Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms

Prior studies have shown that pancreatic -cells can transdifferentiate into {beta}-cells, and that {beta}-cells de-differentiate and are prone to acquire an -cell phenotype in type 2 diabetes (T2D). However, the specific human -cell and {beta}-cell subtypes that are involved in -to-{beta}-cell and {beta}-to--cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into -{beta} cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG-expressing human -cell subclusters [1, 2, -{beta}-transition 1 (AB-Tr1), -{beta}-transition 2 (AB-Tr2), and -{beta} (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The 1, 2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for -cell function while AB cells are enriched in genes related to pancreatic progenitor and {beta}-cell pathways; 4) Trajectory inference analysis of extracted - and {beta}-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both - and {beta}-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards {beta}-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards -cells. 6) Remarkably, in contrast to the events in vitro, the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from -to-{beta}-cells in vivo. 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from {beta}-to--cells compatible with dedifferentiation or conversion into -cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro, in vivo, in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between - and {beta}-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo. Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.