DNA-Based Engineering Strategies to Dissect Complex Signaling Environments within the Adult Neural Stem Cell Niche

Stem cell niches are discrete anatomical microenvironments that present a rich collection of extrinsic factors to govern stem cell behavior. In particular, these niche signals – including soluble cues, extracellular matrix (ECM) associated signals, and cues from neighboring cell types – play critical roles in balancing stem cell self-renewal and differentiation. This balance ensures that tissues can maintain homeostatic tissue turnover while also adapting to external demands, such as injury, inflammation, and infection, to name but a few. Recapitulating the complex signaling dynamics of the stem cell niche in vitro has proven to be a challenging, yet necessary, task for dissecting and understanding the underlying mechanisms that instruct stem cell fate decisions. The resulting biological insight, in turn, accelerates stem cell applications to the clinic by informing the development of cell replacement therapies to regenerate injured or diseased cell types./nThe emergence of innovative engineering strategies within the field has helped elucidate both the key signaling components and mechanisms of niche-directed stem cell behavior (Chapter 1). As these diverse networks continue to be explored, engineering strategies are evolving concurrently to facilitate the study of more complex niche signaling environments in vitro, where multiple inputs are coordinating with each other across time and space to guide resident stem cells. Mapping the dynamic relationships between niche signaling nodes requires developing methods and platforms that provide multiplexed, spatiotemporal control. In this dissertation, we address this technological challenge by drawing inspiration from one of biology’s most robust, functional nano-building block materials, i.e. DNA, as a means to achieve such control. We demonstrated the resulting technology’s utility for investigating stem cell biology by applying these DNA-based engineering strategies to study hippocampal adult neural stem cells (NSCs), a powerful cell type within the mammalian brain that gives rise to adult neurogenesis and holds promising therapeutic potential for treating neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Specifically, we capitalized on DNA’s rapid yet highly specific Watson-Crick base-pairing, ease of programmability, remarkable stability, and the added advantage that biology has evolved already a collection of enzymes for targeting and modifying DNA./nWe first highlighted the unprecedented multiplexing capabilities imparted by DNA, assembling heterogeneous cell communities (up to four distinct cell types) with single-cell precision. We spotted onto a glass surface unique 20-base pair oligonucleotides that hybridize with complementary strands that are each tethered to the surface of different cell types. With this capability, we were able to position strategically single NSCs alongside different astrocyte neighbors that present opposing juxtacrine cues and, thus, tease apart the juxtacrine signaling hierarchy within the NSC niche (Chapter 2). While this approach offers a simple solution for multiplexing by simply modifying the sequence of surface-patterned oligo, we drastically improved the throughput and resolution of DNA surface patterns through the use of photolithography, converting our previously time-intensive and serial method to an inherently parallel one that captures the spatial dimension of niche-driven signaling due to the tight spatial control afforded by lithography (Chapter 3). Additionally, in this work, we expanded the patterning capabilities to include solid-phase cues in addition to cells, empowering additional investigations into the role that spatial presentation plays in how single NSCs resolve competing solid-phase ligands. Finally, we concluded by presenting parallel strategies for integrating temporal control into our DNA-based system by implementing various classes of nucleases and programming nuclease-targeting sequences into our patterned oligo strands (Chapter 4)./nIn summary, we have developed a repertoire of DNA-instructive engineering methods that we employed to elucidate the complex signaling dynamics of the NSC niche but can be widely applied to other stem cell microenvironments or translated to other tissue systems. Together, these tools assemble more mimetic in vitro models through multiplexed control over different cell types and solid-phase ligands as well as robust spatial and temporal control.

Advisor: Schaffer, David V
Sohn, Lydia L