Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell... Show moreMuch of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes. Show less
Gurp, L. van; Muraro, M.J.; Dielen, T.; Seneby, L.; Dharmadhikari, G.; Gradwohl, G.; ... ; Koning, E.J.P. de 2019
During pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into alpha-, beta-,gamma- and delta-cells. The... Show moreDuring pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into alpha-, beta-,gamma- and delta-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on alpha- and beta-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev. Pseudotime analysis revealed specific branches for developing alpha- and beta-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into alpha- and beta-cells. Show less
Dharmadhikari, G.; Stolz, K.; Hauke, M.; Morgan, N.G.; Varki, A.; Koning, E. de; ... ; Maedler, K. 2017
