Exploiting the power of CyTOF/Mass Cytometry (MC) to elucidate the complex interactions of islet and immune cells in human type 1 diabetes pancreata
Contact PI: Dirk Homann, MD, Icahn School of Medicine at Mount Sinai
Andrew Stewart, MD, Investigator, Icahn School of Medicine at Mount Sinai
Clayton Mathews, PhD, Investigator, University of Florida
Al Powers, MD, Investigator, Vanderbilt University
Seung Kim, MD, PhD, Stanford University
Direct interrogation of the human pancreas has been constrained by the twin challenges of procuring suitable tissue samples, and the limitation of discrete analytical readouts that can be obtained from scarce biological samples. To overcome these obstacles, we propose to harness the power of CyTOF/mass cytometry (MC) for the creation of an integrated analysis platform that will permit the generation of an inclusive perspective onto the cellular identities, properties and functionalities of normal (non-diabetic) and type 1 diabetic human islet and associated cell populations.
The principal aim of this proposal is the development of a basic “islet cell MC” staining panel focused on major islet cell hormones, transcription factors, cell surface antigens, and markers for distinction of exocrine and ductal cells. To this end, we will employ a structured experimental approach to antibody selection, testing and validation that builds on initial flow cytometry-based assays, incorporates a variety of technical controls and will identify suitable antibodies before proceeding to MC staining panel design, metal conjugation and overall optimization/consolidation of staining protocols. This core “islet cell MC” staining panel will then serve as a “backbone” for the development of higher-order staining panels that draw on an array of independently established or yet to be developed probes for characterization immune cell subsets, inflammatory mediators, viral traces, cell cycle checkpoints, and other parameters. Execution of this proposal and its future continuation are contingent on ready access to healthy and diseased human islets and we have therefore established interactions with various co- investigators, collaborators, facilities, institutions, programs and in particular the HIRN consortia that together will ensure the continued availability of these precious tissues and their broadly collaborative interrogation at the level of experimental design, practical implementation and data analysis.
In summary, we expect to establish “islet cell MC” as a flexible analysis platform for the elucidation, stratification and integration of complex protein expression patterns that will effectively parse islet cell identities, subset composition and phenotypic/functional properties in pancreatic health and disease; beyond the immed iate experimental questions addressed in the present and related future work, “islet MC” may also find application in the larger field of human islet cell biology including studies of beta cell de- and trans-differentiation, pluripotent cell differentiation and pancreas development, refinement of drug discovery strategies, and other areas.
Opportunity Pool Project Sponsored by CMAI & CTAR