The Experts below are selected from a list of 9102 Experts worldwide ranked by ideXlab platform
Alysson R Muotri - One of the best experts on this subject based on the ideXlab platform.
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systematic optimization of human pluripotent stem cells media using design of experiments
Scientific Reports, 2015Co-Authors: Paulo Andre Nobrega Marinho, Thanathom Chailangkarn, Alysson R MuotriAbstract:Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell Reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epiGenetic status after Genetic Reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.
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evidence for premature aging due to oxidative stress in ipscs from cockayne syndrome
Human Molecular Genetics, 2012Co-Authors: Luciana Nogueira De Sousa Andrade, Carlos Frederico Martins Menck, Alysson R Muotri, Jason L Nathanson, Gene W YeoAbstract:Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to Genetic Reprogramming. However, iPSCs derived from CSB patient’s fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.
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transcriptional signature and memory retention of human induced pluripotent stem cells
PLOS ONE, 2009Co-Authors: Maria C Marchetto, Gene W Yeo, Osamu Kainohana, Martin Marsala, Fred H Gage, Alysson R MuotriAbstract:Genetic Reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The Reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal Reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.
Vania Broccoli - One of the best experts on this subject based on the ideXlab platform.
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efficient Genetic Reprogramming of unmodified somatic neural progenitors uncovers the essential requirement of oct4 and klf4
Stem Cells and Development, 2009Co-Authors: Bruno Di Stefano, Alessandro Prigione, Vania BroccoliAbstract:Significant breakthroughs have been recently achieved in Reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of Reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epiGenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether Genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell–like state with a 2-fold efficiency increase, faster kinetic, and with a low...
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efficient Genetic Reprogramming of unmodified somatic neural progenitors uncovers the essential requirement of oct4 and klf4
Stem Cells and Development, 2009Co-Authors: Bruno Di Stefano, Alessandro Prigione, Vania BroccoliAbstract:Significant breakthroughs have been recently achieved in Reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of Reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epiGenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether Genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell-like state with a 2-fold efficiency increase, faster kinetic, and with a lower number of viral integrations. Quantification analysis of Reprogramming-associated genes revealed that NSCs endogenously expressed high levels of Sox2 and c-Myc. Accordingly, NSCs could be successfully induced to pluripotency through the ectopic viral expression of the other two factors (Oct4 and Klf4). These findings suggest that endogenous expression of Reprogramming genes could help the Reprogramming process and somatic stem cells might be more prone to Reprogramming due to their specific Genetic background. Genetic-based somatic cell screening might provide essential information for the selection of alternative cell sources more suitable to direct Reprogramming.
Bruno Di Stefano - One of the best experts on this subject based on the ideXlab platform.
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efficient Genetic Reprogramming of unmodified somatic neural progenitors uncovers the essential requirement of oct4 and klf4
Stem Cells and Development, 2009Co-Authors: Bruno Di Stefano, Alessandro Prigione, Vania BroccoliAbstract:Significant breakthroughs have been recently achieved in Reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of Reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epiGenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether Genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell–like state with a 2-fold efficiency increase, faster kinetic, and with a low...
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efficient Genetic Reprogramming of unmodified somatic neural progenitors uncovers the essential requirement of oct4 and klf4
Stem Cells and Development, 2009Co-Authors: Bruno Di Stefano, Alessandro Prigione, Vania BroccoliAbstract:Significant breakthroughs have been recently achieved in Reprogramming somatic cells to a pluripotent embryonic state by the ectopic expression of specific transcription factors. One of the major drawbacks of Reprogramming strategies lays in the low efficiency of the process. It is likely that the required complex epiGenetic-remodeling events could be cell-type specific and more rational approaches to cell source selection might help to improve the outcome of the procedure. Because the use of somatic stem cells, and specifically neural stem cells (NSCs), as nuclear donors significantly increased the efficiency of somatic cell nuclear transfer, we aimed to determine whether Genetically unmodified somatic NSCs could be more easily reprogrammed to pluripotency than unmodified mouse embryonic fibroblasts. Retroviral transduction of the factors Oct4, Sox2, Klf4, and c-Myc successfully reverted NSCs to a pluripotent embryonic stem cell-like state with a 2-fold efficiency increase, faster kinetic, and with a lower number of viral integrations. Quantification analysis of Reprogramming-associated genes revealed that NSCs endogenously expressed high levels of Sox2 and c-Myc. Accordingly, NSCs could be successfully induced to pluripotency through the ectopic viral expression of the other two factors (Oct4 and Klf4). These findings suggest that endogenous expression of Reprogramming genes could help the Reprogramming process and somatic stem cells might be more prone to Reprogramming due to their specific Genetic background. Genetic-based somatic cell screening might provide essential information for the selection of alternative cell sources more suitable to direct Reprogramming.
