Objective
The principal objective of this proposal is to create the foundation for a “single-cell atlas” of the non-diabetic human pancreas, and to build a modular analysis tool set that will be made available to the T1D research community at large. We envision that such an atlas will serve as a universal resource for investigations into the healthy and diseased pancreas in general, and as a specific foil for T1D research in particular. Furthermore, the proposed development of novel investigative tools will furnish the T1D research community with access to validated and considerably enhanced analysis modalities.
Background Rationale
Identifying, understanding and therapeutically targeting pertinent pathological alterations associated with T1D development and progression requires the study of the T1D pancreas at a level of analytical detail that needs to be matched by corresponding investigations into the non-diabetic pancreas. While technological advances made in the wake of the current “single-cell science revolution” can therefore be leveraged for the enhanced interrogation and comprehensive characterization of pancreatic single-cell populations, applying such strategies to T1D research, especially considering the challenges and complexities of single-cell data science, requires the establishment of a corresponding reference using healthy, non-diabetic human pancreata. Accordingly, we here propose the arguably first interrogation of the normal human pancreas using a combined gene expression (“transcriptional”) and protein expression profiling by means of CITE-seq technology. Furthermore, we will harness relevant insights gained in our CITE-seq studies for the creation of novel practical tools that are predicted to greatly enhance the analytical modalities of future T1D research including both our own work as well as that of interested T1D investigators with whom we intend to share these insights, strategies and reagents.
Description of Project
Type 1 diabetes (T1D) is a major autoimmune disease that poses significant problems to afflicted individuals, the development of effective therapeutic interventions, and public health initiatives at large. Initiated and perpetuated by a complex interplay of genetic and environmental risk factors, insulin-secreting pancreatic beta-cells are progressively destroyed by aberrant immune responses leading to elevated blood glucose levels as well as serious disturbances of protein, fat and carbohydrate metabolism. Currently, no cure or effective prevention is available, and despite insulin treatment, serious long-term complications are frequent.
Due to the considerable logistical challenges to procure primary human pancreas tissues, broad and diversified investigations into the T1D target organ gained momentum only over the past ~15 years, and now are poised to benefit from remarkable technology advances that have nothing less than revolutionized biomedical research. In particular, “single-cell data science” is in the process of profoundly reshaping our perspective onto the human pancreas in health and disease. Often organized in large collaborative consortia that support, coordinate, collate and/or communicate a diverse range of efforts, the striking complexities of individual pancreatic cells are emerging in troves of data sets that provide unparalleled opportunities for collaborative analysis efforts. However, an outstanding challenge pertains to the development of investigative strategies that leverage such insights generated in “single-cell studies” for the targeted interrogation of larger cohorts of non-diabetic control and T1D tissue donors.
Here, we propose a two-tiered research project that combines a discovery component with the development of focused analytical tool sets that are expected to be of broad utility for the T1D research community. Specifically, we will harness so-called CITE-seq technology for the interrogation of the non-diabetic pancreas, a state-of-the-art methodology that to our knowledge has not yet been deployed for analysis of human pancreatic single-cells. The unique advantage of the CITE-seq platform is that it combines comprehensive gene expression analyses of single-cells (this part is identical to single-cell RNA-seq studies) with a targeted proteomic profiling of the same cells; for the latter part, we have secured access to a custom panel of ~285 antibodies that will permit an unprecedented phenotypic characterization of pancreatic cells. While the required tissue specimens, live pancreatic islets, can be readily procured from non-diabetic pancreas donors, corresponding T1D samples are very challenging to obtain yet we are prepared to include them in our CITE-seq analyses should they become available as part of a new effort by the network for pancreatic organ donors with diabetes (nPOD). Importantly, for the second component of our proposal, we will use the insights generated in our CITE-seq studies as well as our considerable experience with mass cytometry to develop tailored antibody staining panels for the targeted annotation as well as phenotypic and functional characterization of pancreatic cells; moreover, these panels can readily be moved to conventional or spectral flow cytometry platforms for facilitated and more wide-spread application by the research community.
The overarching goal of these efforts is four-fold: 1., to define, arguably for the first time, the combined transcriptomic and proteomic properties of human pancreatic cells (e.g., beta cells, other endocrine cells, acinar cells, ductal cells etc.). 2., to leverage pertinent insights for the development of targeted antibody staining panels to be deployed in various cytometry applications. 3., to generate a resource for high-dimensional pancreatic single-cell properties that can directly inform the applied design and focus of multiple other investigations (e.g., imaging studies, in vitro beta cell studies, drug testing etc.). And 4., to establish a non-diabetic pancreatic single-cell atlas that will serve as a direct foil for future studies, including our own, with pancreatic tissue specimens from T1D donors.
Anticipated Outcome
The proposed creation of a combined gene and protein expression atlas of pancreatic single-cells will constitute a novel resource of both conceptual and practical utility. To date, it remains unclear how exactly pancreatic single cell gene expression relates to corresponding protein expression, i.e. which precise proteins are in fact expressed and if so, what the average expression levels are in the non-diabetic pancreas. Such insights will be of considerable value for a multitude of other investigations that seek to interrogate phenotypic properties of pancreatic cell types in health and disease. Moreover, based on our experience with the application of mass cytometry to the study of the human pancreas as well as our preliminary data, we will now design a set of robust analysis tools that will be shared with the wider T1D research community. This applied component of our proposal is therefore expected to considerably advance T1D research in the near future.
Relevance to T1D
The combined investment into T1D research, management and care over the past decades has produced a wealth of new insights, tangible improvements and practical advances, and promising new leads for future treatment strategies. Nevertheless, progress in understanding T1D pathogenesis in all its complexity and heterogeneity has been hampered by considerable knowledge gaps pertaining to the precise cellular composition and properties of the human pancreas. Although this proposal is largely focused on non-diabetic tissue samples, its significance for T1D research is at least five-fold: 1., our work will provide a comprehensive reference for correlated single-cell transcriptomes and proteomes (expression patterns of ~285 cell surface proteins) in the non-diabetic pancreas. 2., our in-depth analyses will define the phenotypic complexity and potential heterogeneity of pancreatic cell types with unparalleled detail. 3., precise cellular protein expression patterns, many of which are expected to be novel, are revealed with extensively validated antibodies and thus can inform and guide a broad range of other investigations (e.g., imaging studies) where reagent validation is more challenging, precise target selection can be difficult, and expected expression patterns may remain unclear. 4., the development of modular mass cytometry staining panels will generate practical tools for targeted investigations into the nature and extent of pathological alterations including those specifically associated with T1D pathogenesis. 5., validated mass cytometry staining panels can be readily moved to flow cytometry platforms allowing for more wide-spread utilization, including the sorting of live pancreatic cell subsets according to novel cell surface markers to be identified here for a multiplicity of downstream applications. Finally, we have made arrangements to accommodate CITE-seq analyses of recent-onset T1D samples should such rare specimens (live islets) become available in the course of the present and/or related future projects.