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ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Korea University College of Medicine

The migration and differentiation of adult neural stem cells (aNSCs) are believed to be strongly influenced by the spatial distribution of extracellular matrix (ECM) proteins in the stem cell niche. In vitro culture platform, which involves the specific spatial distribution of ECM protein, could offer novel tools for better understanding of aNSC behavior in the spatial pattern of ECM proteins. In this work, we applied soft-lithographic technique to design simple and reproducible laminin (LN)-polylysine cell culture substrates and investigated how aNSCs respond to the various spatial distribution of laminin, one of ECM proteins enriched in the aNSC niche. We found that aNSC preferred to migrate and attach to LN stripes, and aNSC-derived neurons and astrocytes showed significant difference in motility towards LN stripes. By changing the spacing of LN stripes, we were able to control the alignment of neurons and astrocytes. To the best of our knowledge, this is the first time to investigate the differential cellular responses of aNSCs on ECM protein (LN) and cell adhesive synthetic polymer (PDL) using surface micropatterns. Our findings would provide a deeper understanding in astrocyte-neuron interactions as well as ECM-stem cell interactions.

Effects of ECM protein micropatterns on the migration and differentiation of adult neural stem cells.

Neurogenesis occurs spontaneously in the subventricular zone (SVZ) of the lateral ventricle in adult rodent brain, but it has long been debated whether there is sufficient adult neurogenesis in human SVZ. Subcallosal zone (SCZ), a posterior continuum of SVZ closely associated with posterior regions of cortical white matter, has also been reported to contain adult neural stem cells (aNSCs) in both rodents and humans. However, little is known whether SCZ-derived aNSC (SCZ-aNSCs) can produce cortical neurons following brain injury. We found that SCZ-aNSCs exhibited limited neuronal differentiation potential in culture and after transplantation in mice. Neuroblasts derived from SCZ initially migrated toward injured cortex regions following brain injury, but later exhibited apoptosis. Overexpression of anti-apoptotic bcl-xL in the SCZ by retroviral infection rescued neuroblasts from cell death in the injured cortex, but neuronal maturation was still limited, resulting in atrophy. In combination with Bcl-xL, infusion of brain-derived neurotropic factor rescued atrophy, and importantly, a subset of such SCZ-aNSCs differentiated and attained morphological and physiological characteristics of mature, excitatory neurons. These results suggest that the combination of anti-apoptotic and neurotrophic factors might enable the use of aNSCs derived from the SCZ in cortical neurogenesis for neural replacement therapy.

Promotion of Cortical Neurogenesis from the Neural Stem Cells in the Adult Mouse Subcallosal Zone.

Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.

Tissue clearing technology is currently one of the fastest growing fields in biomedical sciences. Tissue clearing techniques have become a powerful approach to understand further the structural information of intact biological tissues. Moreover, technological improvements in tissue clearing and optics allowed the visualization of neural network in the whole brain tissue with subcellular resolution. Here, we described an overview of various tissue-clearing techniques, with focus on the tissue-hydrogel mediated clearing methods, and discussed the main advantages and limitations of transparent tissue for clinical diagnosis.

See-Through Technology for Biological Tissue: 3-Dimensional Visualization of Macromolecules.

Surface micropatterns have been widely used as chemical cues to control the microenvironment of cultured neurons, particularly for neurobiological assays and neurochip designs. However, the cell-type dependency on the interactions between neurons and underlying micropatterns has been rarely investigated despite the inherent differences in the morphology of neuronal types. In this study, we used surface-printed microdot arrays to investigate the effect of the same micropatterns on the growth of mouse spinal interneuron, mouse hippocampal neurons, and rat hippocampal neurons. While mouse hippocampal neurons showed no significantly different growth on control and patterned substrates, we found the microdot arrays had different effects on early neuronal growth depending on the cell type; spinal interneurons tended to grow faster in length, whereas hippocampal neurons tended to form more axon collateral branches in response to the microdot arrays. Although there was a similar trend in the neurite length and branch number of both neurons changed across the microdot arrays with the expanded range of size and spacing, the dominant responses of each neuron, neurite elongation of mouse spinal interneurons and branching augmentation of rat hippocampal neurons were still preserved. Therefore, our results demonstrate that the same design of micropatterns could cause different neuronal growth results, raising an intriguing issue of considering cell types in neural interface designs.

Cell-Type Dependent Effect of Surface-Patterned Microdot Arrays on Neuronal Growth.

New phenyl and phenylthienyl derivatives end-functionalized with carbazole and α-carboline, 9-(4- (9H-carbazol-9-yl)phenyl)-9H-carbazole, 9-(4-(9H-carbazol-9-yl)phenyl)-9H-pyrido[2,3-b]indole, 9-(3-(9H-carbazol-9-yl)phenyl)-9H-carbazole, 9-(3-(9H-carbazol-9-yl)phenyl)-9H-pyrido[2,3-b]indole, 9-(4-(5-(9H-carbazol-9-yl)thiophen-2-yl)phenyl)-9H-carbazole, and 9-(3-(5-(9H-carbazo-9-yl) thiophen-2-yl)phenyl)-9H-carbazole were synthesized and characterized as organic semiconductors for organic thin-film transistors (OTFTs). Most compounds exhibited p-channel characteristics with carrier mobility as high as 1.7 x 10⁻⁵ cm²/Vs and a current on/off ratio of 10²-10⁴ for top-contact/bottom-gate OTFT devices.

Novel Organic Semiconductors Based on Phenyl and Phenylthienyl Derivatives for Organic Thin-Film Transistors.

New benzothiadiazole derivatives end-functionalized with carbazole and a-carboline, 4,7-di(9H-carbazol-9-yl)benzo[c][1,2,5]thiadiazole (1) and 4-(9H-carbazol-9-yl)-7-(9H-pyrido[2,3-bindol-9-yl) benzo[c][1,2,5]thiadiazole (2) were synthesized and characterized as organic semiconductors for organic thin-film transistors (OTFTs). Thermal, optical, and electrochemical properties of the corresoponding compounds were characterized. Thin films of compound 1 exhibited p-channel characteristics with carrier mobility as high as 10⁻⁴ cm²/Vs and a current on/off ratio of 10⁵ for top-contact/bottom-gate OTFT devices.

Eunsoo Lee

Department of Anatomy

Korea University College of Medicine

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging