Concepts and limitations for learning developmental trajectories from single cell genomics.

Single cell genomics has become a popular approach to uncover the cellular heterogeneity of progenitor and terminally differentiated cell types with great precision. This approach can also delineate lineage hierarchies and identify molecular programmes of cell-fate acquisition and segregation. Nowadays, tens of thousands of cells are routinely sequenced in single cell-based methods and even more are expected to be analysed in the future. However, interpretation of the resulting data is challenging and requires computational models at multiple levels of abstraction. In contrast to other applications of single cell sequencing, where clustering approaches dominate, developmental systems are generally modelled using continuous structures, trajectories and trees. These trajectory models carry the promise of elucidating mechanisms of development, disease and stimulation response at very high molecular resolution. However, their reliable analysis and biological interpretation requires an understanding of their underlying assumptions and limitations. Here, we review the basic concepts of such computational approaches and discuss the characteristics of developmental processes that can be learnt from trajectory models.

Comprehensive single cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis.

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7260 profiled Ngn3-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis in vivo.

A transcriptional cross species map of pancreatic islet cells.

OBJECTIVE: Pancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions. METHODS: In this study, we used single-cell RNA sequencing to profile more than 50'000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and ß-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions. RESULTS: We showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and ß-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and ß-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data. CONCLUSIONS: Here, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

T cells modulate the microglial response to brain ischemia.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations - with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

Synaptotagmin-13 orchestrates pancreatic endocrine cell egression and islet morphogenesis.

During pancreas development endocrine cells leave the ductal epithelium to form the islets of Langerhans, but the morphogenetic mechanisms are incompletely understood. Here, we identify the Ca2+-independent atypical Synaptotagmin-13 (Syt13) as a key regulator of endocrine cell egression and islet formation. We detect specific upregulation of the Syt13 gene and encoded protein in endocrine precursors and the respective lineage during islet formation. The Syt13 protein is localized to the apical membrane of endocrine precursors and to the front domain of egressing endocrine cells, marking a previously unidentified apical-basal to front-rear repolarization during endocrine precursor cell egression. Knockout of Syt13 impairs endocrine cell egression and skews the α-to-ß-cell ratio. Mechanistically, Syt13 is a vesicle trafficking protein, transported via the microtubule cytoskeleton, and interacts with phosphatidylinositol phospholipids for polarized localization. By internalizing a subset of plasma membrane proteins at the front domain, including α6ß4 integrins, Syt13 modulates cell-matrix adhesion and allows efficient endocrine cell egression. Altogether, these findings uncover an unexpected role for Syt13 as a morphogenetic driver of endocrinogenesis and islet formation.

Sfaira accelerates data and model reuse in single cell genomics.

Single-cell RNA-seq datasets are often first analyzed independently without harnessing model fits from previous studies, and are then contextualized with public data sets, requiring time-consuming data wrangling. We address these issues with sfaira, a single-cell data zoo for public data sets paired with a model zoo for executable pre-trained models. The data zoo is designed to facilitate contribution of data sets using ontologies for metadata. We propose an adaption of cross-entropy loss for cell type classification tailored to datasets annotated at different levels of coarseness. We demonstrate the utility of sfaira by training models across anatomic data partitions on 8 million cells.

CD81 marks immature and dedifferentiated pancreatic ß-cells.

OBJECTIVE: Islets of Langerhans contain heterogeneous populations of insulin-producing ß-cells. Surface markers and respective antibodies for isolation, tracking, and analysis are urgently needed to study ß-cell heterogeneity and explore the mechanisms to harness the regenerative potential of immature ß-cells. METHODS: We performed single-cell mRNA profiling of early postnatal mouse islets and re-analyzed several single-cell mRNA sequencing datasets from mouse and human pancreas and islets. We used mouse primary islets, iPSC-derived endocrine cells, Min6 insulinoma, and human EndoC-ßH1 ß-cell lines and performed FAC sorting, Western blotting, and imaging to support and complement the findings from the data analyses. RESULTS: We found that all endocrine cell types expressed the cluster of differentiation 81 (CD81) during pancreas development, but the expression levels of this protein were gradually reduced in ß-cells during postnatal maturation. Single-cell gene expression profiling and high-resolution imaging revealed an immature signature of ß-cells expressing high levels of CD81 (CD81high) compared to a more mature population expressing no or low levels of this protein (CD81low/-). Analysis of ß-cells from different diabetic mouse models and in vitro ß-cell stress assays indicated an upregulation of CD81 expression levels in stressed and dedifferentiated ß-cells. Similarly, CD81 was upregulated and marked stressed human ß-cells in vitro. CONCLUSIONS: We identified CD81 as a novel surface marker that labels immature, stressed, and dedifferentiated ß-cells in the adult mouse and human islets. This novel surface marker will allow us to better study ß-cell heterogeneity in healthy subjects and diabetes progression.

Non-canonical Wnt/PCP signalling regulates intestinal stem cell lineage priming towards enteroendocrine and Paneth cell fates.