Sheng Ding - One of the best experts on this subject based on the ideXlab platform.
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small molecules big roles the chemical manipulation of stem cell fate and somatic cell Reprogramming
Journal of Cell Science, 2012Co-Authors: Yu Zhang, Timothy Laurent, Sheng DingAbstract:Despite the great potential of stem cells for basic research and clinical applications, obstacles - such as their scarce availability and difficulty in controlling their fate - need to be addressed to fully realize their potential. Recent achievements of cellular Reprogramming have enabled the generation of induced pluripotent stem cells (iPSCs) or other lineage-committed cells from more accessible and abundant somatic cell types by defined Genetic factors. However, serious concerns remain about the efficiency and safety of current Genetic approaches to cell Reprogramming and traditional culture systems that are used for stem cell maintenance. As a complementary approach, small molecules that target specific signaling pathways, epiGenetic processes and other cellular processes offer powerful tools for manipulating cell fate to a desired outcome. A growing number of small molecules have been identified to maintain the self-renewal potential of stem cells, to induce lineage differentiation and to facilitate Reprogramming by increasing the efficiency of Reprogramming or by replacing Genetic Reprogramming factors. Furthermore, mechanistic investigations of the effects of these chemicals also provide new biological insights. Here, we examine recent achievements in the maintenance of stem cells, including pluripotent and lineage-specific stem cells, and in the control of cell fate conversions, including iPSC Reprogramming, conversion of primed to naive pluripotency, and transdifferentiation, with an emphasis on manipulation with small molecules.
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generation of rat and human induced pluripotent stem cells by combining Genetic Reprogramming and chemical inhibitors
Cell Stem Cell, 2009Co-Authors: Wei Wei, Saiyong Zhu, Jinliang Zhu, Yan Shi, Tongxiang Lin, Ergeng Hao, Alberto Hayek, Hongkui Deng, Sheng DingAbstract:(Cell Stem Cell 4, 16–19; January 9, 2009)In our recent article, we unfortunately misquoted the findings in a recent study by Ying et al. (2008)xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al. (2008). When describing previous work using combination of the MEK inhibitor PD0325901 and the GSK3b inhibitor CHIR99021, our statement “Recent studies demonstrated that addition of the FGFR inhibitor PD173074 to the above cocktail is sufficient to maintain mESC pluripotency in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008).” was not accurate. Ying et al., in fact, used PD0325901 to replace PD1730474 and demonstrated maintenance of mESCs in the absence of LIF with these two factors only.The corrected section reads “Indeed, after serial passages, the growth of putative riPSCs treated with only PD0325901 and CHIR99021 declined, and the culture deteriorated due to the expansion of differentiated cells, although recent studies demonstrated that this combination of inhibitors can be used to maintain mESC self-renewal in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008). Because the TGFβ/Activin A/Nodal signaling cascade is essential to maintain undifferentiated hESCs and EpiSCs, but dispensable for mESC self-renewal, we tested whether the addition of an inhibitor of the type 1 TGFβ receptor, ALK5 (A-83-01), could help stabilize our riPSC cultures.”In addition, we omitted to mention that while our study was under review, Silva et al. (2008)xPromotion of Reprogramming to Ground State Pluripotency by Signal Inhibition. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. PLoS Biol. 2008; 6: 2237–2247Crossref | Scopus (456)See all ReferencesSilva et al. (2008) also published the use of these two inhibitors in the generation and propagation of mouse iPSCs.We apologize for any confusion caused.
John M. Mariadason - One of the best experts on this subject based on the ideXlab platform.
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Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis.
