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Forrest M.P.,University of Cardiff | Waite A.J.,University of Cardiff | Martin-Rendon E.,University of Oxford | Martin-Rendon E.,Stem Cell Research Laboratory | Blake D.J.,University of Cardiff
PLoS ONE | Year: 2013

Haploinsufficiency of TCF4 causes Pitt-Hopkins syndrome (PTHS): a severe form of mental retardation with phenotypic similarities to Angelman, Mowat-Wilson and Rett syndromes. Genome-wide association studies have also found that common variants in TCF4 are associated with an increased risk of schizophrenia. Although TCF4 is transcription factor, little is known about TCF4-regulated processes in the brain. In this study we used genome-wide expression profiling to determine the effects of acute TCF4 knockdown on gene expression in SH-SY5Y neuroblastoma cells. We identified 1204 gene expression changes (494 upregulated, 710 downregulated) in TCF4 knockdown cells. Pathway and enrichment analysis on the differentially expressed genes in TCF4-knockdown cells identified an over-representation of genes involved in TGF-β signaling, epithelial to mesenchymal transition (EMT) and apoptosis. Among the most significantly differentially expressed genes were the EMT regulators, SNAI2 and DEC1 and the proneural genes, NEUROG2 and ASCL1. Altered expression of several mental retardation genes such as UBE3A (Angelman Syndrome), ZEB2 (Mowat-Wilson Syndrome) and MEF2C was also found in TCF4-depleted cells. These data suggest that TCF4 regulates a number of convergent signaling pathways involved in cell differentiation and survival in addition to a subset of clinically important mental retardation genes. © 2013 Forrest et al.

Telias M.,Stem Cell Research Laboratory | Telias M.,Tel Aviv University | Segal M.,Weizmann Institute of Science | Ben-Yosef D.,Stem Cell Research Laboratory | Ben-Yosef D.,Tel Aviv University
Developmental Biology | Year: 2013

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability, caused by developmentally regulated inactivation of FMR1, leading to the absence of its encoded protein FMRP. We have previously shown that undifferentiated Fragile X human Embryonic Stem Cells (FX-hESCs) express FMRP, despite the presence of the full FMR1 mutation (>200 CGG repeats). We describe here, for the first time, in-vitro differentiation of FX-hESCs into neurons progressively inactivating FMR1. Abnormal neurogenesis and aberrant gene expression were found already during early stages of differentiation, leading to poor neuronal maturation and high gliogenic development. Human FX neurons fired action potentials but displayed poor spontaneous synaptic activity and lacked reactivity to glutamate. Our dynamic FX-hESCs model can contribute to the understanding of the sequence of developmental events taking place during neurogenesis and how they are altered in FXS individuals, leading to intellectual disability. Furthermore, it may shed light over the striking phenotypic features characterizing FXS in human. © 2012 Elsevier Inc.

Shetty R.,Narayana Nethralaya | Shetty R.,India Stem Cell Research Laboratory | Shetty R.,Maastricht University | Shetty R.,Stem Cell Research Laboratory | Shetty R.,GROW Laboratory
The British journal of ophthalmology | Year: 2015

AIM/BACKGROUND: To compare the effects of accelerated corneal collagen cross-linking (ACXL) and corneal collagen cross-linking (CXL) on ex vivo-cultured limbal epithelial cells (LECs).METHODS: Day 14 cultured LECs were either unexposed (control) or exposed to different intensities of ultraviolet-A (UV-A) irradiance for different durations (3 mW for 30 min, 9 mW for 10 min, 18 mW for 5 min and 30 mW for 3 min) in the presence and absence of riboflavin. These cells were further processed for quantitative real-time PCR, vital staining, immunofluorescence staining and fluorescence-activated cell sorting (FACS) staining to evaluate the apoptotic status. Statistical analysis was performed using a Student t test.RESULTS: Vital staining showed a significantly higher (p=0.004) dead cell population with 3 mW for 30 min when compared with 30 mW for 3 min exposure (p=0.225). Quantitative PCR results revealed significantly reduced abcg2 and Δnp63 mRNA levels, while FACS analysis showed an increase in ABCG2-Annexin V positive population in cells exposed to 3 mW for 30 mins. Neither reduction of mRNA expression of abcg2 and Δnp63 nor increase in FACS-stained ABCG2-Annexin V positivity was detected in cells exposed to 30 mW for 3 min. Additionally, enhanced caspase activity was detected with fluorochrome inhibitor of caspases staining and mRNA expression of caspase 3 and 9 was upregulated in cells exposed to 3 mW for 30 min, but not at 30 mW for 3 min.CONCLUSIONS: The 30 mW UV-A irradiation used in ACXL appears to be safe on cultured LECs in comparison with 3 mW used in CXL. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Epsztejn-Litman S.,Stem Cell Research Laboratory | Eiges R.,Stem Cell Research Laboratory
Methods in molecular biology (Clifton, N.J.) | Year: 2010

One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes and studying their function, and are now being initiated in human ES (HESC) cells. This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose.

Matalia H.,Stem Cell Research Laboratory | Shetty R.,Narayana Nethralaya | Dhamodaran K.,Stem Cell Research Laboratory | Subramani M.,Stem Cell Research Laboratory | And 2 more authors.
British Journal of Ophthalmology | Year: 2012

Aim/background: To study the effects of ultraviolet-A (UV-A) irradiation, in the presence or absence of riboflavin, on ex vivo cultured limbal epithelial cells (LECs). Methods: The study was carried out in a super specialty ophthalmic hospital. Ex vivo cultured LECs were grown on denuded amniotic membranes and exposed to similar levels of UV-A radiation used during corneal cross-linking (CXL), in the presence or absence of the photosensitiser, riboflavin. These cells were then used for extraction of RNA, cDNA conversion, and antibody staining. Quantitative PCR and immuno fluorescence staining were performed to evaluate the apoptotic state of treated and non-treated LECs. Statistical analyses were evaluated using a Student's t test. Results: We found that bcl-2, an antiapoptotic gene, was downregulated, whereas, bax, a proapoptotic gene, was upregulated. After LECs were exposed to UV-A radiation, a significant upregulation of both caspase 3 and caspase 9 was observed in treated cells when compared with untreated LECs. Conclusions: These results indicate that exposure of LECs to UV-A dosages similar to those used in the CXL procedure promotes the expression of genes known to promote apoptosis. In the presence of riboflavin, the damage caused by UV-A treatment was marginalised, but not totally blocked.

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