Maruotti, J. | bioRxiv Science

bioRxiv2020

A high-throughput screening platform for pigment regulating agents using pluripotent stem cell derived melanocytes.

In this study, we describe a simple and straight-forward assay using induced pluripotent stem cell derived melanocytes and high-throughput flow cytometry, to screen and identify pigment regulating agents. The assays is based on the correlation between forward light-scatter characteristics and melanin content, with pigmented cells displaying high light absorption/low forward light-scatter, while the opposite is true for lowly pigmented melanocytes, as a result of genetic background or chemical treatments. Orthogonal validation is then performed by regular melanin quantification. Such approach was validated using a set of 80 small molecules, and yielded a confirmed hit. The assay described in this study may prove a useful tool to identify modulators of melanogenesis in human melanocytes.

cell biology↗

bioRxiv2018

Axon Guidance Signaling Modulates Epithelial to Mesenchymal Transition in Stem Cell-Derived Retinal Pigment Epithelium

The critical role of epithelial to mesenchymal transition (EMT) in embryonic development, malignant transformation, and tumor progression has been well studied in normal and cancerous tissues and cells. Interestingly, EMT has also been reported to play a key role in the early progression of several retinal degenerative diseases, including scarring associated proliferative vitro-retinopathy (PVR), choroidal neo-vascularization induced \"wet\" age-related macular degeneration (AMD) and diabetic retinopathy (DR). Despite these studies, many questions remain unexplored regarding EMT-associated retinal pigment epithelium (RPE) degeneration and dysfunction. We hypothesize that RPE cells undergo EMT prior to cell death during the progression of atrophic \"dry\" AMD. Utilizing human stem cell-derived RPE (hRPE) as a model to study RPE EMT, we optimized two independent but complementary RPE EMT induction systems: 1) enzymatic dissociation of hRPE monolayer cultures and 2) co-treatment of hRPE monolayer cultures with transforming growth factor beta (TGF-{beta}) and the inflammatory cytokine, tumor necrosis factor alpha (TNF-). To further understand the molecular mechanisms of RPE EMT regulation, we performed an RNA-Sequencing (RNA-Seq) time course examination across 48 hours beginning with EMT induction. Our transcriptome profiling provides a comprehensive quantification of dynamic signaling events and associated biological pathways underlying RPE EMT and reveals an intriguing significance for widespread dysregulation of multiple axon guidance molecules in this process.

genomics↗