Jamison L. Nourse

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

Significance Stem cells make lineage-choice decisions based on a combination of internal and external signals, including mechanical cues from the surrounding environment. Here we show that Piezo1, an ion channel opened by membrane tension, plays an important role in transducing matrix mechanical information to intracellular pathways affecting differentiation in neural stem cells. Piezo1 activity influences whether neural stem cells differentiate along a neuronal or astrocytic lineage. One of the barriers to successful neural stem cell transplantation therapy for neurological disorders lies in directing the fate of transplanted cells. Pharmacological agents aimed at modulating Piezo1 activity may be useful in directing the fate of transplanted neural stem cells toward the desired lineage. Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brain-derived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca2+, a known modulator of differentiation, in a substrate-stiffness–dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells.

Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential

In the stem cell field there is a lack of non invasive and fast methods to identify stem cell’s metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors.

Static stretch affects neural stem cell differentiation in an extracellular matrix-dependent manner.

Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been reported. We delivered a 10% static equibiaxial stretch to NSPCs and examined effects on differentiation. We found static stretch specifically impacts NSPC differentiation into oligodendrocytes, but not neurons or astrocytes, and this effect is dependent on particular extracellular matrix (ECM)-integrin linkages. Generation of oligodendrocytes from NSPCs was reduced on laminin, an outcome likely mediated by the α6 laminin-binding integrin, whereas similar effects were not observed for NSPCs on fibronectin. Our data demonstrate a direct role for tensile strain in dictating the lineage choice of NSPCs and indicate the dependence of this phenomenon on specific substrate materials, which should be taken into account for the design of biomaterials for NSPC transplantation.

Membrane Biophysics Define Neuron and Astrocyte Progenitors in the Neural Lineage

Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self‐renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes, and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here, we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA‐NCAM, which is considered an NP marker. Additionally, cell surface N‐linked glycosylation was found to significantly affect cell fate‐specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface. Stem Cells 2014;32:706–716

Increasing label-free stem cell sorting capacity to reach transplantation-scale throughput.

Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP.

How cells channel their stress: Interplay between Piezo1 and the cytoskeleton

Cells constantly encounter mechanical stimuli in their environment, such as dynamic forces and mechanical features of the extracellular matrix. These mechanical cues are transduced into biochemical signals, and integrated with genetic and chemical signals to modulate diverse physiological processes. Cells also actively generate forces to internally transport cargo, to explore the physical properties of their environment and to spatially position themselves and other cells during development. Mechanical forces are therefore central to development, homeostasis, and repair. Several molecular and biophysical strategies are utilized by cells for detecting and generating mechanical forces. Here we discuss an important class of molecules involved in sensing and transducing mechanical forces - mechanically-activated ion channels. We focus primarily on the Piezo1 ion channel, and examine its relationship with the cellular cytoskeleton.

Laurdan Monitors Different Lipids Content in Eukaryotic Membrane During Embryonic Neural Development

Abstract We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.

Immunocytochemical Analysis of Human Stem Cells

Antibodies can recognize antigens that are protein, glycolipid (such as the SSEA-4 epitope), carbohydrate, small molecule, or DNA. Staining of cells by a particular antibody is often described as “weakly positive” or “strongly positive.” If a new antibody or cellular sample gives only a weakly positive signal, it is essential to confirm the presence of the antigen using another method. If the antigen is a protein, it is usually sufficient to demonstrate the presence in the same cells of the specific coding transcript, using a method such as RT-PCR. Other methods used for confirmation of antibody staining include the use of a second antibody that recognizes another epitope on the same molecule, and immunoblots (Western blots), in which molecular weight information revealed by SDS gel electrophoresis adds confirmation of antigen identity. Here we provide a straight forward, reliable protocol for immuno-cytochemical staining of hPSCs.

Piezo1 calcium flickers localize to hotspots of cellular traction forces

Piezo channels transduce mechanical stimuli into electrical and chemical signals, and in doing so, powerfully influence development, tissue homeostasis, and regeneration. While much is known about how Piezo1 responds to external forces, its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that actomyosin-based cellular traction forces generate spatially-restricted Ca2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched in regions proximal to force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that diffusion allows channel molecules to efficiently respond to transient, local mechanical stimuli.Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, homeostasis, and regeneration. Due to their location in the plasma membrane, they are positioned to transduce both external forces and internal forces generated by cells. While much is known about how Piezo1 responds to external forces, its response to cell-generated forces that are vital for cellular and organismal physiology is poorly understood. Here we show that Ca2+ flickers generated by endogenous Piezo1 in human neural stem cells and in fibroblasts are stimulated by actomyosin-based traction forces. Further, although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, flicker activity is enriched in spatially constrained regions at force-producing adhesions. We propose that Piezo1 Ca2+ flickers allow spatial segregation of mechanotransduction events, and that Piezo1 diffusion allows channel molecules to efficiently respond to transient and localized mechanical stimuli throughout the cell surface.

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

Summary 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.