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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2019)

The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by spatially and temporally varying morphogen signals. Such dynamic morphogen patterns drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. In embryonic stem cell (ESC) models for early development, for example, analogous tissue patterns spontaneously emerge but mechanistic insight into this self-organization has been limited. In this dissertation, we combine engineered methods for optogenetic stimulation and biological analysis to photoactivate Wnt/B-catenin signaling and to study morphogen dynamics and self-organization of human pluripotent stem cells.

First, we show that optogenetic Wnt/B-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns in human ESCs (hESCs). We expressed a fusion of the plant blue-light photoreceptor Cryptochrome 2 (Cry2) to the Wnt co-receptor LRP6 (the optoWnt system) in hESCs and induce Cry2-LRP6c oligomerization and subsequent canonical Wnt/B-catenin activation using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). Such LAVA devices are a LED-based, programmable illumination system for photostimulation of multi-well plates that can be readily incorporated into the workflow of routine cell culture and allow controlled and quantitative spatiotemporal light patterning. We optimized the LAVA board optical configuration for illumination uniformity and achieve programmable photostimulation of independent wells of 24-well or 96-well culture plates kept in standard 37C tissue culture incubators. Each well can be wirelessly programmed through a graphical user interface (GUI) at user-defined intensities (0 -- 20 uWmm-2, 0.005 uWmm-2 resolution), temporal sequences (10 ms resolution), and spatial patterns (100 um resolution). We demonstrate LAVA board performance by modulating the intensity, timing, and spatial location of canonical Wnt/$\beta$-catenin signaling in hESC cultures using the optoWnt optogenetic system. We show that Wnt pathway activation and hESC differentiation is dose-responsive to light intensity and duration of illumination, and that spatial patterning can be used to simulate the embryonic, spatially polarized presentation of the Wnt ligand.

In parallel, we engineered a microscope capable of dynamic photostimulation and high-resolution imaging with structured illumination microscopy (SIM). A digital micromirror device (DMD) is used to project user-defined photostimulation sequences onto the sample with diffraction-limited spatial resolution (800 nm). For fluorescence imaging, a series of multi-spot patterns is projected onto the sample to reconstruct a high-resolution SIM image with a 2-fold resolution improvement over the diffraction limit. Such multi-color, user-defined photostimulation and SIM imaging (opto-SIM) allows live-cell timelapse imaging while localizing Wnt/B-catenin photoactivation to specific regions within hESC cultures. Unlike LED-based illumination devices, such as the LAVA board system, the opto-SIM system enables pattern projection at subcellular resolution. Furthermore, projected patterns can be dynamically updated based on feedback from measurements acquired during experiment progression. The opto-SIM system also enables temporal control of signaling at a resolution of 60 Hz, as well as 8-bit greyscale modulation to achieve dose-dependent optoWnt activation. Such flexibility in spatiotemporal patterning gives the unprecedented ability to stimulate stem-cell based model systems, organoids, or embryos with time-varying patterns of morphogen gradients.

Lastly, we implement optogenetic control of canonical Wnt signaling to determine whether differential Wnt signaling can model human gastrulation and lead to emergence of organized shape and structure through collective cell rearrangement. We achieve optogenetic control of Wnt signaling in hESCs by illuminating hESC cultures expressing the optoWnt system with LAVA devices. Wnt/B-catenin signaling was activated over a high dynamic range (>500-fold) and drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. We thus developed an hESC model for studying Wnt-mediated morphogenesis in early development. Using this gastrulation model in combination with transcriptomic analysis and single-cell migration studies, we show that Wnt signaling is sufficient for inducing self-organization of cells in an EMT-dependent manner. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for early embryogenesis.

In summary, we developed and utilized engineered methods for spatial and temporal light patterning to photoactivate Wnt signaling in a precise location and at a given time within embryonic stem cell cultures. Such optogenetic activation of morphogen signaling in specific cell subpopulations allows studies of cell-cell interactions and signaling dynamics that regulate early embryonic development.

