Many immune-mediated diseases-infectious diseases like HIV and autoimmune diseases like multiplesclerosis or diabetes-mediate pathology in specific tissues, yet most of our knowledge about them hasresulted from studying cells circulating in blood. These cells have been a convenient proxy because blood isthe most accessible compartment and the number of cells recovered can be large. Increasing evidencesuggests, however, that the biology of diseases in affected tissues can vary substantially from that in the blood,and understanding these differences may be critical to develop new drugs, vaccines, and diagnostics toimprove patient care. The significant heterogeneities among cells resident in tissues necessitatescharacterizing such samples with single-cell resolution, but existing technologies routinely employed by clinicalimmunologists (flow cytometry, ELISpot) typically require an excess of cells to use for analysis. Theirinefficiencies have hindered the ability to pursue science understanding the human biology of diseases andtreatments in tissues because biopsies yield very few cells. This research will optimize, validate, and deploy aunique nanowell-based platform to address this unmet need for characterizing single cells from clinicalbiopsies with minimal manipulations. The project is a collaboration amongst: the Love and LauffenburgerLabs (MIT) with expertise in applying microfabricated technologies to resolve single-cell heterogeneities and indeveloping computational tools for analyzing such data; the Kwon and Walker Labs (Ragon Institute) withexpertise on the clinical immunology of HIV and vaccines; the Mesirov and Wong Labs (Broad Institute) withexpertise in developing software tools for data analysis and means of visualizing complex data; and theRoederer Lab (NIH VRC) with expertise in single-cell technologies for characterizing immunophenotypes andgene expression. Together, this interdisciplinary team spanning engineering, computational biology, clinicalimmunology, and data visualization will 1) improve the experience of end-users using nanowells to study cellsfrom biopsies by increasing the number of samples each user can process through engineering andautomation, by streamlining the process for extracting, integrating, analyzing and viewing data, and byenhancing the ability to recover rare cells; 2) validate modular nanowell-based operations for determining thetypes of cells present (cytometry) and their secreted proteins (microengraving) and the efficiencies ofrecovering cells and genes expressed relative to current standards; and 3) deploy the platform as a core facilityat the Ragon Institute, making the technology broadly available for the first time to the community of end-users(scientists and physicians studying phenotypic diversity in clinical samples). The success of the project willyield a quantitative increase in the number of samples analyzed in nanowells per user, define protocols forexecuting assays comparable to conventional technologies, and establish a publicly-accessible platform forend-users, opening up new biology in all areas of human cellular disease and treatments.
This research will optimize, validate, and deploy a new platform for single-cell analysis of cells from clinical samples (e.g. tissue biopsies, pediatric samples, body fluids, etc) ex vivo that combines simple nanoliter-scale arrays of wells (similar to ice cube trays) with principles from engineering to enable automated processes for comprehensively characterizing human cells with minimal manipulation. This capability addresses the widespread need of physicians and researchers to understand how the biology of human disease and treatments in affected tissues differs from that in the blood. The novel technology for single-cell analysis developed here will transform the ability to conduct science at the source of diseases and generate new knowledge to improve the development of drugs and vaccines, as well as diagnostics, in infectious diseases, autoimmunity, and cancer, among others.
| Gierahn, Todd M; Wadsworth 2nd, Marc H; Hughes, Travis K et al. (2017) Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods 14:395-398 |
| Chattopadhyay, Pratip K; Gierahn, Todd M; Roederer, Mario et al. (2014) Single-cell technologies for monitoring immune systems. Nat Immunol 15:128-35 |
| Gierahn, Todd M; Loginov, Denis; Love, J Christopher (2014) Crossword: a fully automated algorithm for the segmentation and quality control of protein microarray images. J Proteome Res 13:362-71 |
| Yamanaka, Yvonne J; Szeto, Gregory L; Gierahn, Todd M et al. (2012) Cellular barcodes for efficiently profiling single-cell secretory responses by microengraving. Anal Chem 84:10531-6 |
