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Open Access Publications from the University of California

Scholarly Works (3 results)

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

Embryonic development is a complex process that relies on a set of signaling molecules called morphogens. Morphogen diffusion creates spatial concentration gradients across a field of cells to direct the patterning of gene expression. It remains unclear, however, exactly how noisy processes such as morphogen secretion result in robust pattern formation. In vitro systems allow for focused studies of morphogen gradients in a quantitative manner with increased precision. The goal of this dissertation was to develop a platform that enables in vitro reconstitution of morphogen gradients for precise observation and interrogation of transport mechanisms contributing to gradient robustness.

In this work, cell micropatterning technologies are employed to establish in vitro cocultures of morphogen sending and receiving cells. To address limitations of existing patterning tools, we first developed a novel cellular interface patterning device to enable sharp interfaces with minimal cross-contamination. We demonstrate the utility of our device in monitoring stem cell migration during a border competition assay as well as establishing reproducible morphogen gradients. Next, to isolate the effect of diffusion on gradient formation, we introduce a coculture patterning method within microchannels. Bone morphogenetic protein (BMP) cocultures are placed under convection-dominated perfusion to study the role of extracellular diffusion in gradient formation, with our results ruling out free diffusion as a controlling factor. Finally, we employ stencil micropatterning to reconstitute in vitro gradients of an essential morphogen, Sonic Hedgehog (Shh), to investigate mechanisms of gradient robustness. We observe that while gradient amplitude and length increases with greater production of Shh as expected, there is a threshold beyond which the gradient remains stable even as Shh secretion rate continues to increase. We hypothesize that this limiting profile results from saturation of the Shh carrying capacity of the system, which arises due to heparan sulfate proteoglycan (HSPG)-dependent transport. This is supported by our observations that Shh transport is cell-contact dependent, robust to perfusion and can be perturbed by disrupting HSPGs. In summary, we experimentally recapitulated a regime of gradient robustness and our results supports a HSPG-mediated mechanism behind this phenomenon.

    Cover page: An In Vitro Platform for the Study of Morphogen Gradients
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2020)
    We present a simple method to pattern multiple cell populations inside a microfluidic channel. The microchannel is partially filled with a cell suspension, and the position of the liquid boundary remains pinned by surface tension. Cells then adhere only in the filled portion of the channel, producing a very sharp boundary. The process can be performed in an unmodified microfluidic channel with only a manual syringe and can be repeated multiple times to pattern cocultures or tricultures. We demonstrate the patterning method with two different mammalian cell types, 3T3 fibroblasts and NMuMG epithelial cells, and channel heights of 1.5 mm and 0.5 mm. We anticipate that this method will be useful for studies of cell-cell interactions where precise control of the fluidic microenvironment is required.
      Cover page: Cell patterning by surface tension pinning in microfluidic channels
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2017)
      Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.
        Cover page: Patterning of sharp cellular interfaces with a reconfigurable elastic substrate
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