    Cover page: Optogenetic control of morphogen signaling in embryonic stem cells
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2019)
    High-content biological microscopy targets high-resolution imaging across large fields-of-view, often achieved by computational imaging approaches. Previously, we demonstrated 2D multimodal high-content microscopy via structured illumination microscopy (SIM) with resolution > 2 × the diffraction limit, using speckle illumination from Scotch tape. In this work, we extend the method to 3D by leveraging the fact that the speckle illumination is in fact a 3D structured pattern. We use both a coherent and an incoherent imaging model to develop algorithms for joint retrieval of the 3D super-resolved fluorescent and complex-field distributions of the sample. Our reconstructed images resolve features beyond the physical diffraction-limit set by the systems objective and demonstrate 3D multimodal imaging with ∼ 0.6 × 0.6 × 6   μ m3 resolution over a volume of ∼ 314 × 500 × 24   μ m3.
      Cover page: Speckle-structured illumination for 3D phase and fluorescence computational microscopy.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2018)
      Through three-dimensional STORM super-resolution microscopy, we resolve the spectrin-actin-based membrane cytoskeleton of neural stem cells (NSCs) and NSC-derived neurons, astrocytes, and oligodendrocytes. We show that undifferentiated NSCs are capable of forming patches of locally periodic, one-dimensional (1D) membrane cytoskeleton with ∼180 nm periodicity. Such periodic structures become increasingly ordered and long-ranging as the NSCs mature into terminally differentiated neuronal and glial cell types, and, during this process, distinct 1D periodic "strips" dominate the flat 2D membranes. Moreover, we report remarkable alignment of the periodic cytoskeletons between abutting cells at axon-axon and axon-oligodendrocyte contacts and identify two adhesion molecules, neurofascin and L1CAM, as candidates to drive this nanoscale alignment. We thus show that a conserved 1D periodic membrane cytoskeletal motif serves as a nanoscale scaffold and ruler to mediate the physical interactions between cell types of the NSC lineage.
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        Optical diffraction tomography (ODT) reconstructs a samples volumetric refractive index (RI) to create high-contrast, quantitative 3D visualizations of biological samples. However, standard implementations of ODT use interferometric systems, and so are sensitive to phase instabilities, complex mechanical design, and coherent noise. Furthermore, their reconstruction framework is typically limited to weakly scattering samples, and thus excludes a whole class of multiple-scattering samples. Here, we implement a new 3D RI microscopy technique that utilizes a computational multi-slice beam propagation method to invert the optical scattering process and reconstruct high-resolution (NA > 1.0) 3D RI distributions of multiple-scattering samples. The method acquires intensity-only measurements from different illumination angles and then solves a nonlinear optimization problem to recover the samples 3D RI distribution. We experimentally demonstrate the reconstruction of samples with varying amounts of multiple-scattering: a 3T3 fibroblast cell, a cluster of C. elegans embryos, and a whole C. elegans worm, with lateral and axial resolutions of ≤ 240 nm and ≤ 900 nm, respectively. The results of this work lays groundwork for future studies into using optical wavelengths to probe 3D RI distributions of highly scattering biological organisms.
          Cover page: High-resolution 3D refractive index microscopy of multiple-scattering samples from intensity images.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2020)
          Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.
            Cover page: Engineered Illumination Devices for Optogenetic Control of Cellular Signaling Dynamics
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2021)
            Biophysical cues in the extracellular matrix (ECM) regulate cell behavior in a complex, nonlinear, and interdependent manner. To quantify these important regulatory relationships and gain a comprehensive understanding of mechanotransduction, there is a need for high-throughput matrix platforms that enable parallel culture and analysis of cells in various matrix conditions. Here we describe a multiwell hyaluronic acid (HA) platform in which cells are cultured on combinatorial arrays of hydrogels spanning a range of elasticities and adhesivities. Our strategy utilizes orthogonal photopatterning of stiffness and adhesivity gradients, with the stiffness gradient implemented by a programmable light illumination system. The resulting platform allows individual treatment and analysis of each matrix environment while eliminating contributions of haptotaxis and durotaxis. In human mesenchymal stem cells, our platform recapitulates expected relationships between matrix stiffness, adhesivity, and cell mechanosensing. We further applied the platform to show that as integrin ligand density falls, cell adhesion and migration depend more strongly on CD44-mediated interactions with the HA backbone. We anticipate that our system could bear great value for mechanistic discovery and screening where matrix mechanics and adhesivity are expected to influence phenotype.
              Cover page: Multiwell Combinatorial Hydrogel Array for High-Throughput Analysis of Cell–ECM Interactions
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