Stemness of the Organ of Corti Relates to the Epigenetic Status of Sox2 Enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti

The molecular basis of genistein-induced mitotic arrest and exit of self-renewal in embryonal carcinoma and primary cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic properties. The issue of genistein as a potential anti-cancer drug has been addressed in some papers, but comprehensive genomic analysis to elucidate the molecular mechanisms underlying the effect elicited by genistein on cancer cells have not been performed on primary cancer cells, but rather on transformed cell lines. In the present study, we treated primary glioblastoma, rhabdomyosarcoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with μ-molar concentrations of genistein and assessed mitotic index, cell morphology, global gene expression, and specific cell-cycle regulating genes. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common genistein-dependent transcriptional changes and accompanying signaling cascades.</p> <p>Methods</p> <p>We treated primary cancer cells and NCCIT cells with 50 μM genistein for 48 h. Thereafter, we compared the mitotic index of treated versus untreated cells and investigated the protein expression of key regulatory self renewal factors as OCT4, SOX2 and NANOG. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools.</p> <p>Results</p> <p>We found that cancer cells treated with genistein undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, <it>PBK</it>, <it>BUB1</it>, and <it>CDC20 </it>as determined by microarray-analysis and verified by Real-Time PCR. In contrast, human NCCIT cells showed over-expression of <it>GADD45 A </it>and <it>G </it>(growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, genistein induced the expression of apoptotic and anti-migratory proteins p53 and p38 in all cell lines. Genistein also up-regulated steady-state levels of both <it>CYCLIN A </it>and <it>B</it>.</p> <p>Conclusion</p> <p>The results of the present study, together with the results of earlier studies show that genistein targets genes involved in the progression of the M-phase of the cell cycle. In this respect it is of particular interest that this conclusion cannot be drawn from comparison of the individual genes found differentially regulated in the datasets, but by the rather global view of the pathways influenced by genistein treatment.</p

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2-/- embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7-21% form blastocysts compared to 76.2-83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages

Specific control of cell-material interactions: Targeting cell receptors using ligand-functionalized polymer substrates

Cells respond to their environment in complex and sometimes poorly understood ways. Protein, peptide and synthetic peptidomimetic ligands may all be used to stimulate cells via receptor signaling, using interactions that are often highly specific. Polymer substrates that present these ligands provide a promising way to control cell development, both for applications in biotechnology and for fundamental studies of cell biology. Here we review a large range of techniques that have been employed to create and characterize ligand-functionalized substrates, with a particular focus on techniques that allow specific and consistent stimulation

Implantation of functionalized thermally gelling xyloglucan hydrogel within the brain: associated neurite infiltration and inflammatory response

To develop neural tissue engineering strategies that are useful for repairing damaged neural pathways in the central nervous system, it is essential to control and optimise neurone and neurite interactions with functional scaffolds. In this study, the suitability of thermally gelling xyloglucan hydrogels, along with xyloglucan-graft-poly-d-lysine (PDL) hydrogels, was assessed through their implantation within the caudate putamen of adult rats. The ability of the hydrogel scaffolds to encourage the infiltration of axons in a controlled manner was investigated, as was the inflammatory response associated with the implantation. The microglia reaction was the same for unmodified xyloglucan and the xyloglucan-graft-PDL scaffolds, peaking after 3 days before decreasing back to homeostatic levels after ∼28 days. Penetration of the microglia into the scaffold was not observed, with these cells accumulating at the scaffold-tissue interface. For astrocytes, the other type of glial cell with migratory capacity, the peak activation occurred between 14 and 21 days. This reaction subsided more rapidly for the unmodified scaffold compared to the xyloglucan-graft-PDL scaffolds, which remained elevated 21-28 days before returning to homeostatic levels within 60 days. Most noteworthy was the discovery of increased infiltration levels for astrocytes and neurites with higher concentrations of grafted PDL. The timing of the astrocyte migration coincided with neurite infiltration within the scaffolds, suggesting that astrocytes may have facilitated this infiltration, possibly due to the secretion of laminin

Neurite infiltration and cellular response to electrospun polycaprolactone scaffolds implanted into the brain

Assessment of axonal infiltration and guidance within neural tissue engineering scaffolds, along with the characterisation of the inflammatory response, is critical in determining these scaffolds' potential for facilitating neural repair. In this study, the extent of microglial and astrocytic response was measured following implantation of electrospun poly(ε-caprolactone) (PCL) scaffolds into the caudate putamen of the adult rat brain. The inflammation peaked at around 4 days (microglia) and 7 days (astrocytes) and subsided to homeostatic levels by 60 days. There was no evidence of microglial encapsulation and indeed neurites had infiltrated the implants, evidence of scaffold-neural integration. Whilst the inflammatory response was uninfluenced by the degree of PCL fibre alignment, the extent of neurite entry was. Large porosity, as was the case with the randomly orientated polymer fibres, enabled neurite infiltration and growth within the scaffold. However, neuronal processes could not penetrate scaffolds when fibres were partially aligned and instead, preferentially grew perpendicular to the direction of PCL fibre alignment at the implant-tissue interface i.e. perpendicular, not parallel, contact guidance was provided. This investigation shows that electrospun PCL fibres are compatible with brain tissue and provide preliminary insights regarding the influence of microglia and astrocytes in neural integration within such scaffolds

Surface and bulk characterisation of electrospun membranes: Problems and improvements

Electrospun membranes are used in a variety of applications, including filtration systems and sensors for chemical detection, and have attracted increased interest in the field of tissue engineering and regenerative medicine. Successful integration of these materials into a specific technology will require understanding of the fibres' surface, bulk and architectural properties. Detailed characterisation of these properties is frequently overlooked, particularly in specialised interdisciplinary fields such as tissue engineering. In this article we have reviewed the current status of the characterisation of electrospun membranes, while recommending improvements in using these techniques to better understand these very interesting nanostructured materials. Crow