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to treat chronic intestinal diseases. However, the different models of ISC lineage hierarchy1-6 and segregation7-12 are subject to debate. Here, we have discovered non-canonical Wnt/planar cell polarity (PCP)-activated ISCs that are primed towards the enteroendocrine or Paneth cell lineage. Strikingly, integration of time-resolved lineage labelling with single-cell gene expression analysis revealed that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells7. Finally, Wnt/PCP-activated Lgr5+ ISCs are molecularly indistinguishable from Wnt/ß-catenin-activated Lgr5+ ISCs, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we redefine the mechanisms underlying ISC lineage hierarchy and identify the Wnt/PCP pathway as a new niche signal preceding lateral inhibition in ISC lineage priming and segregation.

Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice.

Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.

Targeted pharmacological therapy restores ß-cell function for diabetes remission.

Dedifferentiation of insulin-secreting ß cells in the islets of Langerhans has been proposed to be a major mechanism of ß-cell dysfunction. Whether dedifferentiated ß cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study ß-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with ß-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in ß cells and restores maturation and function for diabetes remission. Additional ß-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases ß-cell survival and regeneration. GLP-1-oestrogen also protects human ß cells against cytokine-induced dysfunction. This study not only describes mechanisms of ß-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated ß cells for diabetes remission.

Establishment of a high-resolution 3D modeling system for studying pancreatic epithelial cell biology in vitro.

OBJECTIVE: Translation of basic research from bench-to-bedside relies on a better understanding of similarities and differences between mouse and human cell biology, tissue formation, and organogenesis. Thus, establishing ex vivo modeling systems of mouse and human pancreas development will help not only to understand evolutionary conserved mechanisms of differentiation and morphogenesis but also to understand pathomechanisms of disease and design strategies for tissue engineering. METHODS: Here, we established a simple and reproducible Matrigel-based three-dimensional (3D) cyst culture model system of mouse and human pancreatic progenitors (PPs) to study pancreatic epithelialization and endocrinogenesis ex vivo. In addition, we reanalyzed previously reported single-cell RNA sequencing (scRNA-seq) of mouse and human pancreatic lineages to obtain a comprehensive picture of differential expression of key transcription factors (TFs), cell-cell adhesion molecules and cell polarity components in PPs during endocrinogenesis. RESULTS: We generated mouse and human polarized pancreatic epithelial cysts derived from PPs. This system allowed to monitor establishment of pancreatic epithelial polarity and lumen formation in cellular and sub-cellular resolution in a dynamic time-resolved fashion. Furthermore, both mouse and human pancreatic cysts were able to differentiate towards the endocrine fate. This differentiation system together with scRNA-seq analysis revealed how apical-basal polarity and tight and adherens junctions change during endocrine differentiation. CONCLUSIONS: We have established a simple 3D pancreatic cyst culture system that allows to tempo-spatial resolve cellular and subcellular processes on the mechanistical level, which is otherwise not possible in vivo.

Systematic single-cell analysis provides new insights into heterogeneity and plasticity of the pancreas.

BACKGROUND: Diabetes mellitus is characterized by loss or dysfunction of insulin-producing ß-cells in the pancreas, resulting in failure of blood glucose regulation and devastating secondary complications. Thus, ß-cells are currently the prime target for cell-replacement and regenerative therapy. Triggering endogenous repair is a promising strategy to restore ß-cell mass and normoglycemia in diabetic patients. Potential strategies include targeting specific ß-cell subpopulations to increase proliferation or maturation. Alternatively, transdifferentiation of pancreatic islet cells (e.g. α- or δ-cells), extra-islet cells (acinar and ductal cells), hepatocytes, or intestinal cells into insulin-producing cells might improve glycemic control. To this end, it is crucial to systematically characterize and unravel the transcriptional program of all pancreatic cell types at the molecular level in homeostasis and disease. Furthermore, it is necessary to better determine the underlying mechanisms of ß-cell maturation, maintenance, and dysfunction in diabetes, to identify and molecularly profile endocrine subpopulations with regenerative potential, and to translate the findings from mice to man. Recent approaches in single-cell biology started to illuminate heterogeneity and plasticity in the pancreas that might be targeted for ß-cell regeneration in diabetic patients. SCOPE OF REVIEW: This review discusses recent literature on single-cell analysis including single-cell RNA sequencing, single-cell mass cytometry, and flow cytometry of pancreatic cell types in the context of mechanisms of endogenous ß-cell regeneration. We discuss new findings on the regulation of postnatal ß-cell proliferation and maturation. We highlight how single-cell analysis recapitulates described principles of functional ß-cell heterogeneity in animal models and adds new knowledge on the extent of ß-cell heterogeneity in humans as well as its role in homeostasis and disease. Furthermore, we summarize the findings on cell subpopulations with regenerative potential that might enable the formation of new ß-cells in diseased state. Finally, we review new data on the transcriptional program and function of rare pancreatic cell types and their implication in diabetes. MAJOR CONCLUSION: Novel, single-cell technologies offer high molecular resolution of cellular heterogeneity within the pancreas and provide information on processes and factors that govern ß-cell homeostasis, proliferation, and maturation. Eventually, these technologies might lead to the characterization of cells with regenerative potential and unravel disease-associated changes in gene expression to identify cellular and molecular targets for therapy.