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Gene Expression and Cell Identity
Alexander Diehl
ImmPort Science Talk3/20/14
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Understanding the Nature of Entities in Reality
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Depends on What Parts We See
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Reality is Often More Complex Than at First Glance
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And Perspective is Important
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What are Cells?
• Cell are physical entities that exist in reality.
• We understand aspects of cells based on results of experimental assays.
• Our knowledge of cell types is necessarily incomplete even as we attempt to understand their nature.
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How We Represent Cells in the Cell Ontology
• Morphology• Surface marker expression, singly or in
combination• Transcription factor expression or expression
of other internal protein• By lineage• By function or capability
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Types of Evidence Behind the Representation of Cells in CL
• Microscopy, with or without staining (histology).• Immunofluorescence in situ or in vitro• Flow cytometry or CyTOF• Colony formation assays• In vivo/in vitro lineage tracking• Directs assays of cellular function, typically in vitro• Indirect assays of cellular function in vivo• And rarely, assays of gene expression.
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9
Experimental Data from Multiple Sources Is Synthesized into a Single Definition in CL
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Challenges in Ontology Building
• We want to represent both general cell types and specific cell types.
• Many cell types are considered equivalent across species in their general characteristics such as surface marker expression or functions.
• Hematopoietic cell types in different species, such as mouse and human, sometimes are called the same name but are defined by different sets of surface markers.
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Challenges in Ontology Building
• We need to provide unique representations via logical definitions for each cell type.
• We need to recognize that in some cases, different combinations of markers may identify the same cell type.
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The HIPC Lyoplate Project
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The HIPC Lyoplate Project
• Standardization of human PBMC immunophenotyping to enhance reproducibility across different facilities.
• Use of eight color flow cytometry with standardized antibody panels
• Use of standardized sample preparation• Use of standardized instrument settings• Use of standardized data analysis
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The HIPC Lyoplate Project
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HIPC-Defined Cell Types in CL
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HIPC-Defined Cell Types in CL
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HIPC-Defined Cell Types in CL
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HIPC-Defined Cell Types in CL
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HIPC-Defined Cell Types in CL
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“effector CD8+ T cell”
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“effector CD8+ T cell”
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“effector CD8+ T cell”
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A request to CL…
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TEMRA = a memory T cell without CD45RO expression.
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Are These the Same Cell Type?
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Are These the Same Cell Type?
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Are These the Same Cell Type?
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But… “effector CD4+ cell”?
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The Gene Expression Part
Questions:Can we use the structure of the Cell Ontology as a framework for comparing gene expression data tied to specific cell types?
Can we use the CL framework to identify genes that distinguish one cell type from closely related cell types?
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The Immunological Genome Project
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Linking IGP data to CL cell types.
• IGP provides gene expression data based on sorted mouse immune cell types developed according to standardized methods.
• We mapped IGP cell types to cell type terms in the Cell Ontology.
• The CL structure was used to guide comparisons between gene expression profiles of different cell types.
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Linking IGP data to CL cell types.
• 88 cell types were compared in a pairwise fashion.
• Separate gene sets were created for genes whose expression differed by greater than or less than 1.5 fold, respectively, for each pairwise comparison.
• 7656 gene sets resulted.
• An ontological framework was created to map these gene sets to Cell Ontology classes.
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Workflow of CL-IGP Project
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Searching for Pairwise
Comparisons Mapped to CL
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Summary of Upregulated and Downregulated Genes by Cell Type
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Novel Genes Identified for Specific Cell Types and Confirmed by IGP
Scd1 is an enzymeinvolved in biosynthesis of monounsaturated fatty acids whose expression is restricted to mature B cells types in comparison to other immune cell types.
I830077J02Rik, a single-pass transmembrane protein, otherwise uncharacterized, is widely expressed among myeloid cells. In lymphocytes, expression of this protein is restricted to marginalzone B cells.
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Novel Genes Identified for Specific Cell Types and Confirmed by IGP
Scd1 is an enzymeinvolved in biosynthesis of monounsaturated fatty acids whose expression is restricted to mature B cells types in comparison to other immune cell types.
I830077J02Rik, a single-pass transmembrane protein, otherwise uncharacterized, is widely expressed among myeloid cells. In lymphocytes, expression of this protein is restricted to marginalzone B cells.
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Candidate Genes
Involved in the Unique
Functions of Germinal
Center B cells
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Conclusions
• Gene expression comparisons placed in an ontology framework can provide details about genes uniquely expression in particular immune cell subtypes.
• Results of our approach has been validated against non-ontologically based analyses of IGP data, for instance for NK cells, and similar results are seen.
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Acknowledgements• Barry Smith• Alan Ruttenberg• Ryan Brinkman• Raphael Gottardo• Richard Scheuermann• David Dougall• Holden Maeckler• Philip McCoy
• Terry Meehan
• Nicole Vasilevsky• Chris Mungall• Melissa Haendel• Judy Blake
• And many other contributors to the CL