UC Berkeley

The signaling environment experienced by a single cell is highly dependent on its interactions with its neighbors, which may secrete locally acting factors or act via membrane bound receptor-ligand systems. Stem cells are particularly sensitive to these signals, which act in concert to regulate self-renewal and discrete transitions into distinct fates. In the case of the adult neural stem cell, decades of in vivo and in vitro work have illustrated specific roles for different types of niche cells and some of the molecular mediators of their instructive roles. However, these studies have not investigated the strengths of these fate-inducing or fate-repressing cues as presented by single neighboring cells at endogenous expression levels. In this work, we have developed and applied microengineered tools to examine the transmission of signals between small populations of cells and to assess their impact on neural stem cell fate.

First, we measured gap junction coupling between cells by using microfluidic flow focusing of calcein/AM dye and timelapse imaging to measure the dynamics of dye transfer through gap-junction coupled glioma cells. Secondly, we developed single-cell micropatterning methods to investigate the potential for single neural progenitor cells to influence their neighbor's fate. Size-matched circular and hourglass shaped polystyrene microwells achieved up to 80% efficiency in capturing single and paired neural progenitor cells, a large improvement over Poisson-distributed random seeding. In conjunction, we also developed a method to fabricate and align PDMS meshes to corral patterned cells into non-connected arenas so that single cells and cell pairs can be grown in isolation for six days before immunostaining for fate markers. Lastly, we applied a simplified micropatterning technique in conjunction with automated high-throughput imaging to enforce persistent interactions between initially patterned cells. We discovered that single neural progenitor cells can significantly bias their neighbor's fate in mixed differentiation medium. Although the initial number of cells on a pattern did not affect the overall distribution of different fates, we saw reduced fate asymmetry on patterns with initially paired cells, indicating that single cells may be inducing a similar fate in their neighbors, or repressing differentiation. By tracking cell populations on micropatterns on every day of the experiment via brightfield imaging, we also ascertained that the initial patterning state was maintained for approximately two days, suggesting that fate specification or repression occurred early in the process. Our discovery that a single as-yet undifferentiated neural stem cell can significantly bias its neighbor's fate paves the road for future work in elucidating how conflicting signals from various types of niche cells are arbitrated in a single recipient stem cell.