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Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue
The ciliary margin of the eye functions as a source of multipotent progenitor cells in certain organisms but whether it plays this role in humans has not been easy to study. Here the authors culture human embryonic stem cells that self-organize into retinal tissue, and show that ciliary margin-like growth zones emerge from the developing human retinal tissue and contain stem cell niches.
- Atsushi Kuwahara
- , Chikafumi Ozone
- & Yoshiki Sasai
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| Open Access
Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets
Antigenic peptides are loaded into major histocompatibility complex class I (MHC I) molecules after entering the ER via the TAP transporter. Here, Fischbach et al. develop a flow cytometry assay to monitor TAP-dependent peptide translocation that is sensitive enough to use on scarce primary cell subsets.
- Hanna Fischbach
- , Marius Döring
- & Robert Tampé
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Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy
Tumour microenvironment affects the outcome of pharmacological anticancer treatments. Here, Kenny et al. show that organotypic cultures of ovarian cancer cells can recapitulate metastasis. They identify several new compounds that block cancer invasion and metastasis and improve survival in mouse models.
- Hilary A. Kenny
- , Madhu Lal-Nag
- & Ernst Lengyel
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Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles
Individual viral particles can display heterogeneity in their composition of lipids, proteins or nucleic acids. Here, the authors apply advanced flow cytometry techniques to sort Junin virus particles and show that particle size and envelope glycoprotein concentration correlate with infectivity.
- Raphaël Gaudin
- & Natasha S Barteneva
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| Open Access
DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts
Cancer cells within the same tumour are heterogeneous in their tumorigenic potential, differentiation status and sensitivity to treatments. Here Nguyen et al. use a sensitive DNA barcoding method to characterize the diversity of clonal growth behaviour within human breast tumours.
- Long V. Nguyen
- , Claire L. Cox
- & Connie J. Eaves
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High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry
Flow cytometry allows high-throughput analysis of multiple proteins in individual cells, but relies on availability of antibodies. Here Porichis et al.report a sensitive method for multi-parameter flow cytometric and imaging detection of proteins together with mRNA or miRNA at the single-cell level.
- Filippos Porichis
- , Meghan G. Hart
- & Daniel E. Kaufmann
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Localized light-induced protein dimerization in living cells using a photocaged dimerizer
Protein localization in cells can yield much information about the spatial arrangement of cellular processes and the participating groups. Here, the authors present a membrane-permeable and photoactive agent for localized protein dimerization in cells.
- Edward R. Ballister
- , Chanat Aonbangkhen
- & David M. Chenoweth
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RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways
Large-scale genetic screens combined with phenotype assays can identify novel regulators of specific cellular functions. Here, Seo et al. use shRNA-based genetic selection to identify novel genes regulating cell migration, functioning within the PI 3-kinase/PTEN/AKT signalling pathway.
- Minchul Seo
- , Shinrye Lee
- & Kyoungho Suk
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Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria
Research on the roles played by gut microbes in human health is hampered by our inability to isolate and study them in pure cultures. Here, the authors describe an approach that allows targeted cultivation and rapid characterization of a significant proportion of human gut bacteria.
- Elizabeth A. Rettedal
- , Heidi Gumpert
- & Morten O.A. Sommer
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Poking cells for efficient vector-free intracellular delivery
The incorporation of foreign objects into cells can be used in various avenues of biological research, although crossing the cell membrane can be challenging. Here, the authors use a diamond nanoneedle array for enhanced delivery of various particles into cells, including neurons.
- Ying Wang
- , Yang Yang
- & Peng Shi
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| Open Access
Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells
Numerous obstacles posed by cellular subcompartments and structures constrain protein transport in the cell. Here, Baum et al.map the intracellular topology from a diffusing protein’s point of view by measuring the diffusive movements of fluorescently labelled reporter proteins in living cells on multiple time and length scales.
- Michael Baum
- , Fabian Erdel
- & Karsten Rippe
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| Open Access
3D niche microarrays for systems-level analyses of cell fate
3D cell culture matrices more closely resemble the natural microenvironments of stem cells than 2D systems. Here, the authors present a 3D cell culture approach to screen for the influence of environmental parameters on self-renewal and differentiation of single mouse embryonic stem cells.
- A. Ranga
- , S. Gobaa
- & M. P. Lutolf
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Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis
Many tissues can be grown as 3D spheroid models in hanging drops of media. Here, Frey et al. develop a microfluidic, interconnected hanging drop network to facilitate inter-drop communication, and demonstrate that pro-drug conversion by liver spheroids can limit the growth of cancer spheroids in adjacent drops.
- Olivier Frey
- , Patrick M. Misun
- & Andreas Hierlemann
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| Open Access
Generation of organized germ layers from a single mouse embryonic stem cell
The three germ layers are formed from the inner cell mass of the mammalian embryo during gastrulation. Here, the authors present a method by which a single mouse embryonic stem cell, derived from inner cell mass, differentiates into the three germ layers in a self-organized manner when cultured in soft fibrin gel.
- Yeh-Chuin Poh
- , Junwei Chen
- & Ning Wang
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Estimating the effective density of engineered nanomaterials for in vitro dosimetry
The assessment of nanomaterial toxicity can be hampered by difficulties in determining cell dosage. Here, the authors present a simple method for determination of nanomaterial agglomerate density in liquid media, enabling accurate calculation of dose delivered to cells in an in vitrosystem.
- Glen DeLoid
- , Joel M. Cohen
- & Philip Demokritou
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Microfluidic probe for single-cell analysis in adherent tissue culture
Cellular heterogeneity is a feature of diverse disease processes, yet is masked in typical biochemical assays. Sarkar et al.develop a microfluidic device to access the contents of single cells in adherent culture, allowing biochemical measurements to be connected with phenotypic information.
