Hyperosmolarity induces formation of NETs by neutrophils. This NETosis mechanism may explain the presence of excessive NETs on the ocular surface of patients with dry eye disease. Because they reduce hyperosmolarity-induced NETosis, FPR2 agonists may have therapeutic potential in these patients.

Primary neuronal cultures have been widely used to study neuronal morphology, neurophysiology, neurodegenerative processes, and molecular mechanism of synaptic plasticity underlying learning and memory. Yet, the unique behavioral properties of neurons make them challenging to study -with phenotypic differences expressed as subtle changes in neuronal arborization rather than easy to assay features such as cell count. The need to analyze morphology, growth, and intracellular transport has motivated the development of increasingly sophisticated microscopes and image analysis techniques. Due to its high-contrast, highspecificity output, many assays rely on confocal fluorescence microscopy, genetic methods, or antibody staining techniques. These approaches often limit the ability to measure quantitatively dynamic activity such as intracellular transport and growth. In this work, we describe a method for label-free live-cell cell imaging with antibody staining specificity by estimating the associated fluorescent signals via quantitative phase imaging and deep convolutional neural networks. This computationally inferred fluorescence image is then used to generate a semantic segmentation map, annotating subcellular compartments of live unlabeled neural cultures. These synthetic fluorescence maps were further applied to study the time-lapse development of hippocampal neurons, highlighting the relationships between the cellular dry mass production and the dynamic transport activity within the nucleus and neurites. Our implementation provides a high-throughput strategy to analyze neural network arborization dynamically, with high specificity and without the typical phototoxicity and photobleaching limitations associated with fluorescent markers.

PurposeCorneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes.MethodsSpheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes.ResultsStromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids.ConclusionStemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.