Technical Reports

Building on their recent work establishing long-term cultures from Lgr5⁺ cells of the small intestine and stomach, Shiro Yui and colleagues describe a new colonic stem cell culture and expansion method generating organoids from single Lgr5⁺ stem cells. The study provides proof of principle that the cultured Lgr5⁺ cells can be used for stem cell therapy to repair superficially damaged epithelium in a dextran sulfate sodium (DSS)-induced acute colitis mouse model.

There are currently no good ways to track human cells in vivo in a clinical setting using magnetic resonance imaging (MRI) based on superparamagnetic iron-oxide nanoparticles. Mya Thu and colleagues have introduced a simple magnetic cell labeling approach that combines three currently US Food and Drug Administration–approved drugs—ferumoxytol, heparin and protamine—to form self-assembling nanocomplexes of about 150 nm in size that effectively label cells for MRI. The approach was shown to effectively label three types of stem cells and two types of immune cells.

Targeting mesenchymal stem cells (MSCs), progenitors of osteoblasts, to bone has been a long-standing goal but has had limited success so far. Here, Min Guan and her colleagues deliver a peptidomimetic integrin ligand against integrin α4β1 conjugated to the bone-seeking agent bisphosphonate alendronate as a means of attracting infused and/or endogenous MSCs to the bone surface to stimulate bone formation. The approach was tested in both xenotransplantation and immunocompetent mice, as well as in mouse models of trabecular bone loss induced by aging and estrogen deficiency (ovariectomy).

Zhang and colleagues have developed a new targeted delivery system for RNA interference–based bone anabolic therapy. Using dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine (AspSerSer)₆, the system provided selective enrichment of the encapsulated osteogenic siRNA in osteogenic lineage cells at the bone formation surface and the subsequent depletion of the target gene, encoding the bone formation inhibitor casein kinase-2 interacting protein-1 (PLEKHO1, also known as CKIP-1), leading to the promotion of bone formation in healthy and osteoporotic rats.

Mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) in the majority of people with grade 2 and 3 gliomas is associated with elevated levels of 2-hydroxyglutarate (2HG) within the tumor. As harboring IDH1 or IDH2 mutations confers a considerable survival benefit in these individuals, there has been considerable interest in studying this metabolite as a potential biomarker. Here, Changho Choi et al. report the successful noninvasive detection of 2HG in 30 subjects with gliomas using a proton magnetic resonance spectroscopy approach.

Kejia Cai et al. describe a method to non-invasively detect glutamate (Glu) concentrations in the brain with MRI at high resolution. The approach is based on the pH-dependent chemical exchange saturation transfer (CEST) effect between the amino group of Glu and bulk water and offers advantages over proton magnetic resonance spectroscopy. Feasibility of GluCEST was demonstrated in rat brain after middle cerebral artery occlusion stroke and in a rat brain tumor model, as well as in healthy human brain at 7 Tesla.

Cell-surface glycans are known to alter as Barrett's esophagus progresses to adenocarcinoma, leading to specific changes in lectin binding patterns. Bird-Lieberman and her colleagues have exploited this knowledge to develop a new endoscopic approach that uses fluorescent-labeled lectins to visualize pre-cancerous, high-grade dysplastic lesions in Barrett's esophagus that cannot be detected by conventional endoscopy. The method uses commonly available endoscopic equipment, provides a wide field of view and is shown here in ex vivo esophageal tissue.

Ali Ertürk and colleagues present a novel tetrahydrofuran-based histological tissue clearing procedure that renders fixed and unsectioned adult CNS tissue (spinal cord and brainstem) transparent and, as such, fully amenable to a range of different optical imaging techniques. This three-dimensional imaging method can be used for studying axon regeneration and glial reactions.

New methods for analyzing ligand-receptor binding under physiologic conditions in cell-based assays and living animals are much needed for the testing and validation of candidate therapeutic agents targeting ligand-receptor binding. Here, Kathryn Luker and her colleagues developed a molecular imaging assay for ligand-receptor binding based on Gaussia luciferase complementation, focusing on chemokine CXCL12 and its chemokine receptors CXCR4 and CXCR7, although the assay could be applied more broadly to any receptor with a protein ligand.

