Aligning Single-Cell Developmental and Reprogramming Trajectories Identifies Molecular Determinants of Myogenic Reprogramming Outcome

Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy and for revealing principles of gene regulation. However, most reprogramming systems are inefficient, converting only a fraction of cells to the desired state. Here, we analyze MYOD-mediated reprogramming of human fibroblasts to myotubes, a well-characterized model system for direct conversion by defined factors, at pseudotemporal resolution using single-cell RNA-seq. To expose barriers to efficient conversion, we introduce a novel analytic technique, trajectory alignment, which enables quantitative comparison of gene expression kinetics across two biological processes. Reprogrammed cells navigate a trajectory with branch points that correspond to two alternative decision points, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Analysis of these branch points revealed insulin and BMP signaling as crucial molecular determinants of reprogramming. Single-cell trajectory alignment enables rigorous quantitative comparisons between biological trajectories found in diverse processes in development, reprogramming, and other contexts

Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization.

Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1–neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined “neutralization fingerprints” for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1–infected donors and a chimeric siman-human immunodeficiency virus–infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies

Multiplex single-cell RNA sequencing for chemical genomics and spatial transcriptomics

Thesis (Ph.D.)--University of Washington, 2021Each of us begins life as a single fertilized cell. Following a seemingly predetermined set of cell divisions, the single cell morphs into a rough mass, then a hollowed tube, and finally becomes a recognizable neonatal form. How the information contained within a single cell si- multaneously specifies an organism’s anatomy, the construction of its organs, and the ability to cogitate on this very question, remains one of biology’s open questions. Although centuries of careful experiments devoted to characterizing development have revealed many important genes and mechanisms, the results of these experiments span different model organisms, developmental stages, cell populations and measurement modalities. Integrating this knowledge base into coher- ent representation requires a cellular scaffold that charts an organism’s development over the axes of time and space. Preliminary unified representations of developing organisms (e.g. C. Elegans, Zebrafish and Mouse) have been created by large-scale single cell RNA sequencing (scRNA-seq) efforts. These efforts have characterized the set of intermediates through which differentiating cells transit and have profiled the large number of cell types present in a developing organism. Although scRNA-seq data have proven powerful in cataloging cellular states, they lack crucial context: i) the experimental context afforded by the comparison of multiple conditions (e.g. wild-type vs. perturbation) and ii) a cell’s spatial context, a crucial factor driving its behavior. To address these knowledge gaps, over the course of my PhD I have developed two scRNA-seq technologies: 1) sci- Plex, a generalizable strategy to label cell populations and 2) sci-Space, a methodology to record acell’s spatial position in conjunction with its single cell transcriptome. (1) First I developed the sci-Plex protocol, an inexpensive and efficient method to label single cells through the chemical fixation of unmodified single stranded oligos to nuclei prior to scRNA- seq library preparation. To demonstrate proof-of-concept of the sci-Plex protocol, I performed a high-throughput, high-content drug screen at single cell resolution in 3 cancer cell lines; effectively conducting 4,500 independent scRNA-seq experiments at once. The resulting dataset enabled characterization of a drug’s potency, class, mechanism of action, and the heterogeneity of cellular responses induced upon drug treatment. For example, our scRNA-seq data showed that histone deacetylase inhibitors likely lead to cell death by trapping valuable acetyl molecules on chromatin. (2) Next, I extended the application of the sci-Plex protocol and developed the sci-Space method to capture spatial information from sectioned tissue. The fast and scalable sci-Space method uses patterned oligonucleotide barcodes in a regular array such that each spot contains a unique set of sequences. Then, to mark each nucleus’ coordinates on the grid, the barcodes are stamped onto a tissue section prior to disaggregation and library preparation. To showcase the power of sci-Space, I collected a dataset comprising over 120,000 cells originating from 14 sections of a single E14 mouse embryo. The resulting data uncovers the genes that drive the devel- oping organism’s body plan and reveals a widespread migration signature within neurons that form the developing brain. These data also provide a quantitative assessment of how cell state relates to spatial position within the developing embryo. Specifically, our estimates indicate that 25% of the variance in gene expression observed is attributable to spatial position. It is my hope that this technology will power the generation of a unified scaffold of development akin to the reference genome. I believe that such a unified representation will be instrumental in amassing data, accel- erating discovery and facilitating translation through the training of machine learning models of cellular state

The Energy Absorption Capability of Composite Materials and Structures: Influence of Impact Loading

In this paper, the energy absorption capability of composite materials and performance-critical structures made from using these materials under conditions of impact loading is presented and discussed. An overview is provided of the key events associated with detonation and decomposition of an explosive that eventually culminates in the generation of a shock wave that exerts an impact type of loading on structures both in contact and in the immediate vicinity. The blast loads that culminate from an explosive often tend to generate very high strain rates in the range of 102/sec to 104/sec. The resultant high rate of loading does tend to exert an influence on dynamic mechanical properties of the targeted structure besides exerting an influence on damage mechanisms experienced by the structural element. The progressive use of composite materials for structures, such as sandwich panels, that essentially comprise of a mixture of composite face sheets and foam cores was shown by researchers, based on actual field test data, to offer few to many advantages over usual metal counterparts when it came to the purpose of offering acceptable blast resistance. Thus, it became both essential and desirable to assess the blast response of composite structures made using appropriate selection of composite materials. Through the years several independent research studies have been conducted to understand the influence of blast loading on the response kinetics of sandwich panels. Key highlights of the research done and resultant findings obtained from these studies is presented and briefly discussed The importance of both material selection and resultant structure for providing adequate protection against an impact type of loading caused by an explosive device, such as Improvised Explosive Device (IED), is highlighted through appropriate summary of research conducted and published in the open literature. The need and necessity for developing new and improved materials, composite in nature, that can be used for performance-critical structures that can also offer an enhanced level of safety to all personal involved in emphasized

Contained Mycobacterium tuberculosis infection induces concomitant and heterologous protection

Progress in tuberculosis vaccine development is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Although the M72/ASOE1 trial yielded encouraging results (54% efficacy in subjects with prior exposure to Mtb), a highly effective vaccine against adult tuberculosis remains elusive. We show that in a mouse model, establishment of a contained and persistent yet non-pathogenic infection with Mtb ("contained Mtb infection", CMTB) rapidly and durably reduces tuberculosis disease burden after re-exposure through aerosol challenge. Protection is associated with elevated activation of alveolar macrophages, the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma. Systems approaches, as well as ex vivo functional assays and in vivo infection experiments, demonstrate that CMTB reconfigures tissue resident alveolar macrophages via low grade interferon-γ exposure. These studies demonstrate that under certain circumstances, the continuous interaction of the immune system with Mtb is beneficial to the host by maintaining elevated innate immune responses

High-capacity sample multiplexing for single cell chromatin accessibility profiling

Abstract Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation

High‐throughput, microscope‐based sorting to dissect cellular heterogeneity

Abstract Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence‐activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single‐cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs

Systematic reconstruction of cellular trajectories across mouse embryogenesis.

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type\u27s specification, as well as \u27cell-type homologs\u27 across vertebrate evolution