High-throughput Format Research Articles

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

Multiplex Detection of Five Canine Viral Pathogens for Dogs as Laboratory Animals by the Luminex xTAG Assay.

More and more dogs have been used as a disease model for medical research and drug safety evaluation. Therefore, it is important to make sure that the dogs and their living houses are special pathogen free. In this study, the development and evaluation of a Luminex xTAG assay for simultaneous detection of five canine viruses was carried out, including canine distemper virus, canine parvovirus, canine parainfluenza virus, canine adenovirus, and rabies virus. Assay specificity was accomplished by targeting conserved genomic regions for each virus. Hybridization between multiplexed PCR products and the labeled fluorescence microspheres was detected in a high throughput format using a Luminex fluorescence reader. The Luminex xTAG assay showed high sensitivity with limits of detection for the five viruses was 100 copies/μL. Specificity of the xTAG assay showed no amplification of canine coronavirus, pseudorabies virus and canine influenza virus indicating that the xTAG assay was specific. Seventy-five clinical samples were tested to evaluate the xTAG assay. The results showed 100% coincidence with the conventional PCR method. This is the first report of a specific and sensitive multiplex Luminex xTAG assay for simultaneous detection of five major canine viral pathogens. This assay will be a useful tool for quality control and environmental monitoring for dogs used as laboratory animals, may even be applied in laboratory epidemiological investigations.

Validating outsourced high throughput automated qPCR for increased research outputs from forest pathology trials

Needle diseases of Pinus radiata caused by Phytophthora pluvialis and Phythophthora kernoviae have been increasingly recognised since the discovery of red needle cast in 2008. There is a need for rapid diagnostic screening of numerous samples, but sample processing time, equipment and staff availability limit the throughput and utilisation of diagnostic qPCR analysis in the research environment. Automated and high-throughput capable DNA extraction and real-time PCR provides the opportunity to expand the capacity of research trial analysis and a potential alternative to laborious isolation and plating but must be thoroughly validated before results can be used with confidence. The use of a high-throughput format for qPCR assays targeting Phytophthora pluvialis and Phythophthora kernoviae was validated on a robotic platform, proving to be consistently more sensitive than isolation, achieving qPCR detection down to 1% diluted inoculated material for Phytophthora kernoviae and 10% for Phytophthora pluvialis. Plating results yielded a 60% detection rate of Phythophthora pluvialis in inoculated needle fragments, whereas qPCR yielded a 100% detection on the same material. High throughout automated qPCR can therefore be utilised with confidence in forest pathology research trial analyses in future.

Abstract 3868: Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells

Abstract Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays composed of both endothelial and stroma-producing cells were significantly more heterogeneous and more closely resembled capillaries than mono cell culture models utilizing only endothelial cells to generate tubules in an extracellular matrix. Current co-culture models using primary cells have donor variability and inconsistent results due to lot-to-lot variation. In this study, we established an in vitro co-culture model system consisting of an assay-ready mixture of an aortic endothelial cell line TeloHAEC-GFP and an hTERT immortalized adipose-derived mesenchymal stem cell line in a specially formulated medium containing VEGF supplement. Both cell lines were immortalized by hTERT alone and have been well-characterized; showing that the cells retain the most important characteristic of their primary counterparts. The new co-culture system forms functional tubular structures in less than 7 days, additionally, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining, indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, ramucirumab. More importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as sunitinib and bevacizumab. We also tested four HIF-1 inhibitors identified in previous high-throughput screens and found that those compounds inhibited tubule formation in the co-culture system. These results suggest that the co-culture system can mimic the hypoxic environment in solid tumors. Previously the authors optimized the system for 384-well performance and here we report further optimization of the system into a 1536-well high-throughput format and a shortening of the assay time frame to 3 days. Using this format, we evaluated 2816 drugs from The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC), and 35 potent inhibitors (IC50 ≤1 μM) were identified. Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study. Among the inhibitors, some compounds were proven to be involved in the hypoxia-inducible factor-1α and the nuclear factor-kappa B pathways. These results demonstrate that the co-culture model described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening. Citation Format: Chaozhong Zou, Shuaizhang LI, Chia-Wen Hsu, Menghang Xia, Metewo S. Enuameh. Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3868.

Hollow Micropillar Array Method for High-Capacity Drug Screening on Filter-Grown Epithelial Cells.

New high-throughput assay formats and innovative screening technologies are needed for miniaturized screens using small quantities of near-native, patient-derived cells. Here, we developed a hollow micropillar array method to screen compounds using epithelial cells cultured on a porous support, with the goal of screening thousands of compounds using a single 24 mm diameter transwell filter containing cultured cells. Test compounds (∼1 nL) in an alginate hydrogel were printed by microinjection in hollow cylindrical micropillars (height = 150 μm, inner diameter = 100 μm) spaced 300 μm apart in a square array configuration. Compounds were delivered by positioning the array near the surface of a cell layer, with 5-10 μm of distance between the micropillars and cell surface. Micropillar array geometry, and the viscosity of the hydrogel and overlying solutions, were optimized computationally and experimentally to produce sustained exposure of cells to test compounds with minimal cross-talk from compounds in neighboring micropillar wells. The method was implemented using a 10 × 10 micropillar array (size = 3 × 3 mm) on CFTR-expressing epithelial cells, in which CFTR chloride channel function was measured from fluorescence in response to iodide addition using a genetically encoded cytoplasmic yellow fluorescent protein halide indicator. The hollow micropillar array platform developed here should be generally applicable for high-capacity drug screening using small numbers of cells cultured on solid or porous supports.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament.

The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z’-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens.

Nitrogen Cycle Evaluation (NiCE) Chip for Simultaneous Analysis of Multiple N Cycle-Associated Genes.

