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Cláudia C Miranda, Tiago G Fernandes, M Margarida Diogo, Joaquim M S Cabral
The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21...
October 18, 2016: Biotechnology Journal
Fen Wei, Jie Chen, Xinye Chen, Baolong Bao
Flatfish with left-right eye asymmetry are the most significant among vertebrates. However, the genetic basis for the control of this characteristic is still unclear. We propose that the gene(s) for eye asymmetry initially control minor differences in cell number in the tissues around the eyes during development. This minor difference is then amplified, causing eye migration during metamorphosis. Therefore, comparing the neurula transcriptomes between flatfish species with different eye-reversal mutants may provide very useful information to screen for genes involved in eye asymmetry...
October 14, 2016: Gene
V Pibiri, A Ravarino, C Gerosa, M C Pintus, V Fanos, G Faa
The aim of this study was to analyze, by immunohistochemistry, the occurrence of stem/progenitor cells localized in the different niches of the developing human cerebellum. To this end, cerebellar samples were obtained from 3 fetuses and 3 newborns ranging, respectively, from 11 to 24 and from 30 to 38 weeks of gestation. Specimens were 10% formalin-fixed, routinely processed and paraffin-embedded; 3 μm-tick sections were immunostained with anti-SOX2 and PAX6 antibodies. Our study evidenced SOX2 and PAX6 immunoreactivity in precursors cells in all six developing human cerebella...
September 19, 2016: European Journal of Histochemistry: EJH
Balachandar Radhakrishnan, A Alwin Prem Anand
MicroRNAs (miRNAs) are a class of small regulatory RNAs involved in gene regulation. The regulation is effected by either translational inhibition or transcriptional silencing. In vertebrates, the importance of miRNA in development was discovered from mice and zebrafish dicer knockouts. The miRNA-9 (miR-9) is one of the most highly expressed miRNAs in the early and adult vertebrate brain. It has diverse functions within the developing vertebrate brain. In this article, the role of miR-9 in the developing forebrain (telencephalon and diencephalon), midbrain, hindbrain, and spinal cord of vertebrate species is highlighted...
2016: Journal of Experimental Neuroscience
Ashley Alldredge, Sabine Fuhrmann
Purpose: The scaffold protein Axin2 is an antagonist and universal target of the Wnt/β-catenin pathway. Disruption of Axin2 may lead to developmental eye defects; however, this has not been examined. The purpose of this study was to investigate the role of Axin2 during ocular and extraocular development in mouse. Methods: Animals heterozygous and homozygous for a Axin2lacZ knock-in allele were analyzed at different developmental stages for reporter expression, morphology as well as for the presence of ocular and extraocular markers using histologic and immunohistochemical techniques...
October 1, 2016: Investigative Ophthalmology & Visual Science
Wei-Lun Chang, Wu-Wei Lai, I-Ying Kuo, Chien-Yu Lin, Pei-Jung Lu, Bor-Shyang Sheu, Yi-Ching Wang
BACKGROUND: Prognosis of esophageal squamous cell carcinoma (ESCC) patients remains poor, and the chemoradiotherapy (CRT) applied to ESCC patients often failed. Therefore, development of biomarkers to predict CRT response is immensely important for choosing the best treatment strategy of an individual patient. METHODS: The methylation array and pyrosequencing methylation assay were performed in pre-treatment endoscopic biopsies to identify probes with differential CpG methylation levels between good and poor CRT responders in a cohort of 12 ESCC patients...
September 26, 2016: Journal of Gastroenterology
Sara M Duncan, Gail M Seigel
Historically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate. Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate...
2016: Journal of Biological Methods
Martin Miguel Casco-Robles, Md Rafiqul Islam, Wataru Inami, Hibiki Vincent Tanaka, Ailidana Kunahong, Hirofumi Yasumuro, Shiori Hanzawa, Roman Martin Casco-Robles, Fubito Toyama, Fumiaki Maruo, Chikafumi Chiba
The newt, a urodele amphibian, has an outstanding ability- even as an adult -to regenerate a functional retina through reprogramming and proliferation of the retinal pigment epithelium (RPE) cells, even though the neural retina is completely removed from the eye by surgery. It remains unknown how the newt invented such a superior mechanism. Here we show that disability of RPE cells to regenerate the retina brings about a symptom of proliferative vitreoretinopathy (PVR), even in the newt. When Pax6, a transcription factor that is re-expressed in reprogramming RPE cells, is knocked down in transgenic juvenile newts, these cells proliferate but eventually give rise to cell aggregates that uniformly express alpha smooth muscle actin, Vimentin and N-cadherin, the markers of myofibroblasts which are a major component of the sub-/epi-retinal membranes in PVR...
2016: Scientific Reports
Naghmeh Bahrami, Mohammad Bayat, Abdolreza Mohamadnia, Mehrdad Khakbiz, Meysam Yazdankhah, Jafar Ai, Somayeh Ebrahimi-Barough
There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton's jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects...
September 14, 2016: Molecular Neurobiology
Betty Freret-Hodara, Yi Cui, Amélie Griveau, Lisa Vigier, Yoko Arai, Jonathan Touboul, Alessandra Pierani
Loss of neurons in the neocortex is generally thought to result in a final reduction of cerebral volume. Yet, little is known on how the developing cerebral cortex copes with death of early-born neurons. Here, we tackled this issue by taking advantage of a transgenic mouse model in which, from early embryonic stages to mid-corticogenesis, abundant apoptosis is induced in the postmitotic compartment. Unexpectedly, the thickness of the mutant cortical plate at E18.5 was normal, due to an overproduction of upper layer neurons at E14...
