyocytes to nanopatterns, such cells are less able to detect underlying substrate cues, enabling clearer stratification of healthy and diseased phenotypes.

We also show how integration of our nanopatterned substrate technology with established high-throughput electrophysiological assays produces improved functional performance in cultured cardiac monolayers and demonstrate how this improvement can be used for more effective assessment of novel drugs.

Lastly, we show that integration of thermoresponsive polymers with nanopatterns enables the detachment and stacking of multiple intact cell sheets to create organized 3D engineered tissues. The wide variety of nanopatterning applications, and the robust improvement to structural development that these structures produce in cardiomyocytes, could have far-reaching implications for a number of downstream, stem cell-based applications, including cell replacement therapy, drug screening, and disease modeling The Broadened Role of Human iPSC-derived Hepatocytes for Non-Alcoholic Fatty Liver Disease Modeling in Understanding Disease Development and Progression Maddalena Parafati, Sanford Burnham Prebys Medical Discovery Institute Abstract Introduction: Human induced pluripotent stem cell iPSC -derived hepatocytes hiPSC-Hep hold great promise for modeling liver diseases and cell-based therapy.

Non-alcoholic fatty liver disease NAFLD represents a spectrum of disease ranging from hepatocellular steatosis, non-alcoholic steatohepatitis NASH to hepatocellular carcinoma. These diseases can develop via several molecular pathways. We have developed a phenotypic assay to monitor these mechanisms in hiPSC-Hep. Quantification of lipids is achieved by integrated spot analysis to high content images. FFAs overload increased esterification to triglycerides dose-dependently and the ER stress potentiated lipid accumulation driving de novo lipogenesis without cytotoxicity.

We provide a well platform to model liver diseases by dysregulating key processes and proof of concept that the assay is sensitive to nuclear receptor modulation as validated with the clinical compound for NASH. We developed functional spheroids of iCell Cardiomyocytes and iCell Hepatocytes using magnetic 3D bioprinting, by magnetizing cells with a biocompatible nanoparticle assembly NanoShuttlethen aggregating them into spheroids with magnetic forces.

These spheroids are rapid to print, easy to handle, reproducible, and scalable for high- throughput plates Greiner Bio-One. With this method, we built beating spheroids with iCell Cardiomyocytes, and spheroids with higher metabolic activity in 3D than 2D with iCell Hepatocytes.

Overall, representative models can be made by magnetic 3D bioprinting iCell products.

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Because ImmTAC molecules are human specific, animal toxicity studies are not appropriate for safety testing. To assess their safety and specificity, ImmTAC molecules are tested in vitro using a wide range of normal cells representing the different human tissues.

Investigations by Immunocore revealed the engineered TCR to be cross-reactive with a Titin-derived peptide expressed in heart tissue. This peptide was found to be expressed in iPSC-derived cardiomyocytes but not in any of the 2D cultured cardiac cells tested. Further, iPSC-derived cardiomyocytes were killed by the engineered T cells whereas no activity was seen against the normal cells.

ipsac track meet 2015

Thus iPSC-derived cells represent a more biologically relevant model for human tissues than 2D cultured cells. The model is composed of endothelial cells and astrocytes derived from induced pluripotent stem cells Cellular Dynamics Internationaland neural pericytes derived from the brain ScienCell Research Laboratories. The cells are seeded onto synthetic poly ethylene glycol hydrogels that were customized with appropriate stiffness and integrin-binding peptide presentation to induce vascular network formation by endothelial cells seeded alone, or in combination with astrocytes and pericytes.

Functional outcomes of chemical exposure include changes in total area of the vascular networks, changes in network organization, and changes in co-localization of astrocytes and pericytes with endothelial cells of the neurovascular network. We demonstrate the role of astrocytes and pericytes in protecting neurovascular network integrity during exposure to Sunitnib Malate, a known inhibitor of VEGF signaling, or high glucose levels representative of diabetic hyperglycemia.

We aim to utilize this neurovascular unit model in future studies that expose cells to other training chemicals that recapitulate environmental toxicity.

ipsac track meet 2015

The impedance-based cardiac contractility assay has been systematically studied and validated previously Scott et al. While this assay showed comparable sensitivity and specificity using hiPSC-CMs to the well-validated optical-based contractility assay using adult dog cardiomyocytes Harmer et al. In order to address this limitation, we utilized xCELLigence RTCA CardioECR system, which combines impedance measurement and electrical pacing, to evaluate the effect of cardio-modulating compounds under pacing and non-pacing conditions.

