Pseudotime Ordering of Single Human β-Cells Reveals States of Insulin Production and Unfolded Protein Response

Xin, Yurong; Gutiérrez, Giselle Domínguez; Okamoto, Haruka; Kim, Jinrang; Lee, Ann–Hwee; Adler, Christina; Ni, Min; Yancopouloš, George D.; Murphy, Andrew; Gromada, Jesper

doi:10.2337/db18-0365

Cited by 160 publications

(239 citation statements)

References 60 publications

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“…BIP), showed peak expression in moderately gfp expressing cells ( Fig. 5C), consistent with its known relationship with insulin production in human β-cells (Xin et al, 2018). Heat shock protein 90kDa beta 1 (Hsp90b1, a.k.a.…”

Section: Profiling β-Cell States With Single-cell Rna Sequencingsupporting

confidence: 81%

“…Heterogeneity of insulin production is an established phenomenon, and we started our study by staining human pancreas with antibodies to insulin and PDX1, a key transcription factor for βcell survival and function (Johnson et al, 2003;Szabat et al, 2012). As expected based on single-cell RNA sequencing data (Xin et al, 2018) and previous single-cell imaging of human βcells (Johnson et al, 2006), imaging over a large dynamic range enabled the identification of βcells with both high and low insulin protein levels in human pancreas ( Fig. 1A).…”

Section: In Vivo Heterogeneity Of Insulin Content In Human β-Cell Andmentioning

confidence: 94%

“…However, not all β-cells appear the same. Indeed, functional β-cell heterogeneity is well established (Benninger and Hodson, 2018), including cellular specialization for islet cell synchronization, insulin secretion, insulin production, and marker gene expression (Bader et al, 2016;Dorrell et al, 2016;Heimberg et al, 1993;Johnston et al, 2016;Kiekens et al, 1992;Kolic and Johnson, 2016;Ling et al, 1998;Pipeleers, 1992;van der Meulen et al, 2017;Wills et al, 2016;Xin et al, 2018). For example, recent in situ imaging has revealed the existence of extreme β-cells, defined as having >2 fold Ins2 mRNA than the median expression as measured by single-molecule fluorescence in situ hybridization (Benninger and Hodson, 2018;Farack et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Dynamic Ins2 gene activity defines β-cell maturity states

Modi

Chu

Skovsø

et al. 2019

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Word Count: 230 Word Count: 4037 Reference Count: 54 Figure Count: 5 AbstractHeterogeneity within specific cell types is common and increasingly apparent with the advent of single-cell transcriptomics. Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas, including so-called extreme β-cells exhibiting >2 fold higher insulin gene activity. However, it is not yet clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live-cell imaging, with complementary experiments employing FACS and single-cell RNA sequencing, in β-cells from Ins2 GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2 GFP mice confirmed that at a given moment, ~25% of β-cells exhibited significantly higher activity at the conserved insulin gene Ins2(GFP) HIGH . Live-cell imaging captured on and off 'bursting' behaviour in single β-cells that lasted hours to days. Single cell RNA sequencing determined that Ins2(GFP) HIGH βcells were enriched for markers of β-cell maturity and had reduced expression of anti-oxidant genes. Ins2(GFP) HIGH β-cells were also significantly less viable at all glucose concentrations and in the context of ER stress. Collectively, our results demonstrate that the heterogeneity of extreme insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of β-cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.

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Section: Profiling β-Cell States With Single-cell Rna Sequencingsupporting

confidence: 81%

Section: In Vivo Heterogeneity Of Insulin Content In Human β-Cell Andmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic Ins2 gene activity defines β-cell maturity states

Modi

Chu

Skovsø

et al. 2019

Preprint

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“…We thus hypothesize that acinar-I cells represent a different stage of acinar cell activation that could be converted to acinar-S cells following hormonal or nutritional stimulation, resembling the different activation states recently identified in human and mouse β cells 38,39 . Moreover, acinar-I cells might constitute a source of cell replacement for acinar-S cells, which are subject to high level of ER stress and therefore more prone to cell death via apoptosis.…”

mentioning

confidence: 62%

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

Tosti

Hang

Debnath

et al. 2019

Preprint

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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.

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“…4A). We then compared these populations corresponding to primary pancreatic human islets 37 (Fig. 4B).…”

Section: Transcriptome Analysis Shows An Increase In Endocrine Cellsmentioning

confidence: 99%

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Sui

Egli

Xin

et al. 2020

Preprint

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Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown.Understanding proliferation potential is important for the safty of cell replacement therapy with 2 cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing Sphase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

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Pseudotime Ordering of Single Human β-Cells Reveals States of Insulin Production and Unfolded Protein Response

Cited by 160 publications

References 60 publications

Dynamic Ins2 gene activity defines β-cell maturity states

Dynamic Ins2 gene activity defines β-cell maturity states

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

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

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