Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein.

Improving the affinity of protein-protein interactions is a challenging problem that is particularly important in the development of antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize the binding affinity of VNAC1, a single-domain intracellular antibody (intrabody) from the camelid family that was selected for its specific binding to the nonamyloid component (NAC) of human α-synuclein (α-syn), a natively disordered protein, implicated in the pathogenesis of Parkinson's disease (PD) and related neurological disorders. Specifically, we performed ab initio modeling that revealed several possible modes of VNAC1 binding to the NAC region of α-syn as well as mutations that potentially enhance the affinity between these interacting proteins.

Screening and optimization of potential injection vehicles for storage of retinal pigment epithelial stem cell before transplantation.

Retinal pigment epithelial (RPE) cells are highly specialized neural cells that have several functions essential for vision. Progressive deterioration of RPE cells in elderly individuals can result in visual impairment and ultimately the blinding disease age-related macular degeneration. Subretinal transplantation of stem cell-derived RPE cell suspensions is being explored as a strategy to recover the damaged retina and improve vision.

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses.

Regenerating Eye Tissues to Preserve and Restore Vision.

Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing.

Effect of Bmi1 over-expression on gene expression in adult and embryonic murine neural stem cells.

The ability of isolated neural stem cells (NSCs) to proliferate as neurospheres is indicative of their competence as stem cells, and depends critically on the polycomb group (PcG) member Bmi1: knockdown of Bmi1 results in defective proliferation and self-renewal of isolated NSCs, whereas overexpression of Bmi1 enhances these properties. Here we report genome-wide changes in gene expression in embryonic and adult NSCs (eNSCs and aNSCs) caused by overexpression of Bmi1. We find that genes whose expression is altered by perturbations in Bmi1 levels in NSCs are mostly distinct from those affected in other multipotent stem/progenitor cells, such as those from liver and lung, aside from a small core of common targets that is enriched for genes associated with cell migration and mobility.

Staufen2 deficiency leads to impaired response to novelty in mice.

Staufen2 (Stau2) is a double-stranded RNA-binding protein (RBP) involved in posttranscriptional gene expression control in neurons. In flies, staufen contributes to learning and long-term memory formation. To study the impact of mammalian Stau2 on behavior, we generated a novel gene-trap mouse model that yields significant constitutive downregulation of Stau2 (Stau2).

Quantified F-Actin Morphology Is Predictive of Phagocytic Capacity of Stem Cell-Derived Retinal Pigment Epithelium.

With stem cell-derived retinal pigment epithelial (RPE) replacement therapies in clinical testing, establishing potency of RPE prior to transplantation is imperative. Phagocytosis of photoreceptor outer segment fragments (POS) is a key indicator of RPE functionality. Comparing RPE derived from different donor human adult RPE stem cell lines, we found that cells were either high-phagocytic or low-phagocytic despite sharing phagocytic receptors and ligands, junctional ZO-1, and lack of epithelial-mesenchymal transition.

Endothelial Cells Exposed to Fluid Shear Stress Support Diffusion Based Maturation of Adult Neural Progenitor Cells.

Introduction: The neural stem cell (NSC) niche is a highly complex cellular and biochemical milieu supporting proliferating NSCs and neural progenitor cells (NPCs) with close apposition to the vasculature, primarily comprised of endothelial cells (ECs). Current models of the niche incorporate EC-derived factors, but do not reflect the physiologically relevant hemodynamic state of the ECs or the spatial resolution observed between cells within the niche.

Methods: In this work, we developed a novel model of the niche that (1) incorporates ECs cultured with fluid shear stress and (2) fosters paracrine cytokine gradients between ECs and NSCs in a spatiotemporal configuration mimicking the cytoarchitecture of the subventricular niche.

Epimetronomics: m6A Marks the Tempo of Corticogenesis.

Yoon et al. (2017) uncover a key role for the m6A RNA mark in regulating the timing of cerebral cortex development in mouse and human. This discovery opens new avenues of exploration into how the epitranscriptome helps orchestrate central nervous system formation.

Non-monotonic Changes in Progenitor Cell Behavior and Gene Expression during Aging of the Adult V-SVZ Neural Stem Cell Niche.

