Single nucleus andin situRNA sequencing reveals cell topographies in the human pancreas

Abstract

The cellular heterogeneity of the human pancreas has not been previously characterized due to the presence of extreme digestive enzymatic activities, causing rapid degradation of cells and RNA upon resection. Therefore, previous cellular mapping studies based on gene expression were focused on pancreatic islets, leading to a vast underrepresentation of the exocrine compartment. By profiling the transcriptome of more than 110,000 cells from human donors, we created the first comprehensive pancreas cell atlas including all the tissue components. We unveiled the existence of four different acinar cell states and suggest a division of labor for enzyme production within the healthy exocrine pancreas, which has so far been considered a homogeneous tissue. This work provides a novel and rich resource for future investigations of the healthy and diseased pancreas. Main textSingle-cell RNA sequencing (scRNA-seq) has tremendously expanded our understanding of heterogeneous human tissues and made the identification of novel functional cell types in the lung, brain and liver possible 1-5 . The development of single-nucleus RNA-seq (sNuc-seq) has further broadened its application to tissues which are difficult to dissociate or already archived, such as clinical samples 6 . Pancreatic exocrine tissues contain among the highest level of digestive enzymatic activities in the human body 7 , hindering the preparation of undegraded RNA from this organ. Therefore, previous scRNA-seq studies of the human pancreas have been restricted to the islets of Langerhans (the endocrine part of the organ) in order to remove the exocrine compartment, namely the acinar and ductal cells responsible for the production and transport of digestive enzymes. Following their isolation, the endocrine islets were cultured in vitro, enzymatically dissociated and processed on microfluidics devices before next-generation sequencing [8][9][10][11][12][13][14] . While this strategy proved to be successful in generating a draft of the endocrine human pancreas cell atlas, it has distinct disadvantages. For example, only a very small number of exocrine cells have been captured and their numbers are largely underrepresented relative to homeostatic physiological conditions (approximately 5% rather than 95%). Moreover, in vitro culture and dissociation steps are known to introduce technical artefacts in gene expression measurements 15 . In this work we opted to use flash-frozen tissue biopsies isolated from pancreata of six human donors followed by sNuc-seq ( Fig. 1a), avoiding in vitro expansion and dissociation procedures, aiming to obtain an unbiased sampling of the organ.

Single-nucleus and spatial transcriptomics of archival pancreatic cancer reveals multi-compartment reprogramming after neoadjuvant treatment

“…Examining treatment-naïve and neoadjuvant-treated specimens separately ( Figure 1C) (44), nonmalignant cell subsets primarily partitioned by cell type with substantial inter-patient mixing, whereas malignant cells partitioned by patient, as we previously reported for other tumor types (29,31,32,45,46). Among non-malignant cells, we readily annotated diverse immune, endocrine, and acinar cells, and their cell subsets by known gene signatures ( Figures 1B-C) (40,(47)(48)(49).…”

“…However, scRNA-seq in PDAC has lagged behind other cancer types due to high intrinsic nuclease content and dense desmoplastic stroma (33)(34)(35)(36), resulting in reduced RNA quality, low numbers of viable cells, preferential capture of certain cell types at the expense of others, and challenges with dissociating treated tumors. Single nucleus RNA-seq (snRNA-seq) provides a compelling alternative for difficult-to-dissociate specimens or frozen archival samples (37)(38)(39)(40), and can better recover malignant cells and stroma while reducing stress signatures (introduced by dissociation) and maintaining the same spectrum of cell states (41)(42)(43).…”

Benchmarking single-cell RNA-sequencing protocols for cell atlas projects

“…Several additional steps are crucial for the success of single-cell projects, especially sample preparation. Optimization of sample procurement and tissue-processing conditions is of crucial importance to avoid composition biases and gene expression artifacts [32][33][34][35] that could limit the value of a cell atlas. Therefore, dedicated studies are required to define optimal conditions for tissue and organ preparation in healthy and disease contexts.…”

SARS‐CoV‐2 receptorACE2 andTMPRSS2 are primarily expressed in bronchial transient secretory cells

“…Essentially, single nuclei were isolated as described elsewhere (preprint: Tosti et al , ). Briefly, snap‐frozen healthy lung tissue from lung adenocarcinoma patients was cut into pieces with less than 0.3 cm diameter and single nuclei were isolated at low pH by homogenizing the cells in 1 ml of citric acid‐based buffer (Sucrose 0.25 M, Citric Acid 25 mM, Hoechst 33342 1 g/ml) at 4°C using a glass Dounce tissue grinder.…”