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Methods in Molecular Biology

Asako Tajima, Isha Pradhan, Xuehui Geng, Massimo Trucco, Yong Fan
One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progress has been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma...
October 12, 2016: Methods in Molecular Biology
Dustin J Flanagan, Renate H M Schwab, Bang M Tran, Toby J Phesse, Elizabeth Vincan
The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro...
October 5, 2016: Methods in Molecular Biology
Yohei Shimono, Junko Mukohyama, Taichi Isobe, Darius M Johnston, Piero Dalerba, Akira Suzuki
Organoid culture is a three-dimensional culture method that enables ex vivo analysis of stem cell behavior and differentiation. This method is also applicable to the studies on stem cell characters of human cancer stem cells. The components of organoid culture include Matrigel® and a culture medium containing growth factor cocktails that mimic the microenvironments of organ stem cell niches. Here, we describe the basic methods for the organoid culture of dissociated or FACS-sorted human cancer stem cells. Then, we introduce a method to dissociate the organoids for serial passage and propagation...
September 22, 2016: Methods in Molecular Biology
Giulia Nigro, Melissa Hanson, Cindy Fevre, Marc Lecuit, Philippe J Sansonetti
The gut, particularly the colon, is the host of approximately 1000 bacterial species, the so-called gut microbiota. The relationship between the gut microbiota and the host is symbiotic and mutualistic, influencing many aspects of the biology of the host. This homeostatic balance can be disrupted by enteric pathogens, such as Shigella flexneri or Listeria monocytogenes, which are able to invade the epithelial layer and consequently subvert physiological functions. To study the host-microbe interactions in vitro, the crypt culture model, known as intestinal organoids, is a powerful tool...
September 15, 2016: Methods in Molecular Biology
Mahboobe Ghaedi, Laura E Niklason
Induced pluripotent stem (iPS) cells are the product of adult somatic cell reprogramming to an embryonic-like state by inducing a "forced" expression of specific genes. They are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and potency and differentiability. Human iPS cells are invaluable resource for basic research, cell therapy, drug discovery, and human organ tissue engineering. iPS cells can be derived from the patient to be treated and thus are genetically identical cells that may avoid immune rejection...
September 15, 2016: Methods in Molecular Biology
Hayley E Francies, Andrew Barthorpe, Anne McLaren-Douglas, William J Barendt, Mathew J Garnett
Drug sensitivity testing utilizing preclinical disease models such as cancer cell lines is an important and widely used tool for drug development. Importantly, when combined with molecular data such as gene copy number variation or somatic coding mutations, associations between drug sensitivity and molecular data can be used to develop markers to guide patient therapies. The use of organoids as a preclinical cancer model has become possible following recent work demonstrating that organoid cultures can be derived from patient tumors with a high rate of success...
September 15, 2016: Methods in Molecular Biology
Jessica L Forbester, Nicholas Hannan, Ludovic Vallier, Gordon Dougan
Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs)...
August 31, 2016: Methods in Molecular Biology
Yang-Yi Fan, Laurie A Davidson, Robert S Chapkin
Colonic organoids, three-dimensional colonic crypts grown in vitro that show realistic microanatomy, have many potential applications for studying physiology, developmental biology, and pathophysiology of intestinal diseases including inflammatory bowel disease and colorectal cancer. Here, we describe detailed protocols for mouse colonic crypt isolation, organoid culture, and downstream applications. Specific culture strategies including growth factor enriched Matrigel and Wnt and R-spondin conditioned media serve as key factors for enhancing the growth and cost efficiency of colonic organoid cultures...
August 19, 2016: Methods in Molecular Biology
Yong-Guo Zhang, Jun Sun
The intestinal epithelial cells function to gain nutrients, retain water and electrolytes, and form an efficient barrier against foreign microbes and antigens. Researchers employed cell culture lines derived from human or animal cancer cells as experimental models in vitro for understanding of intestinal infections. However, most in vitro models used to investigate interactions between bacteria and intestinal epithelial cells fail to recreate the differentiated tissue components and structure observed in the normal intestine...
August 19, 2016: Methods in Molecular Biology
Nina Bertaux-Skeirik, Jomaris Centeno, Jian Gao, Joel Gabre, Yana Zavros
The culture of organoids has represented a significant advancement in the gastrointestinal research field. Previous research studies have described the oncogenic transformation of human intestinal and mouse gastric organoids. Here we detail the protocol for the oncogenic transformation and orthotopic transplantation of human-derived gastric organoids.
August 19, 2016: Methods in Molecular Biology
Robert E Hynds, Colin R Butler, Sam M Janes, Adam Giangreco
Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells...