Gastroenterology, 2005Co-Authors: John M. Mariadason, Courtney Nicholas, Kaitlin L’italien, Min Zhuang, Helena J.m. Smartt, Barbara G. Heerdt, Wancai Yang, Georgia A. Corner, Andrew J. Wilson, Lidija KlampferAbstract:Background & Aims: To define the Genetic Reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. Methods: Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. Results: A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. Conclusions: This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.
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Genetic Reprogramming in pathways of colonic cell maturation induced by short chain fatty acids comparison with trichostatin a sulindac and curcumin and implications for chemoprevention of colon cancer
Cancer Research, 2000Co-Authors: John M. Mariadason, Georgia A. Corner, Leonard H AugenlichtAbstract:The short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, is a physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063 sequences demonstrated a complex cascade of Reprogramming of SW620 colonic epithelial cells upon treatment with butyrate characterized by the progressive recruitment of gene sets as a function of time. Comparison with the effects of trichostatin A, in conjunction with differences in the kinetics of alteration of histone acetylation induced by butyrate and trichostatin A, identified subsets of induced and repressed genes likely coordinately regulated by altered histone acetylation. The butyrate response was also compared in detail with that of sulindac, a nonsteroidal anti-inflammatory drug with significant chemopreventive activity for colon cancer, and curcumin, a component of mustard and curry structurally and functionally related to sulindac that also has chemopreventive activity. Although gene clusters were identified that showed similar responses to butyrate and sulindac, the data were characterized by the extensive differences in the effects of the two agents. This was striking for functional classes of genes involved in signaling pathways and in cell cycle progression, although butyrate and sulindac induce a similar G 0 -G 1 arrest, elevation of β-catenin-Tcf signaling, and apoptotic cascade. As regards cell cycle arrest, the underlying mechanism in response to butyrate was most similar to that of the Caco-2 cell line that had spontaneously undergone a G 0 -G 1 arrest and least similar to the G 2 -M arrest stimulated by curcumin. Thus, high-throughput microarray analysis of gene expression profiles can be used to characterize and distinguish the mechanisms of response of colonic epithelial cells to physiological and pharmacological inducers of cell maturation. This has important implications for characterization of chemopreventive agents and recognition of potential toxicity and synergies. The data bases, gene clusters, and analyses are available at http://sequence.aecom.yu.edu/genome/.
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Genetic Reprogramming in pathways of colonic cell maturation induced by short chain fatty acids comparison with trichostatin a sulindac and curcumin and implications for chemoprevention of colon cancer
Cancer Research, 2000Co-Authors: John M. Mariadason, Georgia A. Corner, Leonard H AugenlichtAbstract:The short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, is a physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063 sequences demonstrated a complex cascade of Reprogramming of SW620 colonic epithelial cells upon treatment with butyrate characterized by the progressive recruitment of gene sets as a function of time. Comparison with the effects of trichostatin A, in conjunction with differences in the kinetics of alteration of histone acetylation induced by butyrate and trichostatin A, identified subsets of induced and repressed genes likely coordinately regulated by altered histone acetylation. The butyrate response was also compared in detail with that of sulindac, a nonsteroidal anti-inflammatory drug with significant chemopreventive activity for colon cancer, and curcumin, a component of mustard and curry structurally and functionally related to sulindac that also has chemopreventive activity. Although gene clusters were identified that showed similar responses to butyrate and sulindac, the data were characterized by the extensive differences in the effects of the two agents. This was striking for functional classes of genes involved in signaling pathways and in cell cycle progression, although butyrate and sulindac induce a similar G0-G1 arrest, elevation of beta-catenin-Tcf signaling, and apoptotic cascade. As regards cell cycle arrest, the underlying mechanism in response to butyrate was most similar to that of the Caco-2 cell line that had spontaneously undergone a G0-G1 arrest and least similar to the G2-M arrest stimulated by curcumin. Thus, high-throughput microarray analysis of gene expression profiles can be used to characterize and distinguish the mechanisms of response of colonic epithelial cells to physiological and pharmacological inducers of cell maturation. This has important implications for characterization of chemopreventive agents and recognition of potential toxicity and synergies. The data bases, gene clusters, and analyses are available at http:// sequence.aecom.yu.edu/genome/.