Biophysical regulation of stem cell behavior within the niche.

Stem cells reside within most tissues throughout the lifetimes of mammalian organisms. To maintain their capacities for division and differentiation and thereby build, maintain, and regenerate organ structure and function, these cells require extensive and precise regulation, and a critical facet of this control is the local environment or niche surrounding the cell. It is well known that soluble biochemical signals play important roles within such niches, and a number of biophysical aspects of the microenvironment, including mechanical cues and spatiotemporally varying biochemical signals, have also been increasingly recognized to contribute to the repertoire of stimuli that regulate various stem cells in various tissues of both vertebrates and invertebrates. For example, biochemical factors immobilized to the extracellular matrix or the surface of neighboring cells can be spatially organized in their placement. Furthermore, the extracellular matrix provides mechanical support and regulatory information, such as its elastic modulus and interfacial topography, which modulate key aspects of stem cell behavior. Numerous examples of each of these modes of regulation indicate that biophysical aspects of the niche must be appreciated and studied in conjunction with its biochemical properties

Nox2 redox signaling maintains essential cell populations in the brain.

Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. NADPH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins have cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H(2)O(2)), we show that adult hippocampal stem/progenitor cells (AHPs) generate H(2)O(2) through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations

AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems.

Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications

Stochastic Gene Expression in a Lentiviral Positive Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity

Stochastic gene expression has been implicated in a variety of cellular processes, including cell differentiation and disease. In this issue of Cell, Weinberger et al. (2005) take an integrated computational-experimental approach to study the Tat transactivation feedback loop in HIV-1 and show that fluctuations in a key regulator, Tat, can result in a phenotypic bifurcation. This phenomenon is observed in an isogenic population where individual cells display two distinct expression states corresponding to latent and productive infection by HIV-1. These findings demonstrate the importance of stochastic gene expression in molecular "decision-making."Comment: Supplemental data available as q-bio.MN/060800

Single-cell western blotting.

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution

Microarraying for Mechanosensivitity

In a recent issue of Science, Gilbert et al. combine biomaterial microarrays with lineage tracking to demonstrate that muscle stem cell survival is sensitive to the microenvironment's mechanical stiffness (Gilbert et al., 2010). This work enhances the breadth and depth of knowledge in stem cell mechanobiology

In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ

Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent in vivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs