Investigation of Adult Hippocampal Neural Stem Cell Behavior in Response to Complex Biomolecular Signaling Logic

Adult hippocampal neural stem cells (NSCs) retain the capacity to self-renew and differentiate down multiple cell lineages throughout adulthood. These and other cell fate decisions are regulated by the complex microenvironment that comprises the NSC niche. Understanding how biomolecular signals of the niche direct cell behavior is a necessary element in elucidating the biology of NSCs, and it also holds important implications for tissue engineering and regenerative medicine strategies. While previous work has revealed the influence of the presence or absence—that is, the simple binary logic—of several isolated factors in driving NSC fate, less is known about how more complex signaling typical of the niche is interpreted by cells to direct their behavior. In this dissertation I discuss our recent work, including the development of a novel culture platform, to expand the scope of our understanding of how biomolecular cues govern NSC fate beyond their simple binary logic, and instead through a more complex signaling logic.First, I will present our work exploring competing cues that drive mutually exclusive behaviors. Our current understanding of the neural stem cell niche details a highly active signaling microenvironment in which neural stem cells send and receive a multitude of biomolecular cues simultaneously. For example, Eph/ephrin signaling via ephrin-B2/EphB4 instructs cells to neuronally differentiate. In contrast, Notch signaling directs stem cell self-renewal and maintenance. How might neural stem cells integrate these two mutually exclusive cues to decide which fate to choose? To address this question, we developed a novel single-cell patterning platform to recreate model niche microenvironments in which single neural stem cells are presented with these two opposing signals. By observing downstream fate decisions, our data reveal that neural stem cells display a preference for the self-renewal cue of Notch signaling over the Eph/ephrin signal to neuronally differentiate when presented with both cues simultaneously.Second, I will present our results that reveal how the strength of biomolecular signaling pathway activation is a factor in the conditional logic that informs downstream cell behavior. Biomolecular signals fluctuate in strength in the niche, so understanding how cells might differentially respond to weak, mid-level, or strong pathway activation is crucial to fully elucidating a signal’s role in driving fate decisions. We focus on the canonical Wnt pathway and find that it operates through a strength-of-pathway-activation signaling logic to regulate NSC behavior. Using in vitro assays, we reveal proliferation and neuronal differentiation saturate at inequivalent activation strengths of the canonical Wnt/β-catenin pathway. While stronger pathway activation yields greater proportions of cells selecting a neuronal fate, mid-level activation strength drives greater cell proliferation over an extended time period. This proliferation largely occurs in neuronal-committed cells, thus mid-level Wnt activation strength yields greater total numbers of immature neurons than strong activation of the pathway. Further, single-cell tracking reveals this effect at the single-cell level, with mid-level Wnt enabling the greatest proliferative expansion and highest output of immature neurons from single NSCs. Lastly, I will discuss evidence and propose further factors that may contribute to complex logic of adult hippocampal neural stem cell fate decisions in response to biomolecular signaling. I explore how varying cell density may differentially regulate stem cell behavior and discuss possible mechanisms through which cell density is communicated to and sensed by cells. Next, I discuss the possible influence that cell cycle phase during signaling has on determining cellular fate decisions. As signaling mechanisms broadly fluctuate throughout different phases of the cell cycle, this offers an attractive research direction.In summary, we provide evidence to elucidate how several biomolecular signaling cues—Notch, Ephrin/Eph, and Wnt in particular—instruct NSC fate decisions through a complex logic. Beyond their simple presence, additional factors like the activation of other pathways and the strength to which a pathway is turned on contribute to how a cell responds to these biomolecular cues. Our results have implications for regenerative medicine, and further provides a deeper framework through which to explore the influence of biomolecular signaling on stem cell behavior