Our data clearly demonstrate: The methylation status of specific CpGs can be variable across individuals but stable over time within the same individual [ 63 ]. DNMT is expressed during neural development and in adult brain in a tissue- and cell-specific manner including areas of active neurogenesis [ 64 ] and adult stem cell niches [ 65 ] where they have been involved in neural survival and plasticity [ 66 ].

Of these, DNMT3b has been specifically involved in the specification of the neural crest [ 69 ]. Once methylation is established, proteins of the methyl-CpG-binding domain MBD family are recruited to methylated loci to elicit the recruitment of histone modulatory factors such as histone deacetylases HDAC [ 7071 ] indicating a synergistic coordination of different epigenetic marks [ 48 ].

The MBD proteins have also been involved in developmental and adult brain functions [ 72 ]. The most common consequence of DNA methylation is the silencing of genes and noncoding genomic regions, especially when affecting gene promoters. But DNA methylation can also be associated with enhanced expression by mechanisms that yet remain poorly understood [ 7273 ].

Other enzymes such as the oxidoreductases of the Ten-Eleven Translocation TET family are responsible for the oxidation of 5mC to 5-hydroxymethylcytosine 5hmC [ 75 ]. Whereas 5mC correlates positively with age and, in general, negatively with gene expression in the brain [ 76 ], 5hmC despite of correlating also positively with age [ 77 ] has been shown to associate positively with expression [ 6178 ]. In addition, the 5hmC mark seems to be particularly abundant in tissues with low cell renewal rates such as the cerebellum and cortex [ 79 ] where it has been shown to be highly dynamic and susceptible to age-related changes [ 8081 ].

In neurons, the global balance among DNA methylation, demethylation, and hydroxymethylation determines neurobiological processes such as neural plasticity, memory, or learning, and their deregulation can be associated with neurodegenerative disorders [ 58 ].

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Histone Modifications In addition to DNA methylation, the conformation of the chromatin is also regulated by histone posttranslational modifications. In eukaryotic chromatin, the genomic DNA is packed around histone proteins forming the so-called nucleosome, which consists of base pairs of DNA wrapped around a histone octamer containing 2 copies each of the core histones H2A, H2B, H3, and H4 [ 83 ].

The nucleosome represents the fundamental unit of eukaryotic chromatin which folds through a series of successively higher order structures to form the chromosome. Ultimately, the nuclear organization of the chromatin is given by the balance between condense inactive heterochromatin and open active euchromatin [ 85 ]. Ultimately, the transcriptional regulation of genes is primarily controlled by physical access of the RNA polymerase II to promoter regions.

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Nonetheless, gene expression is also regulated by cis-elements termed enhancers which can be distally located upstream or downstream of promoters and whose epigenetic regulation is required for gene expression [ 86 — 88 ].

Thus, in addition to methylation, posttranslational modifications of histones at both promoters and enhancers critically regulate the conformation of the chromatin and the transcriptional state of specific genes [ 89 ]. There are more than different histone posttranslational modifications which can affect different histone amino acid residues including lysine Karginine Rserine Sthreonine Tand glutamate E [ 48 ]. Of these, acetylation and methylation of lysine residues are the most well-known histone modifications [ 90 ].

These histone marks are also specifically recognized by chromatin-binding proteins involved in transcriptional activation or repression and DNA replication and repair. Conversely, methylation of H3K4 normally marks active enhancers whereas acetylation of H3K4, H3K9, and H3K27 correlates with transcriptional activation [ 93 — 95 ]. In addition, the H3K27ac mark has been found to specifically distinguish active enhancers from poised enhancers in embryonic stem cells ESCs in genes which are relevant during development [ 86 ].

In general, acetylation of -amino groups of lysine residues of histones neutralizes their positive charge thereby relaxing chromatin structure [ 91 ] commonly favoring the protein binding of transcriptional activators [ 96 ]. Per contrary, histone deacetylation favors chromatin compaction and transcriptional repression [ 97 ]. Recently, reference functional chromatin states have been defined in humans for a wide variety of tissues including the central nervous system CNS providing a valuable resource for future epigenetic studies [ 99 ].