Various microorganisms play key roles in the nitrogen (N) cycle. Quantitative PCR (qPCR) and PCR amplicon sequencing of N cycle functional genes allow us to analyze the abundance and diversity of microbes responsible for N-transforming reactions in various environmental samples. However, analysis of multiple target genes can be cumbersome and expensive. PCR-independent analysis, such as metagenomics and metatranscriptomics, is useful but expensive, especially when we analyze multiple samples and try to detect N cycle functional genes present at a relatively low abundance. Here, we present the application of microfluidic qPCR chip technology to simultaneously quantify and prepare amplicon sequence libraries for multiple N cycle functional genes as well as taxon-specific 16S rRNA gene markers for many samples. This approach, named the nitrogen cycle evaluation (NiCE) chip, was evaluated by using DNA from pure and artificially mixed bacterial cultures and by comparing the results with those obtained by conventional qPCR and amplicon sequencing methods. Quantitative results obtained by the NiCE chip were comparable to those obtained by conventional qPCR. In addition, the NiCE chip was successfully applied to examine the abundance and diversity of N cycle functional genes in wastewater samples. Although nonspecific amplification was detected on the NiCE chip, this can be overcome by optimizing the primer sequences in the future. As the NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes, this tool should advance our ability to explore N cycling in various samples.IMPORTANCE We report a novel approach, namely, the nitrogen cycle evaluation (NiCE) chip, by using microfluidic qPCR chip technology. By sequencing the amplicons recovered from the NiCE chip, we can assess the diversities of N cycle functional genes. The NiCE chip technology is applicable to analysis of the temporal dynamics of N cycle gene transcription in wastewater treatment bioreactors. The NiCE chip can provide a high-throughput format to quantify and prepare sequence libraries for multiple N cycle functional genes. While there is room for future improvement, this tool should significantly advance our ability to explore the N cycle in various environmental samples.

AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish.

The aryl hydrocarbon receptor (AHR) is a conserved ligand-activated transcription factor required for proper vertebrate development and homeostasis. The inappropriate activation of AHR by ubiquitous pollutants can lead to adverse effects on wildlife and human health. The zebrafish is a powerful model system that provides a vertebrate data stream that anchors hypothesis at the genetic and cellular levels to observations at the morphological and behavioral level, in a high-throughput format. In order to investigate the endogenous functions of AHR, we generated an AHR2 (homolog of human AHR)-null zebrafish line (ahr2osu1) using the clustered, regulatory interspaced, short palindromic repeats (CRISPR)-Cas9 precision genome editing method. In zebrafish, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated toxicity requires AHR2. The AHR2-null line was resistant to TCDD-induced toxicity, indicating the line can be used to investigate the biological and toxicological functions of AHR2. The AHR2-null zebrafish exhibited decreased survival and fecundity compared to the wild type line. At 36 weeks, histological evaluations of the AHR2-null ovaries revealed a reduction of mature follicles when compared to wild type ovaries, suggesting AHR2 regulates follicle growth in zebrafish. AHR2-null adults had malformed cranial skeletal bones and severely damaged fins. Our data suggests AHR2 regulates some aspect(s) of neuromuscular and/or sensory system development, with impaired behavioral responses observed in larval and adult AHR2-null zebrafish. This study increases our understanding of the endogenous functions of AHR, which may help foster a better understanding of the target organs and molecular mechanisms involved in AHR-mediated toxicities.

Development of a high-throughput assay for rapid screening of butanologenic strains

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to “ethanol interference”. To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP+ to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

Next generation high throughput DNA damage detection platform for genotoxic compound screening

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

Yeast-based genotoxicity tests for assessing DNA alterations and DNA stress responses: a 40-year overview.

By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.

Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format.

Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic

Acidic hydrolysis is commonly used as a first step to break down oligo- and polysaccharides into monosaccharide units for structural analysis. While easy to set up and amenable to mass spectrometry detection, acid hydrolysis is not without its drawbacks. For example, ring-destruction side reactions and degradation products, along with difficulties in optimizing conditions from analyte to analyte, greatly limits its broad utility. Herein we report studies on a hydrogen peroxide/CuGGH metallopeptide-based glycosidase mimetic design for a more efficient and controllable carbohydrate hydrolysis. A library of methyl glycosides consisting of ten common monosaccharide substrates, along with oligosaccharide substrates, was screened with the artificial glycosidase for hydrolytic activity in a high-throughput format with a robotic liquid handling system. The artificial glycosidase was found to be active towards most screened linkages, including alpha- and beta-anomers, thus serving as a potential alternative method for traditional acidic hydrolysis approaches of oligosaccharides.

A cell death assay for assessing the mitochondrial targeting of proteins

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.

Identification of Novel "Inks" for 3D Printing Using High-Throughput Screening: Bioresorbable Photocurable Polymers for Controlled Drug Delivery.

A robust methodology is presented to identify novel biomaterials suitable for three-dimensional (3D) printing. Currently, the application of additive manufacturing is limited by the availability of functional inks, especially in the area of biomaterials; this is the first time when this method is used to tackle this problem, allowing hundreds of formulations to be readily assessed. Several functional properties, including the release of an antidepressive drug (paroxetine), cytotoxicity, and printability, are screened for 253 new ink formulations in a high-throughput format as well as mechanical properties. The selected candidates with the desirable properties are successfully scaled up using 3D printing into a range of object architectures. A full drug release study and degradability and tensile modulus experiments are presented on a simple architecture to validating the suitability of this methodology to identify printable inks for 3D printing devices with bespoke properties.

SOX10 Single Transcription Factor-Based Fast and Efficient Generation ofOligodendrocytes from Human Pluripotent Stem Cells.

SummaryScarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.