September 12, 2016: Cerebral Cortex
Jian Sun, Yilin Zhao, Rebecca McGreal, Yamit Cohen-Tayar, Shira Rockowitz, Carola Wilczek, Ruth Ashery-Padan, David Shechter, Deyou Zheng, Ales Cvekl
BACKGROUND: Pax6 is a key regulator of the entire cascade of ocular lens formation through specific binding to promoters and enhancers of batteries of target genes. The promoters and enhancers communicate with each other through DNA looping mediated by multiple protein-DNA and protein-protein interactions and are marked by specific combinations of histone posttranslational modifications (PTMs). Enhancers are distinguished from bulk chromatin by specific modifications of core histone H3, including H3K4me1 and H3K27ac, while promoters show increased H3K4me3 PTM...
2016: Epigenetics & Chromatin
Christina Bachmann, Huong Nguyen, Joachim Rosenbusch, Linh Pham, Tamara Rabe, Megha Patwa, Godwin Sokpor, Rho H Seong, Ruth Ashery-Padan, Ahmed Mansouri, Anastassia Stoykova, Jochen F Staiger, Tran Tuoc
Neurogenesis is a key developmental event through which neurons are generated from neural stem/progenitor cells. Chromatin remodeling BAF (mSWI/SNF) complexes have been reported to play essential roles in the neurogenesis of the central nervous system. However, whether BAF complexes are required for neuron generation in the olfactory system is unknown. Here, we identified onscBAF and ornBAF complexes, which are specifically present in olfactory neural stem cells (oNSCs) and olfactory receptor neurons (ORNs), respectively...
September 2016: PLoS Genetics
Marta Grabiec, Hana Hříbková, Miroslav Vařecha, Dana Střítecká, Aleš Hampl, Petr Dvořák, Yuh-Man Sun
This study elucidated the stage-specific roles of FGF2 signaling during neural development using in-vitro human embryonic stem cell-based developmental modeling. We found that the dysregulation of FGF2 signaling prior to the onset of neural induction resulted in the malformation of neural rosettes (a neural tube-like structure), despite cells having undergone neural induction. The aberrant neural rosette formation may be attributed to the misplacement of ZO-1, which is a polarized tight junction protein and shown co-localized with FGF2/FGFR1 in the apical region of neural rosettes, subsequently led to abnormal neurogenesis...
August 27, 2016: Stem Cell Research
Cindy Tzu-Ling Huang, Yunlong Tao, Jianfeng Lu, Jeffrey R Jones, Lucas Fowler, Jason P Weick, Su-Chun Zhang
The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction...
2016: Scientific Reports
Shanli Tsui, Jie Wang, Ling Wang, Wei Dai, Luo Lu
BACKGROUND: The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. METHODS: Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells...
2016: PloS One
Alexandra Kerbl, José M Martín-Durán, Katrine Worsaae, Andreas Hejnol
BACKGROUND: Annelida is a morphologically diverse animal group that exhibits a remarkable variety in nervous system architecture (e.g., number and location of longitudinal cords, architecture of the brain). Despite this heterogeneity of neural arrangements, the molecular profiles related to central nervous system patterning seem to be conserved even between distantly related annelids. In particular, comparative molecular studies on brain and anterior neural region patterning genes have focused so far mainly on indirect-developing macrofaunal taxa...
2016: EvoDevo
Joanna Yeung, Thomas J Ha, Douglas J Swanson, Dan Goldowitz
UNLABELLED: Pax6 is a prominent gene in brain development. The deletion of Pax6 results in devastated development of eye, olfactory bulb, and cortex. However, it has been reported that the Pax6-null Sey cerebellum only has minor defects involving granule cells despite Pax6 being expressed throughout cerebellar development. The present work has uncovered a requirement of Pax6 in the development of all rhombic lip (RL) lineages. A significant downregulation of Tbr1 and Tbr2 expression is found in the Sey cerebellum, these are cell-specific markers of cerebellar nuclear (CN) neurons and unipolar brush cells (UBCs), respectively...
August 31, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Zahra Motahari, Reyna I Martinez-De Luna, Andrea S Viczian, Michael E Zuber
Vertebrate eye formation begins in the anterior neural plate in the eye field. Seven eye field transcription factors (EFTFs) are expressed in eye field cells and when expressed together are sufficient to generate retina from pluripotent cells. The EFTF Tbx3 can regulate the expression of some EFTFs; however, its role in retina formation is unknown. Here, we show that Tbx3 represses bmp4 transcription and is required in the eye field for both neural induction and normal eye formation in Xenopus laevis Although sufficient for neural induction, Tbx3-expressing pluripotent cells only form retina in the context of the eye field...
October 1, 2016: Development
Qingxiang Zhou, Dana F DeSantis, Markus Friedrich, Francesca Pignoni
The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli...
October 1, 2016: Developmental Biology
Petr Walczysko, Ann M Rajnicek, J Martin Collinson
PURPOSE: Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. METHODS: In vivo corneal epithelial wounding was performed in adult wild-type and Pax6(+/-) mice, and the healing migration rates were compared...
2016: Molecular Vision
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