Corneal endothelial expansion using human umbilical cord mesenchymal stem cell-derived conditioned medium.

Rabbit corneal endothelial cells are frequently used in pharmacological experiments and are useful for corneal transplant experiments. We performed the present study to analyze the effect of conditioned medium (CM) derived from human umbilical cord mesenchymal stem cells (HUMSCs) on the growth of rabbit corneal endothelial cells (RCECs) and to establish a program for expansion of RCECs in vitro. RCECs were cultured using a CM derived from HUMSCs (HUMSCs-CM) in vitro. The proliferation ability of RCECs cultured in the presence of HUMSCs-CM was evaluated by conducting 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, colony formation, and scratch migration assays. The proliferation ability of RCECs maintained in HUMSCs-CM was significantly enhanced as compared to RCECs cultivated in the control group. Immunofluorescence indicated that zonula occludens-1 (ZO-1) and N-cadherin were located at intercellular junctions. Real-time PCR and western blot analyses demonstrated that the CEC-relative functional markers were expressed in RCECs maintained in HUMSCs-CM. Flow cytometry analyses demonstrated that HUMSCs-CM promoted the G0/G1 entrance to the S phase in RCECs. Our results demonstrated that HUMSCs-CM induced the proliferation of RCECs in vitro and maintained the necessary characteristic phenotypes. The expanded RCECs may provide a promising cell source for experimental research and clinical therapy.

EPZ015666, a selective protein arginine methyltransferase 5 (PRMT5) inhibitor with an antitumour effect in retinoblastoma.

Retinoblastoma (RB) is the most common intraocular malignant tumour in infants, and chemotherapy has been the primary therapy method in recent years. PRMT5 is an important member of the protein arginine methyltransferase family, which plays an important role in various tumours. Our study showed that PRMT5 was overexpressed in retinoblastoma and played an important role in retinoblastoma cell growth. EPZ015666 is a novel PRMT5 inhibitor, and we found that it inhibited retinoblastoma cell proliferation and led to cell cycle arrest at the G1 phase. At the same time, EPZ015666 regulated cell cycle related protein (P53, P21, P27, CDK2) expression. In brief, our study showed that PRMT5 promoted retinoblastoma growth, the PRMT5 inhibitor EPZ015666 inhibited retinoblastoma in vitro by regulating P53-P21/P27-CDK2 signaling pathways and slowed retinoblastoma growth in a xenograft model.

Identification of neural stem and progenitor cell subpopulations using DC insulator-based dielectrophoresis.

Neural stem and progenitor cells (NSPCs) are an extremely important group of cells that form the central nervous system during development and have the potential to repair damage in conditions such as stroke impairment, spinal cord injury and Parkinson's disease degradation. Current schemes for separation of NSPCs are inadequate due to the complexity and diversity of cells in the population and lack sufficient markers to distinguish diverse cell types. This study presents an unbiased high-resolution separation and characterization of NSPC subpopulations using direct current insulator-based dielectrophoresis (DC-iDEP). The properties of the cells were identified by the ratio of electrokinetic (EK) to dielectrophoretic (DEP) mobilities. The ratio factor of NSPCs showed more heterogeneity variance (SD = 3.4-3.9) than the controlled more homogeneous human embryonic kidney cells (SD = 1.1), supporting the presence of distinct subpopulations of cells in NSPC cultures. This measure reflected NSPC fate potential since the ratio factor distribution of more neurogenic populations of NSPCs was distinct from the distribution of astrogenic NSPC populations (confidence level >99.9%). The abundance of NSPCs captured with different ranges of ratio of EK to DEP mobilities also exhibit final fate trends consistent with established final fates of the chosen samples. DC-iDEP is a novel, label-free and non-destructive method for differentiating and characterizing, and potentially separating, neural stem cell subpopulations that differ in fate.

Separation of neural stem cells by whole cell membrane capacitance using dielectrophoresis.

Whole cell membrane capacitance is an electrophysiological property of the plasma membrane that serves as a biomarker for stem cell fate potential. Neural stem and progenitor cells (NSPCs) that differ in ability to form neurons or astrocytes are distinguished by membrane capacitance measured by dielectrophoresis (DEP). Differences in membrane capacitance are sufficient to enable the enrichment of neuron- or astrocyte-forming cells by DEP, showing the separation of stem cells on the basis of fate potential by membrane capacitance. NSPCs sorted by DEP need not be labeled and do not experience toxic effects from the sorting procedure. Other stem cell populations also display shifts in membrane capacitance as cells differentiate to a particular fate, clarifying the value of sorting a variety of stem cell types by capacitance. Here, we describe methods developed by our lab for separating NSPCs on the basis of capacitance using several types of DEP microfluidic devices, providing basic information on the sorting procedure as well as specific advantages and disadvantages of each device.

Structure-Based Discovery of a Series of 5H-Pyrrolo[2,3-b]pyrazine FGFR Kinase Inhibitors.

Fibroblast growth factor receptors (FGFRs), a subfamily of receptor tyrosine kinases, are aberrant in various cancer types, and considered to be promising targets for cancer therapy. We started with a weak-active compound that was identified from our internal hepatocyte growth factor receptor (also called c-Met) inhibitor project, and optimized it with the guidance of a co-crystal structure of compound 8 with FGFR1. Through rational design, synthesis, and the biological evaluation of a series of 5H-pyrrolo[2,3-b]pyrazine derivatives, we discovered several potent FGFR kinase inhibitors. Among them, compound 13 displayed high selectivity and favorable metabolic properties, demonstrating a promising lead for further development.

Icaritin ameliorates RANKL-mediated osteoclastogenesis and ovariectomy-induced osteoporosis.

A rapidly aging society and longer life expectancy are causing osteoporosis to become a global epidemic. Over the last five decades, a number of drugs aimed at reducing bone resorption or restoring bone mass have been developed, but their efficacy and safety are limited. Icaritin (ICT) is a natural compound extracted from anti-osteoporosis herb Epimedium spp. and has been shown to inhibit osteoclast differentiation. However, the molecular mechanism by which ICT weaken RANKL-induced osteoclast differentiation has not been completely investigated. Here, we evaluated the anti-osteoclastogenic effect of ICT in vitro and the potential drug candidate for treating osteoporosis in vivo. In vitro study, ICT was found to inhibit osteoclast formation and bone resorption function via downregulating transcription factors activated T cell cytoplasm 1 (NFATc1) and c-fos, which further downregulate osteoclastogenesis-specific gene. In addition, the enhanced mitochondrial mass and function required for osteoclast differentiation was mitigated by ICT. The histomorphological results from an in vivo study showed that ICT attenuated the bone loss associated with ovariectomy (OVX). Based on these results, we propose ICT as a promising new drug strategy for osteoporosis that inhibits osteoclast differentiation.

Engineered 3D Immuno-Glial-Neurovascular Human Brain Model.

Patient-specific, human-based cellular models that integrate biomimetic BBB, immune, and myelinated neuron components are critically needed to enable translationally relevant and accelerated discovery of neurological disease mechanisms and interventions. By engineering a brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo- like hallmarks. As proof of principle, here we utilized the miBrain to model Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types in a modular system with unique utility for elucidating cell-type specific contributions to pathogenesis. We here harness this feature to identify that risk factor APOE4 in astrocytes promotes tau pathogenesis and neuronal dysregulation through crosstalk with microglia. One-Sentence Summary: A novel patient-specific brain model with BBB, neuronal, immune, and glial components was developed, characterized, and harnessed to model Alzheimer's Disease-associated pathologies and APOE4 genetic risk.

SP2509, a Selective Inhibitor of LSD1, Suppresses Retinoblastoma Growth by Downregulating ß-catenin Signaling.

Purpose: To study the role of lysine-specific demethylase 1 (LSD1) in retinoblastoma (RB) growth and to determine whether the LSD1 inhibitor SP2509 can inhibit RB progression. Methods: We detected the levels of LSD1 in 12 RB tissue samples, two RB cell lines (Y79 and Weri-RB1), and a retinal pigment epithelium cell line (ARPE-19). Overexpression or knockdown of LSD1 was performed to examine the role of LSD1 in RB cancer cell survival. In vitro and in vivo experiments were conducted to detect the antitumor effect of SP2509, and the antitumor mechanism of SP2509 was examined by RNA sequencing and Western blot. Results: LSD1 is overexpressed in RB tissues and cells and increases RB cancer cell viability and colony formation ability. The LSD1 inhibitor SP2509 inhibits RB cell proliferation in vitro and in vivo. Treatment with SP2509 increases the levels of dimethylated histone 3 lysine 4 (H3K4me2) and inhibits the expression of ß-catenin signaling pathway-related proteins in RB cells. Conclusions: We demonstrated that LSD1 is overexpressed in RB cells and promotes RB cell survival. The LSD1 inhibitor SP2509 exerted strong growth inhibition in vitro and in vivo, which was at least partially mediated by suppression of the ß-catenin pathway.

Label-free enrichment of fate-biased human neural stem and progenitor cells.

Human neural stem and progenitor cells (hNSPCs) have therapeutic potential to treat neural diseases and injuries since they provide neuroprotection and differentiate into astrocytes, neurons, and oligodendrocytes. However, cultures of hNSPCs are heterogeneous, containing cells linked to distinct differentiated cell fates. HNSPCs that differentiate into astrocytes are of interest for specific neurological diseases, creating a need for approaches that can detect and isolate these cells. Astrocyte-biased hNSPCs differ from other cell types in electrophysiological properties, namely membrane capacitance, and we hypothesized that this could be used to enrich these cells using dielectrophoresis (DEP). We implemented a two-step DEP sorting scheme, consisting of analysis to define the optimal sorting frequency followed by separation of cells at that frequency, to test whether astrocyte-biased cells could be separated from the other cell types present in hNSPC cultures. We developed a novel device that increased sorting reproducibility and provided both enriched and depleted cell populations in a single sort. Astrocyte-biased cells were successfully enriched from hNSPC cultures by DEP sorting, making this the first study to use electrophysiological properties for label-free enrichment of human astrocyte-biased cells. Enriched astrocyte-biased human cells enable future experiments to determine the specific properties of these important cells and test their therapeutic efficacy in animal models of neurological diseases.

High-throughput continuous dielectrophoretic separation of neural stem cells.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.

An integrated microfluidic platform for size-selective single-cell trapping of monocytes from blood.

Reliable separation and isolation of target single cells from bodily fluids with high purity is of great significance for an accurate and quantitative understanding of the cellular heterogeneity. Here, we describe a fully integrated single-blood-cell analysis platform capable of size-selective cell separation from a population containing a wide distribution of sizes such as diluted blood sample and highly efficient entrapment of single monocytes. The spiked single U937 cells (human monocyte cell line) are separated in sequence by two different-sized microfilters for removing large cell clumps, white blood cells, and red blood cells and then discriminated by dielectrophoretic force and isolated individually by downstream single-cell trapping arrays. When 2% hematocrit blood cells with a final ratio of 1:1000 U937 cells were introduced under the flow rate of 0.2 ml/h, 400 U937 cells were trapped sequentially and deterministically within 40 s with single-cell occupancy of up to 85%. As a proof-of-concept, we also demonstrated single monocyte isolation from diluted blood using the integrated microfluidic device. This size-selective, label-free, and live-cell enrichment microfluidic single blood-cell isolation platform for the processing of cancer and blood cells has a myriad of applications in areas such as single-cell genetic analysis, stem cell biology, point-of-care diagnostics, and cancer diagnostics.

Discovery of Potent Irreversible Pan-Fibroblast Growth Factor Receptor (FGFR) Inhibitors.

Fibroblast growth factor receptors (FGFR1-4) are promising therapeutic targets in many cancers. With the resurgence of interest in irreversible inhibitors, efforts have been directed to the discovery of irreversible FGFR inhibitors. Currently, several selective irreversible inhibitors are being evaluated in clinical trials that could covalently target a conserved cysteine in the P-loop of FGFR. In this article, we used a structure-guided approach that is rationalized by a computer-aided simulation to discover the novel and irreversible pan-FGFR inhibitor, 9g, which provided superior FGFR in vitro activities and decent selectivity over VEGFR2 (vascular endothelia growth factor receptor 2). In in vivo studies, 9g displayed clear antitumor activities in NCI-H1581 and SNU-16 xenograft mice models. Additionally, the diluting method confirmed the irreversible binding of 9g to FGFR.

Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.