Glycosaminoglycan Compositional Analysis of Relevant Tissues in Zika Virus Pathogenesis and in Vitro Evaluation of Heparin as an Antiviral against Zika Virus Infection.

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 carbohydrate-based antivirals against flaviviral infection.

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

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.

Three-Dimensional Cell-Based Microarrays: Printing Pluripotent Stem Cells into 3D Microenvironments.

Cell-based microarrays are valuable platforms for the study of cytotoxicity and cellular microenvironment because they enable high-throughput screening of large sets of conditions at reduced reagent consumption. However, most of the described microarray technologies have been applied to two-dimensional cultures, which do not accurately emulate the in vivo three-dimensional (3D) cell-cell and cell-extracellular matrix interactions.Herein, we describe the methodology for production of alginate- and Matrigel-based 3-D cell microarrays for the study of mouse and human pluripotent stem cells on two different chip-based platforms. We further provide protocols for on-chip proliferation/viability analysis and the assessment of protein expression by immunofluorescence.

High-Throughput Toxicity and Phenotypic Screening of 3D Human Neural Progenitor Cell Cultures on a Microarray Chip Platform.

A 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.