Molecular Biologist Diya Das deciphers cellular signaling and activation patterns to understand how stem cells regenerate healthy tissue after injury.
Tissues that experience a high degree of environmental stress must be able to respond to both minor stresses and severe injury in order to maintain their structure and function throughout an organism’s life. This is particularly true for epithelial tissues, which maintain physical barriers between different environments. And in all epithelial tissues, this maintenance and repair is driven by adult stem cells.
Adult stem cells are cells that retain the capacity to make more specialized cells and contribute to cell replacement in cases of both minor damage and severe injury. They are found in your skin, lungs, intestine, blood - and in the olfactory epithelium, the home of odor-detecting sensory neurons. These olfactory sensory neurons are susceptible to damage due to their exposure to the air, and if they aren’t replaced on an ongoing basis, the sense of smell can be lost.
Understanding just how stem cells contribute to tissue maintenance under wildly varying conditions is a major undertaking, but one with significant public health implications. One to two percent of North Americans report issues with their sense of smell, and these issues increase with age, affecting approximately 25% of men and 11% of women ages 60-69 (Smell Disorders, National Institute on Deafness and Communication Disorders).
Advances in lineage tracing approaches and sequencing technology have allowed the Ngai Lab and our collaborators to study how olfactory stem cells contribute to tissue maintenance at the level of individual cells. The use of lineage tracing to label and track the fates of individual cells dates back to the late 1800s, but new tracers are genetically encoded and enable the precise labeling and tracking of particular, small populations of cells.
Prior to the advent of single-cell RNA-sequencing, RNA-sequencing only allowed for an "average" measure of gene expression within a population. As stem cells respond to environmental changes, they might not respond in a synchronous or identical manner. Combining lineage tracing with single-cell RNA-sequencing allowed our group to determine how individual olfactory stem cells contribute to tissue maintenance, an approach that has the potential to address similar questions in other regenerating systems (Creating Lineage Trajectory Maps Via Integration of Single-Cell RNA-Sequencing and Lineage Tracing, Fletcher et al, BioEssays, 2018).
We first addressed the question of how olfactory stem cells, termed horizontal basal cells (or HBCs), contribute to maintenance at steady-state (Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution, Fletcher et al, Cell Stem Cell, 2017). We found that HBCs give rise to three lineages, eventually forming sustentacular (support) cells, microvillous cells, and olfactory sensory neurons. Surprisingly, the HBCs are able to form sustentacular cells without dividing first, while both microvillous and neuronal cells arise via a proliferating intermediate.
Measuring transcriptomes of single cells descended from olfactory
stem cells allows us to map lineage trajectories at steady state.
Graphic by Russell Fletcher, from Fletcher et al., 2017.
Lineage tracing also uncovered that individual stem cells usually contribute to either the microvillous/neuronal or the sustentacular cell lineage, but not both. Finally, our single-cell RNA-sequencing results pointed to Wnt signaling (a pathway also involved in epidermal and neuronal cell fate specification during development) as a potential driver of HBC differentiation, and our subsequent experiments revealed its necessity and sufficiency for the formation of neurons.
After establishing the functions of HBCs in steady-state maintenance, we investigated their contributions to regeneration after severe injury (Injury Activates Transient Olfactory Stem Cell States with Diverse Lineage Capacities, Gadye et al, Cell Stem Cell, 2017). Because of its exposure to the air in the nasal cavity, the olfactory epithelium is susceptible to chemical injury. Simply smelling a noxious chemical, such as methyl bromide, is enough to cause cell death. Our manipulations involved the injection of methimazole, an antithyroid drug that can cause similar tissue damage, sparing mostly HBCs to regenerate the tissue.
Olfactory stem cells transit different paths that later converge to maintain
the tissue during steady-state and regeneration after severe injury.
Graphic by Sarah Tronnes, from Gadye et al., 2017.
We discovered the existence of an injury-specific, activated class of HBCs, characterized by expression of "wound response" genes and cell proliferation. We found that all lineages, including the ones that re-form HBCs for future rounds of cell replacement, involve a trip to this activated state. Cells in this activated state exhibit heterogeneity in gene expression, which appears to herald commitment to particular cell fates.
These projects involved collaboration between molecular biologists and neuroscientists (in the Ngai Lab), statisticians (in the groups of BIDS Senior Fellow Sandrine Dudoit and Elizabeth Purdom) and computer scientists (in the group of Nir Yosef). They also involved the development of new software packages and pipelines for analysis of single-cell RNA-sequencing data. Scone filters low-quality data and evaluates data normalization methods based on a panel of data-driven metrics (Performance Assessment and Selection of Normalization Procedures for Single-Cell RNA-Seq. Cole et al, Cell Systems, 2019). ClusterExperiment applies resampling-based ensemble clustering to generate robust and stable clusters (clusterExperiment and RSEC: A Bioconductor package and framework for clustering of single-cell and other large gene expression datasets. Risso et al, PLOS Computational Biology, 2018). Slingshot is a uniquely flexible algorithm that discovers trajectories in single-cell data, and has been found to outperform other lineage inference methods on a number of metrics (Slingshot: Cell lineage and pseudotime inference for single-cell transcriptomics. Street et al, BMC Genomics, 2018) (A comparison of single-cell trajectory inference methods. Saelens et al, Nature Biotechnology, 2019). These algorithms can be integrated in single-cell RNA-sequencing analysis workflows (Analysis of single-cell RNA-seq data: Dimensionality reduction, clustering, and lineage inference. Das et al, BioC 2018, Toronto, Ontario. July 27, 2018).
The combination of wet-lab experimental techniques with high-dimensional data analysis has proven invaluable for increasing our knowledge of how stem cells regenerate tissues. We’re now expanding our forays in this area to understand the molecular regulation of olfactory epithelial regeneration, to lay the groundwork for the development of therapies to treat tissue damage and neurodegeneration.
Diya Das is finishing her Moore-Sloan Data Science Fellowship as a postdoctoral researcher in the Ngai Lab at UC Berkeley, where she was also a graduate student. You can learn more about her work on her website: diyadas.github.io.