This question is not trivial, and attempts to answer it reveal critical gaps in the understanding of human islet cell physiology. In other words, it is not clear exactly what the features are of the target cell type, or cell types, that need to be generated. The development of surrogate β-cells is also hampered by limited understanding of the function and functional heterogeneity in fully responsive human islets from healthy donors. 9 However, the continuous analysis of single living cells, including real-time measurements of signal transduction events, is still required to assess true phenotypic heterogeneity. New breakthroughs in single-cell transcriptomics, epigenomics and proteomics promise to enable snapshots of cell-to-cell heterogeneity. 7 The analyses of mRNA, protein, and metabolites can further assist with the comparison of stem cell derived cultures to human islets, 5,6,8 but these can be misleading as preparations are typically of mixed cell types. 5,6 The reason for assaying Ca 2+ flux is that the main function of a pancreatic β-cell is glucose-stimulated insulin exocytosis, a process that requires voltage-dependent Ca 2+ signals caused by the closure of ATP-sensitive K + channels. To do this, current functional studies typically compare ‘representative example’ Ca 2+ signal traces between stem cell derived cells (or groups of cells) to primary human islet cell preparations of variable quality. 5 This necessitates high-throughput analysis at the single-cell level. 5 Among the many technical challenges is the fact that even the most sophisticated published protocols generate highly heterogeneous cultures of cells. Up to this point, single-cell analysis suggests that only a small percentage of partially functional cells have been produced. 1,2 Clinical studies demonstrate the therapeutic potential of islet cell replacement for diabetes, 3,4 and therefore many academic and industrial laboratories have sought to produce functional β-cells from sources such as embryonic stem cells. We hope that the approaches and tools described here will be helpful for those studying heterogeneity in primary islet cells, as well as excitable cells derived from embryonic stem cells or induced pluripotent cells.ĭiabetes is the result of pancreatic islet β-cell dysfunction and/or death. To the best of our knowledge, this report represents the first unbiased cluster-based analysis of human β-cell functional heterogeneity of simultaneous recordings. Using feature extraction from the Ca 2+ traces on this reference data set, we identified 2 distinct populations of cells with β-like responses to glucose. Using an example data set, we illustrate the utility of these approaches by clustering dynamic intracellular Ca 2+ responses to high glucose in ∼300 simultaneously imaged single islet cells. To enable the analysis of β-cell heterogeneity in an unbiased and quantitative way, we developed model-free and model-based statistical clustering approaches, and created new software called TraceCluster. However, it is unclear what the final product of these efforts should be, as β-cells are thought to be heterogeneous. Worldwide efforts are underway to replace or repair lost or dysfunctional pancreatic β-cells to cure diabetes.
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