The future of imaging is the integration of function and anatomy. Hongki Yoo et al. have successfully done just that by combining two existing intravascular imaging techniques into a single catheter-based system. Their dual-modality intra-arterial catheter uses a combination of optical frequency domain imaging and near-infrared fluorescence imaging to simultaneously provide molecular information in the context of the surrounding three-dimensional microanatomy of the artery wall.

Makoto Mitsunaga et al. have developed a new form of molecular-targeted cancer therapy that provides an alternative to current photodynamic approaches where damage to surrounding healthy cells and tissues can be a problem. They use a target-specific photosensitizer based on a near-infrared phthalocyanine dye, which is conjugated to monoclonal antibodies targeting human epidermal growth factor receptors (HER1 and HER2). Selective treatment using this approach was shown in vivo in subcutaneous cancer xenografts in mice.

The prognosis for patients with advanced stage ovarian cancer is poor. Here, Gooitzen van Dam and colleagues demonstrate the first human application of a tumor-specific intraoperative fluorescence imaging methodology using a folate receptor-α (FR-α)-targeted fluorescent agent that exploits the overexpression of FR-α in the majority of epithelial ovarian cancers. It is hoped this approach may lead to improved intraoperative staging and more radical cytoreductive surgery.

By exploiting the thermoreversible properties of the US Food and Drug Administration–approved poloxamer 407 (triblock polymer) and 2-octylcyanoacrylate bioadhesive, Edward Chang et al. have developed a new method of sutureless vascular anastomosis, even in vessels with a diameter of less than 1.0 mm. This nonmechanical, sutureless approach compared favorably to the standard hand-sewn approach in long-term (two-year) rat studies.

Infection by coagulase-positive Staphylococcus aureus (S. aureus) is the most common cause of acute endocarditis, a destructive and progressive condition of heart valves. Here, Peter Panizzi and his colleagues have developed a targeted, noninvasive fluorescence or positron emission technology imaging strategy that uses an engineered analog of prothrombin that can detect S. aureus in vivo in the endocarditic vegetations that form as a result of bacterial colonization.

Ready access to diagnostic tests that work well under remote field conditions is a major barrier to improving the health of people in the developing world. Here, Curtis Chin and his colleagues have developed a chip-based, microfluidic device and handheld reader for the simultaneous and rapid diagnosis of HIV and syphilis that uses only 1 μl of unprocessed whole blood and that was successfully field tested in Rwanda.

Progress in understanding coronary artery disease has been hampered by the inability of current approaches to interrogate the human coronary wall at cellular-level resolution. Here, Liu and colleagues introduce a second-generation form of OCT, called ↘OCT, that provides three-dimensional images of human coronary atherosclerosis at an axial resolution of only 1 ↘m—an order of magnitude greater than that provided by standard OCT systems.

Using the recently developed phosphorescent probe PtP-C343, in combination with two-photon phosphorescence lifetime microscopy, Lecoq and his colleagues offer a method for mapping oxygen levels in both microvascular and extravascular compartments with high spatial and temporal resolution. They used this set-up to make micron-scale simultaneous measurements of partial pressure of oxygen (PO₂) and blood flow in the rat olfactory bulb vasculature and neuropil.

Current approaches for analyzing the functional heterogeneity of immune cells include ELISPOT and flow cytometry. Chao Ma et al. have introduced an antibody-barcode microchip platform that enables the measurement of a large panel of secreted proteins from several hundred single cells or small cell colonies in parallel. The platform offers advantages over existing technologies in terms of cost, multiplexing capacity, types of proteins that can be measured and experiments performed.

Whether graft rejection occurs by direct or indirect presentation of antigen is still controversial. However, using a mouse ear skin allograft model and a two-photon intravital imaging approach, Susanna Celli and her colleagues have been able to dissect some of the dynamic processes involved in graft rejection, including early- and late-stage events at the transplant site, as well as intermediate events in the draining lymph node.

Studies of the recently discovered human rhinovirus species C (HRV-C) virus have been hampered by an inability to propagate the virus in standard cell culture. Yury Bochkov et al. have now developed a tissue culture system for HRV-C using human organ culture of sinus mucosa. This, together with development of a reverse genetics system for HRV-C, should provide a better understanding of HRV-C biology and facilitate discovery of an HRV-C receptor.