Neural stem cell activity in the ventricular-subventricular zone (V-SVZ) decreases with aging, thought to occur by a unidirectional decline. However, by analyzing the V-SVZ transcriptome of male mice at 2, 6, 18, and 22 months, we found that most of the genes that change significantly over time show a reversal of trend, with a maximum or minimum expression at 18 months. In vivo, MASH1 progenitor cells decreased in number and proliferation between 2 and 18 months but increased between 18 and 22 months.

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies.

Appetite for Neurogenesis.

Adult neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ) produce diverse olfactory bulb (OB) neurons. In a recent paper in Science, Paul et al. (2017) show that hypothalamic propiomelanocortin (POMC) neurons innervate the anterior ventral V-SVZ and regulate deep granule interneuron production depending on feeding behavior.

Persistent Expression of VCAM1 in Radial Glial Cells Is Required for the Embryonic Origin of Postnatal Neural Stem Cells.

During development, neural stem cells (NSCs) undergo transitions from neuroepithelial cells to radial glial cells (RGCs), and later, a subpopulation of slowly dividing RGCs gives rise to the quiescent adult NSCs that populate the ventricular-subventricular zone (V-SVZ). Here we show that VCAM1, a transmembrane protein previously found in quiescent adult NSCs, is expressed by a subpopulation of embryonic RGCs, in a temporal and region-specific manner. Loss of VCAM1 reduced the number of active embryonic RGCs by stimulating their premature neuronal differentiation while preventing quiescence in the slowly dividing RGCs.

Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction.

Recent outbreaks of Zika virus (ZIKV) highlight an urgent need for therapeutics. The protease complex NS2B-NS3 plays essential roles during flaviviral polyprotein processing, and thus represents an attractive drug target. Here, we developed a split luciferase complementation-based high-throughput screening assay to identify orthosteric inhibitors that directly target flavivirus NS2B-NS3 interactions.

The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue.

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction.

Polarized, Cobblestone, Human Retinal Pigment Epithelial Cell Maturation on a Synthetic PEG Matrix.

Cell attachment is essential for the growth and polarization of retinal pigment epithelial (RPE) cells. Currently, surface coatings derived from biological proteins are used as the gold standard for cell culture. However, downstream processing and purification of these biological products can be cumbersome and expensive.

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage.

The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures.

Stem cell therapies for retinal diseases: recapitulating development to replace degenerated cells.

Retinal degenerative diseases are the leading causes of blindness worldwide. Replacing lost retinal cells via stem cell-based therapies is an exciting, rapidly advancing area of translational research that has already entered the clinic. Here, we review the status of these clinical efforts for several significant retinal diseases, describe the challenges involved and discuss how basic developmental studies have contributed to and are needed to advance clinical goals.

Lessons Learned from Pioneering Neural Stem Cell Studies.

As stem cell products are increasingly entering early stage clinical trials, we are learning from experience about how cell products may be best assessed for safety and efficacy. In two papers published in this issue of Stem Cell Reports, a human neural stem cell product, HuCNS-SC, failed to demonstrate efficacy in central nervous system repair in two different animal models (Anderson et al., 2017; Marsh et al.

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype.

Measuring Process Dynamics and Nuclear Migration for Clones of Neural Progenitor Cells.

Neural stem and progenitor cells (NPCs) generate processes that extend from the cell body in a dynamic manner. The NPC nucleus migrates along these processes with patterns believed to be tightly coupled to mechanisms of cell cycle regulation and cell fate determination. Here, we describe a new segmentation and tracking approach that allows NPC processes and nuclei to be reliably tracked across multiple rounds of cell division in phase-contrast microscopy images.

Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ.

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts.

Big Data access and infrastructure for modern biology: case studies in data repository utility.

Big Data is no longer solely the purview of big organizations with big resources. Today's routine tools and experimental methods can generate large slices of data. For example, high-throughput sequencing can quickly interrogate biological systems for the expression levels of thousands of different RNAs, examine epigenetic marks throughout the genome, and detect differences in the genomes of individuals.

LEVER: software tools for segmentation, tracking and lineaging of proliferating cells.

Unlabelled: The analysis of time-lapse images showing cells dividing to produce clones of related cells is an important application in biological microscopy. Imaging at the temporal resolution required to establish accurate tracking for vertebrate stem or cancer cells often requires the use of transmitted light or phase-contrast microscopy. Processing these images requires automated segmentation, tracking and lineaging algorithms.