August 19, 2016: Methods in Molecular Biology
Hiroko Hisha, Hiroo Ueno
A novel lingual epithelial organoid culture system using a three-dimensional (3D) matrix and growth factors has recently been established. In the culture system, organoids with multilayered squamous keratinized epithelium and typical morphological features of filiform papillae are generated from single lingual epithelial cells at a high efficiency. The culture system is created in order to observe the differentiation and maturation process of each lingual epithelial stem cell and to observe abnormal organoid formation from malignant cells obtained from carcinogen-treated mice...
August 19, 2016: Methods in Molecular Biology
Valentina Benedetti, Valerio Brizi, Christodoulos Xinaris
Novel methods in developmental biology and stem cell research have made it possible to generate complex kidney tissues in vitro that resemble whole organs and are termed organoids. In this chapter we describe a technique using suspensions of fully dissociated mouse kidney cells to yield organoids that can become vascularized in vivo and mature and display physiological functions. This system can be used to produce fine-grained human-mouse chimeric organoids in which the renal differentiation potential of human cells can be assessed...
August 19, 2016: Methods in Molecular Biology
Toshio Takahashi
Acetylcholine (ACh) is a neurotransmitter that is present in central, parasympathetic, and neuromuscular synapses of mammals. However, non-neuronal ACh is also predicted to function as a local cell signaling molecule. The physiological significance of the presence of non-neuronal ACh in the intestine remains unclear. Here, experiments using cultured crypt-villus organoids that lack nerve and immune cells led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs)...
August 19, 2016: Methods in Molecular Biology
Guillem Paniagua Soriano, Herman S Overkleeft, Bogdan I Florea
Activity-based protein profiling (ABPP) is a method to highlight enzymatic activities in a biological sample, which uses chemical probes that react covalently with the catalytic nucleophile of the enzyme. To circumvent disadvantages associated with the presence of reporter tags on chemical probes, the probe is equipped with a ligation handle to which the reporter can be reacted at the desired time and place in the ABPP workflow. This chapter demonstrates the power of a triple bioorthogonal ligation strategy which addresses the three activities of the proteasome: the β5-subunit selective norbornene-tagged probe is reacted with fluorescent tetrazine, the β1-selective azide-functionalized probe was addressed with a biotinylated phosphine, followed by an alkyne-substituted pan-reactive probe to label the remaining β2 activity to which an azide-coupled fluorophore was ligated...
2017: Methods in Molecular Biology
Yinliang Yang, Marko Fonović, Steven H L Verhelst
The discovery of the protein targets of small molecule probes is a crucial aspect of activity-based protein profiling and chemical biology. Mass spectrometry is the primary method for target identification, and in the last decade, cleavable linkers have become a popular strategy to facilitate protein enrichment and identification. In this chapter, we provide an overview of cleavable linkers used in chemical proteomics approaches, discuss their different chemistries, and describe how they aid in protein identification...
2017: Methods in Molecular Biology
Jasper H L Claessen, Martin D Witte
Activity-based protein profiling using activity-based probes (ABPs) resulted in the identification of various enzymes that are involved in the onset and progress of diseases. Detection of such proteins, often expressed at low abundance, is greatly enhanced by incorporating chemically cleavable linkers in the ABP of choice. Initial affinity purification, followed by tailored chemical cleavage of the linker, allows for specific release of the captured enzymes and their interaction partners. When the ABPs are delivered directly to semi-permeabilized cells, in contrast to a crude cell lysate, the sensitivity and efficacy of cell impermeable probes can be enhanced even further...
2017: Methods in Molecular Biology
Marc P Baggelaar, Mario Van der Stelt
Competitive activity-based protein profiling is a highly efficient chemical biology technique to determine target engagement and selectivity profiles of enzyme inhibitors in complex proteomes. Fluorophosphonate-based fluorescent inhibitors are widely used as broad-spectrum probes for serine hydrolases. However, diacylglycerol lipase-α is not labeled by fluorophosphonate-based probes. To overcome this problem, we have developed a tailor-made activity-based probe that reacts with diacylglycerol lipase-α. Here we describe a case study in which we apply competitive activity-based protein profiling using a broad-spectrum and a tailor-made activity-based probe to establish selectivity and activity profiles of inhibitors targeting diacylglycerol lipase-α in the mouse brain proteome...
2017: Methods in Molecular Biology
Laura E Edgington-Mitchell, Matthew Bogyo, Martijn Verdoes
Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically "dark" and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy...
2017: Methods in Molecular Biology
Benedikt M Kessler, Sara Bursomanno, Joanna F McGouran, Ian D Hickson, Ying Liu
Posttranslational modification of proteins with the small ubiquitin-like modifier (SUMO) regulates protein function in the context of cell cycle and DNA repair. The occurrence of SUMOylation is less frequent as compared to protein modification with ubiquitin, and appears to be controlled by a smaller pool of conjugating and deconjugating enzymes. Mass spectrometry has been instrumental in defining specific as well as proteome-wide views of SUMO-dependent biological processes, and several methodological approaches have been developed in the recent past...
2017: Methods in Molecular Biology
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