In the CNS, these histone modifications have been associated with neural stem cell NSC maintenance, neural and glial cell type specification, homeostasis, neural plasticity, learning, memory, and aging [ 48 ]. Comprehensive summary of histone epigenetic marks and corresponding functional states of the chromatin. Epigenetic Mechanisms during Neural Cell Differentiation During development, the progression from pluripotent stem cells through progenitors to differentiated cells occurs through an increase of repressive histone marks, DNA methylation, and chromatin compaction [ ].

These repressive epigenetic marks limit the phenotypic plasticity properties of the developing cells and therefore are essential for acquiring a differentiated cell identity [ ]. Little is known about the epigenetic pattering during the development of the human brain but efforts towards its characterization are being conducted including methylome studies for at least certain cell types.

Thus, a pioneer work has identified differentially methylated CpG regions associated with synaptogenesis during brain development in mouse and humans which seem to be enriched in key regulatory regions indicating their putative functional relevance [ 61 ]. In addition, this study revealed that 5hmC marks are present in fetal brain at regions that become activated by losing CG methylation and also that non-CG methylation accumulates in neurons but not in glia during this process.

On the contrary, histone marks of the developing brain [] or global transcriptome alterations involved in the cell-type specification remain poorly explored []. Yet once the neural fate program is activated, the remodeling of the chromatin is driven by cell specification signals such as TFs that interact with target sequences [ ] showing binding site enrichment of the specific TFs whose activity regulates gene expression [ 53].

Conceptually, multiple TFs acting in a coordinated manner orchestrate the remodeling of the epigenome of the differentiating neural cell to acquire specific cell phenotypes []. Of these, OCT4 has been shown to control the expression of H3K9me3 demethylases contributing to preserve the epigenetic marks needed for self-renewal of ESC [ ]. Among genes with bivalent marks are the HOX clusters which are master regulators of embryonic development [ ] and are silenced until cell fate commitment by polycomb repressive complexes PRC.

These PRC promote chromatin condensation by adding H3K27me3 [ ] while keeping a poised state of transcription. The bivalent marks become univalent active ones during ESC commitment towards neural lineage [ ] by the action of specific H3K27me3 [ ] and H3K4me3 demethylases [ ]. In mouse NSC, bivalent marks have been shown to resolve into active H3K4me3 monovalency upon differentiation in GABAergic neurons and into repressive H3K27me3 in non-GABAergic neurons [ ] indicating that genes carrying bivalent marks may lose one type of mark and become active or silenced depending on the direction of the differentiation.

In general, during differentiation, a progressive closure of the chromatin occurs at loci required for differentiation [ ] by a depletion of open chromatin histone marks, mainly H3 and H4 acetylation, and a simultaneous increase of repressive marks such as H3K9me3 []. As part of the Epigenome Roadmap Project, a recent study has shown that cell specification into the three-germ layer derivatives involves dynamic changes of TFs which work coordinately in specific and sequential TF modules which are integrated by individual TFs showing similar binding preferences for common sequences [ ].

In the nervous system, another study characterized the TFs neural regulatory networks involved in differentiation from ESC through neuroepithelial progenitors to radial glial cells [ ]. This study found that different neural stages are characterized by different epigenetic states in which distinct TFs are associated with stage-specific epigenetic changes as observed by shRNA inhibition. Epigenetic Mechanisms during iPSC Reprogramming Whereas the process of cell reprogramming means a truly epigenetic reprogramming [ 7 ], the precise epigenetic mechanisms underlying this process are only partially known.

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The expression of OSKM is needed to overcome epigenetic barriers such as the histone repressive mark H3K9m3 during cell reprogramming [ ]. Once the OSKM factors are expressed and the epigenetic barriers are overcome, pluripotency is stably maintained without the need of further ectopic TF expression. Shortly after the expression of the OSKM factors, human fibroblasts initially downregulate specific markers of their somatic state to subsequently activate genes associated with pluripotency [].

To adopt the epigenome characteristic of a stem cell, the somatic cell has to erase and reorganize its chromatin epigenetic signature [ ]. This process involves the genomewide resetting of histone marks which occurs immediately after the induction of OSKM factors [ 2— ].

Yet DNA demethylation can also occur early since AID is needed to demethylate the OCT4 promoter in fibroblasts and to initiate the process of nuclear reprogramming towards pluripotency [ 82 ]. Recent studies have suggested that the OSK TFs act as pioneer factors in loosening the chromatin into more open accessible forms and allowing the activation of genes relevant to the establishment and maintenance of the induced pluripotent state [ ].

The initial histone posttranslational changes induced by OSKM include acetylation, methylation, phosphorylation, and ubiquitination of histones. Among the earliest processes, an increase of the H3K4me2 mark occurs at promoter and enhancer regions of the genes involved in pluripotency which are enriched for binding sites of the OSKM factors and lack the H3K4me1 and H3K4me3 active marks [ ].

To achieve pluripotency induced by OSKM, recent studies have shown that there are three groups of epigenetic targets. Second, distal regulatory elements showing DNase I hypersensitivity and the enhancer mark H3K4me1 act as permissive enhancers that, after the binding of OSKM, are associated with promoters eliciting nucleosome depletion, chromatin relaxation, and transcriptional activation of lineage-specific genes [ ].

A third group of OSKM targets encompasses core pluripotency genes containing heterochromatic regions enriched for the repressive mark H3K9me3 in which the binding of OSKM leads to the repression of non-lineage-specific genes [ ].

The epigenetic remodeling of chromatin during reprogramming towards pluripotency also requires changes in DNA methylation.

Although DNA methylation is considered as the most stable epigenetic modification conferring permanent gene silencing during development [ ], histone modifications have been shown to typically antedate changes in DNA methylation during development [ ] and consistently this hierarchy of events has also been observed in reprogramming [ ]. Demethylation of pluripotency genes is crucial for faithful reprogramming, and although demethylation can occur either by passive or active mechanisms [ ], active demethylation catalyzed by specific enzymes has been shown to play a more important role in the induction of pluripotency [ ].

In addition, a progressive decrease of DNA methylation and of the H3K27me3 repressive mark at promoters of genes relevant to conversion occurs throughout reprogramming [ ].

Although these changes take place almost exclusively at CpG islands of initiating loci at the beginning of reprogramming process, they later expand outside CpG islands to affect other regions [ ]. This epigenetic memory has been linked to the failure to reverse repressive epigenetic marks associated with cell fate commitment [ ].

To date, epigenetic memory has been regarded as intrinsic limitation of iPSC permitting pluripotency but not totipotency. Epigenetic Research of Neurodegenerative Disorders Using iPSCs From a technical point of view, ESC represents an ideal tool to investigate development and model human disease as they provide a virtually endless resource of cells of interest given their high self-renewal and differentiation capacity. However, the use of ESC has been limited by ethical issues since current isolation protocols of ESC from the blastocyst inner cell mass imply the destruction of the embryo.

In this scenario, in vitro generation of iPSC has contributed to overcome at least in part such an obstacle. Here, we will review the potential of iPSC models as promising cell systems to perform epigenetic research of neurodegenerative disorders in the context of human postmortem brain tissues and animal models which can also implement this new venue of research.

Genomewide Methylation Studies in Patient Postmortem Brain Tissues A recent study investigated the methylome of AD in cortex tissue grey matter using a large number of prospectively collected autopsied brains from patients and controls [ ].

Six of the identified differentially methylated genes connected to a known genetic network of AD susceptibility. Among these, methylation differences in the ANK1 gene were further confirmed in an independent analysis of entorhinal cortex, which is a primary site of AD pathology, as well as in other affected areas including the superior temporal gyrus and the prefrontal cortex [ ]. In PD, one genomewide association study GWAS identified new genetic variants associated with disease and, for a subset of genes, it also found differential methylation levels in PD frontal cortex and cerebellum which overlapped with previously reported genetic associations [ ].

Another genomewide DNA methylation study in PD frontal cortex also identified distinct methylation patterns in PD affecting genetic polymorphisms associated with PD and, interestingly, these differential methylation patterns correlated in brain and blood samples [ ].

Evotec and Celgene expand iPSC collaboration to include additional cell lines - Evotec

Altogether, these studies in AD and PD provide the proof-of-concept that epigenetic deregulation occurs in neurodegenerative disorders and encourage the use of iPSC-based models to conduct epigenetic research in these diseases.

However, DNA methylation changes from these studies were detected despite of the heterogeneous mix of brain cell types, and therefore it is possible that overall epigenetic differences may be underestimated. Similarly, it would be expectable that epigenetic changes associated with disease could be potentially identified using iPSC-based models albeit of the cell population heterogeneity which is inherent to these models.

