SummaryA 3D cell culture chip was used for high-throughput screening of a human neural progenitor cell line. The differential toxicity of 24 compounds was determined on undifferentiated and differentiating NPCs. Five compounds led to significant differences in IC50 values between undifferentiated and differentiating cultures. This platform has potential use in phenotypic screening to elucidate molecular toxicology on human stem cells.

We developed a three-dimensional (3D) cellular microarray platform for the high-throughput (HT) analysis of human neural stem cell (hNSC) growth and differentiation. The growth of an immortalized hNSC line, ReNcell VM, was evaluated on a miniaturized cell culture chip consisting of 60 nl spots of cells encapsulated in alginate, and compared to standard 2D well plate culture conditions. Using a live/dead cell viability assay, we demonstrated that the hNSCs are able to expand on-chip, albeit with lower proliferation rates and viabilities than in conventional 2D culture platforms. Using an in-cell, on-chip immunofluorescence assay, which provides quantitative information on cellular levels of proteins involved in neural fate, we demonstrated that ReNcell VM can preserve its multipotent state during on-chip expansion. Moreover, differentiation of the hNSCs into glial progeny was achieved both off- and on-chip six days after growth factor removal, accompanied by a decrease in the neural progenitor markers. The versatility of the platform was further demonstrated by complementing the cell culture chip with a chamber system that allowed us to screen for differential toxicity of small molecules to hNSCs. Using this approach, we showed differential toxicity when evaluating three neurotoxic compounds and one antiproliferative compound, and the null effect of a non-toxic compound at relevant concentrations. Thus, our 3D high-throughput microarray platform may help predict, in vitro, which compounds pose an increased threat to neural development and should therefore be prioritized for further screening and evaluation.

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

Zika virus (ZIKV) is an enveloped RNA virus from the flavivirus family that can cause fetal neural abnormalities in pregnant women. Previously, we established that ZIKV-EP (envelope protein) binds to human placental chondroitin sulfate (CS), suggesting that CS may be a potential host cell surface receptor in ZIKV pathogenesis. In this study, we further characterized the GAG disaccharide composition of other biological tissues (i.e., mosquitoes, fetal brain cells, and eye tissues) in ZIKV pathogenesis to investigate the role of tissue specific GAGs. Heparan sulfate (HS) was the major GAG, and levels of HS-6-sulfo, HS 0S (unsulfated HS), and CS 4S disaccharides were the main differences in the GAG composition of Aedes aegypti and Aedes albopictus mosquitoes. In human fetal neural progenitor and differentiated cells, HS 0S and CS 4S were the main disaccharides. A change in disaccharide composition levels was observed between undifferentiated and differentiated cells. In different regions of the bovine eyes, CS was the major GAG, and the amounts of hyaluronic acid or keratan sulfate varied depending on the region of the eye. Next, we examined heparin (HP) of various structures to investigate their potential in vitro antiviral activity against ZIKV and Dengue virus (DENV) infection in Vero cells. All compounds effectively inhibited DENV replication; however, they surprisingly promoted ZIKV replication. HP of longer chain lengths more strongly promoted activity in ZIKV replication. This study further expands our understanding of role of GAGs in ZIKV pathogenesis and carbohydratebased antivirals against flaviviral infection.O nce believed to be a benign virus, Zika virus (ZIKV) has cumulatively led to >200000 infections resulting in a global health risk, especially in pregnant women. ZIKV is an

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.