- Aniruddh Sarkar
- , Sarah Kolitz
- & Jongyoon Han
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Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing
Cryopreservation is the method of choice for extending the lifespan of biological samples, although high concentrations of potentially toxic additives are required. Here, the authors show that polymers that mimic antifreeze proteins can enhance cell cryopreservation at only 0.1 wt% concentration.
- Robert C. Deller
- , Manu Vatish
- & Matthew I. Gibson
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| Open Access
Postmortem examination of patient H.M.’s brain based on histological sectioning and digital 3D reconstruction
Studies on Patient H.M. showed that bilateral resection of the hippocampus results in impaired consolidation of long-term memory. Annese et al.create a digital map of Henry Molaison’s brain and find that a significant portion of the posterior hippocampus is actually histologically intact.
- Jacopo Annese
- , Natalie M. Schenker-Ahmed
- & Suzanne Corkin
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| Open Access
Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment
Laminin-511 and its cell-binding domain support long-term self-renewal of human pluripotent stem cells in xeno-free culture conditions. Here the authors show that a xeno-free culture matrix consisting of a full-length laminin-521 and the cell–cell adhesion protein E-cadherin supports clonal expansion of human embryonic stem cells.
- Sergey Rodin
- , Liselotte Antonsson
- & Karl Tryggvason
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Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L
Improving the quality of cotton fibres is important for agriculture and fibre length is influenced by far-red light. Here, the authors knock down the red/far-red photoreceptor Phytocrome A1 of cotton and show that this enhances cotton fibre length.
- Ibrokhim Y. Abdurakhmonov
- , Zabardast T. Buriev
- & Alan E. Pepper
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| Open Access
Visualization and targeted disruption of protein interactions in living cells
Screens for protein–protein interactions and for drugs that disrupt them typically use in vitro assays which fail to capture the complexity of the cell’s interior. By fixing proteins to distinct cellular locations, Herce et al.demonstrate a fluorescent-three-hybrid approach to probe such interactions in their cellular contexts.
- Henry D. Herce
- , Wen Deng
- & M. Cristina Cardoso
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Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage
Proteases of the caspase family are important regulators of apoptosis. Here, Zhang et al. present a genetically encoded protein biosensor that fluoresces if cleaved by caspase-3-like proteases, allowing real-time measurements of apoptosis in cells.
- Jiao Zhang
- , Xin Wang
- & Binghui Li
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Histology and postural change during the growth of the ceratopsian dinosaur Psittacosaurus lujiatunensis
A few dinosaurs have been inferred to have shifted from quadrupedality to bipedality, or vice versa, during growth. Here Zhao et al. use a combination of limb measurements and analysis of limb bone cross-sections to infer a shift towards bipedality in the primitive ceratopsian Psittacosaurus lujiatunensis.
- Qi Zhao
- , Michael J. Benton
- & Xing Xu
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Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells
The potential tumorigenicity of human pluripotent stem cells (hPSCs) limits their application in cell therapies. Ben-David et al.identify the tight-junction protein Claudin-6 as a cell-surface marker of hPSCs, and demonstrate three Claudin-6-based strategies to remove tumorigenic hPSCs from mixed cell cultures.
- Uri Ben-David
- , Neta Nudel
- & Nissim Benvenisty
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| Open Access
A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells
To transfer cultured human embryonic stem cells (hESCs) between culture dishes, cells need to be released using mechanical, enzymatic or chemical means, which can damage cells. Zhanget al. describe a thermomodulatable hydrogel that allows gentle, reagent-free cell passaging for the long-term culture of hESCs.
- Rong Zhang
- , Heidi K. Mjoseng
- & Mark Bradley
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| Open Access
Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells
The use of animal products as culture substrates for human embryonic stem cell and induced pluripotent stem cell culture raises numerous safety concerns in a therapeutic setting. Miyazaki et al.. show that minimal fragments of human laminins provide a more effective support for the culture of these cell types.
- Takamichi Miyazaki
- , Sugiko Futaki
- & Eihachiro Kawase
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Competition–colonization dynamics in experimental bacterial metacommunities
Species coexistence can be explained by the competition–colonization trade-off theory. Here, Livingston et al. illustrate this theory in a metacommunity experiment using two bacterial strains, finding a negative correlation between diversity and productivity when scaled to full metacommunities.
- George Livingston
- , Miguel Matias
- & Nicolas Mouquet
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| Open Access
Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy
Intracellular temperature mapping has not previously been achieved. Now, a fluorescent polymeric thermometer has been developed that can be used in combination with fluorescence-lifetime imaging microscopy to allow thermometry with spatial and temperature resolutions of 200 nm and 0.18–0.58 ° C.
- Kohki Okabe
- , Noriko Inada
- & Seiichi Uchiyama
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Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons
Dorsal root ganglion neurons can regenerate after injury, but the mechanisms underlying axon regrowth are unclear. To address this, an electroporation transfection method is developed that can alter the gene expression of dorsal root ganglion cells in a living adult mouse, providing a tool to study axon regeneration.
- Saijilafu
- , Eun-Mi Hur
- & Feng-Quan Zhou
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An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells
Stem cell-mediated regenerative medicine requires the development of defined culture systems for the maintenance of human embryonic stem cells. Here, feedback system control is used to identify a combination of three small molecule inhibitors that enables long-term human embryonic stem cell maintenance.
- Hideaki Tsutsui
- , Bahram Valamehr
- & Hong Wu
High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq