Cheryl Leong

Neural stem cell specific fluorescent chemical probe binding to FABP7.

Fluorescent small molecules have become indispensable tools for biomedical research along with the rapidly developing optical imaging technology. We report here a neural stem cell specific boron-dipyrromethane (BODIPY) derivative compound of designation red 3 (CDr3), developed through a high throughput/content screening of in-house generated diversity oriented fluorescence library in stem cells at different developmental stages. This novel compound specifically detects living neural stem cells of both human and mouse origin. Furthermore, we identified its binding target by proteomic analysis as fatty acid binding protein 7 (FABP7), also known as brain lipid binding protein) which is highly expressed in neural stem cells and localized in the cytoplasm. CDr3 will be a valuable chemical tool in the study and applications of neural stem cells.

NeuO: a Fluorescent Chemical Probe for Live Neuron Labeling

To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.

Neural stem cell isolation from the whole mouse brain using the novel FABP7-binding fluorescent dye, CDr3

Methods for the isolation of live neural stem cells from the brain are limited due to the lack of well-defined cell surface markers and tools to detect intracellular markers. To date most methods depend on the labeling of extracellular markers using antibodies, with intracellular markers remaining inaccessible in live cells. Using a novel intracellular protein FABP7 (Fatty Acid Binding Protein-7) selective fluorescent chemical probe CDr3, we have successfully isolated high FABP7 expressing cells from the embryonic and adult mouse brains. These cells are capable of forming neurospheres in culture, express neural stem cell marker genes and differentiate into neurons, astrocytes and oligodendrocytes. Characterization of cells sorted with Aldefluor or antibodies against CD133 or SSEA-1 showed that the cells isolated by CDr3 exhibit a phenotype distinct from the cells sorted with conventional methods. FABP7 labeling with CDr3 represents a novel method for rapid isolation of neural stem cells based on the expression of a single intracellular marker.

Physiological Doses of Red Light Induce IL‐4 Release in Cocultures between Human Keratinocytes and Immune Cells

Phototherapy is routinely used for the treatment of various skin conditions and targeted therapy of superficial cancers. However, the molecular mechanisms behind their biological effects and the need for efficacy enhancing photosensitizers are not well addressed. Particularly, not much is known about the inherent effect of light from the visible spectrum on cytokine release and its downstream effects in keratinocytes and immune cells located in skin and therefore exposed to light. To address this, we delivered calibrated doses of well‐defined light qualities (380 to 660 nm) to cocultures of human keratinocytes and macrophage/dendritic cells in the absence or presence of the commonly used photosensitizer 8‐methoxypsoralen (8‐MOP). The experiments identified IL‐4 as a key effector cytokine released by this coculture model with need for 8‐MOP in the UVA1/blue (380 nm) and no requirement for photosensitizer in the red light spectrum (627 nm). 3D organotypic skin cultures treated with IL‐4 showed thickening of the epidermal layer and delayed differentiation. However unlike IL‐4 and UVA1/blue light treatment, red light did not reduce the expression of keratinocyte differentiation markers or increase signs of photo‐oxidative damage. This supports the application of isolated red light as a possible alternative for photo‐immunotherapy without need for additional photosensitizers.

A fluorescent probe for imaging symmetric and asymmetric cell division in neurosphere formation.

We report here a novel fluorescent chemical probe which stains distinct neural stem/progenitor cells (NSPCs) by binding to acid ceramidase in mouse neurospheres. is distributed evenly or unevenly to the daughter cells during multiple mitoses enabling the live imaging of symmetric and asymmetric divisions of isolated NSPCs.

A Fluorescent Probe for Neural Stem/Progenitor Cells with High Differentiation Capability into Neurons

Selection of a specific neural stem/progenitor cells (NSPCs) has attracted broad attention in regenerative medicine for neurological disorders. Here, we report a fluorescent probe, CDg13, and its application for isolating strong neurogenic NSPCs. In comparison to the NSPCs isolated by other biomarkers, CDg13‐stained NSPCs showed higher capability to differentiate into neurons. Target identification revealed that the fluorescence intensity of the probe within cells is inversely proportional to the expression levels of mouse and human Abcg2 transporters. These findings suggest that low Abcg2 expression is a biomarker for neurogenic NSPCs in mouse brain. Furthermore, CDg13 can be used to isolate Abcg2low cells from heterogeneous cell populations.

Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand

Opioids in skin function during stress response, regeneration, ageing and, particularly in regulating sensation. In chronic pruritus, topical treatment with Naltrexone changes μ-opioid receptor (μ-OR) localization to relieve itch. The molecular mechanisms behind the effects of Naltrexone on μ-OR function in reduction of itching behavior has not been studied. There is an immediate need to understand the endogenous complexity of μ-OR dynamics in normal and pathological skin conditions. Here we evaluate real-time behavior of μ-OR-Endomorphine complexes in the presence of agonist and antagonists. The μ-OR ligand Endomorphine-1 (EM) was conjugated to the fluorescent dye Tetramethylrhodamine (TAMRA) to investigate the effects of agonist and antagonists in N/TERT-1 keratinocytes. The cellular localization of the EM-TAMRA was followed through time resolved confocal microscopy and population analysis was performed by flow cytometry. The in vitro analyses demonstrate fast internalization and trafficking of the endogenous EM-TAMRA-μ-OR interactions in a qualitative manner. Competition with Endomorphine-1, Naltrexone and CTOP show both canonical and non-canonical effects in basal and differentiated keratinocytes. Acute and chronic treatment with Naltrexone and Endomorphine-1 increases EM-TAMRA binding to skin cells. Although Naltrexone is clinically effective in relieving itch, the mechanisms behind re-distribution of μ-ORs during clinical treatments are not known. Our study has given insight into cellular mechanisms of μ-OR ligand-receptor interactions after opioid agonist and antagonist treatments in vitro. These findings potentially offer opportunities in using novel treatment strategies for skin and peripheral sensory disorders.