Yet in this scenario, iPSCs models offer the opportunity to characterize the epigenetic profiles of specific cell populations by using techniques such as fluorescence-activated cell sorting FACS as recently shown for the transcriptome characterization of mouse iPSC-derived DAn [ ]. Lessons from Epigenetic Studies in Mouse Models A recent RNA-seq study in the Ck-p25 mouse model of AD identified gene expression upregulation of immune system genes and downregulation of genes associated with neuronal function [ ].

Similar findings were also reported in human AD postmortem hippocampus [ ]. These expression changes correlated with the epigenomic status of promoters, enhancers, and polycomb-repressed regions which showed a specific depletion of neuronal promoter and enhancer marks. Interestingly, this study demonstrated a strong conservation of gene expression and epigenomic signatures between human and mouse with a specific enrichment of AD-associated loci in enhancer orthologs.

Another study in PD compared the transcriptome and the methylome of primary embryonic mesodiencephalic DAn from the knock-in mouse as well as iPSC-derived DAn generated upon embryonic fibroblast reprogramming [ ]. Altogether, these studies in AD and PD illustrate a scenario in which epigenetic research relevant to neurodegenerative diseases is more advanced in iPSC-based models from mouse than humans due to availability reasons.

Yet achievements of these mouse epigenetic studies can be useful for epigenetic research using patient-derived iPSC-based models since mouse studies can provide valid technical data which may help to prevent pitfalls in designing experiments using human iPSCs as well as to generate novel epigenetic knowledge to be explored in human models genuine to the patients.

Although these protocols have been steadily improved by increasing reprogramming and differentiation efficiencies, cell heterogeneity accompanying disease-relevant cell types is still inherent to current iPSC models. This accompanying cell heterogeneity can act as a potential confounder in epigenetic research but, yet, if affecting in an equal manner to iPSC from patients and controls, it may also lead to an underestimation of the observed epigenetic differences, as recently suggested in postmortem epigenetic studies analyzing heterogeneous mix of brain cells [].

Still this cell heterogeneity should be appropriately controlled for epigenetic studies by performing FACS isolation to deliver pure cell populations prior to the epigenetic analyses [].

Alternatively, it could also be possible to control the variability caused by cell heterogeneity by studying the epigenetic profile of iPSC-derived neural types nonenriched in the specific neural type of interest, for example, iPSC-derived neural cultures nonenriched in DAn as a control population for a DAn study in PD. Yet despite of current technical challenges, cellular reprogramming provides conceptually a unique opportunity to generate in vitro human models that will permit to investigate epigenetic regulation and alterations of functional states of the chromatin related to neurodegenerative diseases [ 52 ].

Recently, the epigenetic signature from human tissues has become publically available including multiple brain regions such as the hippocampus or the substantia nigra which are relevant to AD and PD, respectively [ 99 ]. This large multicentre study has implemented the reference human genome sequence and is expected to set the basis for future studies on epigenetic variation and its role in human disease by providing reference maps of histone modifications and DNA methylation, as well as global RNA expression data.

This information will prove instrumental to investigate specific epigenetic alterations and to model in vitro novel epigenetic disease mechanisms using currently available patient-derived iPSC-based models of neurodegenerative diseases which up to date have not been epigenetically characterized [ ]. Interestingly, iPSC-derived neural models preserve the genetic background of the patient and this is relevant since the disease-associated genetic variants were previously shown to be enriched in tissue-specific epigenomic marks suggesting an overlap of genetic and epigenetic alterations which may be associated with human disease [ 99 ].

In addition, iPSC-derived neural models can virtually offer the opportunity to recapitulate the exposome or environmental history of the individual that may be relevant complex diseases with an expected large environmental contribution such as AD, PD, or ALS and also to their monogenic forms Figure 1.

Possible experimental design of epigenomic characterization of neurodegenerative diseases using patient-specific iPSC-derived neural models. To date, although iPSC-based disease modeling has been preferentially performed in mutation-caused monogenic forms of neurodegenerative disease, recent studies in AD and PD have set the proof-of-concept that iPSC-derived models from sporadic patients can exhibit molecular alterations similar to those changes detected in monogenic patients [ 284243 ].

In monogenic cases, iPSC-based systems offer the attractive possibility to perform gene editing [ 29 ] contributing to the elucidation of the molecular events triggering disease through the analysis of the effect of specific pathogenic mutations.