IKKβ Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-β) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied.

High-throughput screening for myelination promoting compounds using human stem cell-derived oligodendrocyte progenitor cells.

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous and genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively.

Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system.

Introduction: Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). Although OLs can be differentiated from human-induced pluripotent stem cells (hiPSCs), the modeling of axon myelination in human cells remains challenging. Brain microphysiological systems (bMPS, e.

Transcriptome Landscape of Epithelial to Mesenchymal Transition of Human Stem Cell-Derived RPE.

Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems.

Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection.

Pluripotent stem cells (PSCs) edited with genetic reporters are useful tools for differentiation analysis and for isolation of specific cell populations for study. Reporter integration into the genome is now commonly achieved by targeted DNA nuclease-enhanced homology directed repair (HDR). However, human PSCs are known to have a low frequency of gene knock-in (KI) by HDR, making reporter line generation an arduous process.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs.

The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment.

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous antitumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms, but also a T cell-intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining tumor-specific dysfunctional TILs, and PD-1 alone is not sufficient.

CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans.

Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood.