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Grapin lab work

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Beta cell formation Questions
Our observations by live imaging and in vivo clonal analysis have shown an important degree of stochasticity in the formation of beta cells. They support the view that progenitors have heterogeneous lineage trees which are not pre-determined but result from the probability that a progenitor encounters differentiation signals (likely escaping Notch ligands).
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Cell ablations Method
We perform ablations of cells during development by genetic means or laser targeting to perturb the system and evaluate how it regenerates. This is a way of testing developmental robustness. 
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Clonal analysis Method
We used clonal analysis to genetically mark individual cells in the pancreas primordium and follow their progeny after several days. Our experiments show that progenitors exhibit very heterogeneous family trees, forming progeny of different sizes and with different ratios of progenitor, acinar and endocrine cells. Together with mathematical modelling and single-cell RNA profiling, we interpret that the founder cells of the pancreas are not pre-determined but depending on the signaling environment they encounter, they will differentiate or remain progenitors.
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Control by medium Questions
Single pancreas progenitors seeded in vitro in 3D in Matrigel form either spheres made of progenitors and endocrine cells or more complex organoids made of progenitors assembled into ducts, acinar and only a few endocrine cells. Thus the culture medium exerts some control over the fate of the seeded cell communities. We are trying to understand how. We are interested in developing culture media that robustly control fate.
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Cysts and diabetes Human condition
There are human conditions that associate enlarged ducts (cysts) in multiple organs (kidney, pancreas, liver) and diabetes. We are developing models to try and understand them. Hnf1b genetic variants can predispose to these conditions in human. Our studies in mouse and human suggest that Bicc1 variants may also predispose to these conditions. We are trying to understand the links of these conditions to planar cell polarity and, cilia defects and create models of these diseases using organoids-
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Diabetes Human condition
Our studies are intended to gain insight into human syndromes impairing pancreas development and they guide the generation of replacement beta cells for Diabetes therapy.
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Differentiation paths Questions
We use single-cell molecular profiling to identify progressive molecular changes that operate in cells as they differentiate from stem or progenitor cells to computationally reconstruct their differentiation trajectories. Using human pluripotent stem cells as a model of human development, we could show that differentiation protocols sequentially activate and repress genes enabling most cells to differentiate. A few cells appear to respond in an aberrant manner in vitro, while most differentiate with a relaxed synchrony. For a few genes the activation sequence relative to other genes can vary (Nkx6.1). With time, the differentiation paths branch, going towards different endocrine lineages. However, all cells produced in vitro remain transcriptionally polyhormonal, which differs from their behavior in the body.
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Genetic perturbations Method
We use the power of mouse genetics to study how gene perturbations alter pancreas development. Though we like the speed and ease of monitoring of in vitro models and the perturbations that can be exerted in these systems, notably via CRISPR/Cas9 targeting, we think it is essential to study the function of genes in the context of the whole body. We focused on mouse mutants causing ductal enlargement and endocrine differentiation defects (cyst-and-diabetes models), notably the planar cell polarity and the Bicc1 gene. We also used mouse models to decipher how transcription factors control pancreas development, notably Ptf1a and Neurog3. We also used electroporation in chick embryos (eggs) to study Neurog3 functions in differentiation and migration of endocrine cells.
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Human pancreas Models and samples
Human samples are rare but they enable to investigate differences between human and mouse
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In silico modelling Method
We use in silico modelling to test or propose concepts of development. It enabled us to show that stochastic endocrine cell induction is a likely mechanism underlying the ratios of symmetric/asymmetric divisions observed from progenitors by live imaging and their family trees observed by clonal analysis.
We proposed scenarios whereby diffusible signals can promote the growth of branches. We recently used it to show that the ducts of the pancreas evolve with development from a mesh to a hierarchical tree, a process likely driven by the early secretion of fluid by exocrine cells (from E12.5) and the flow towards the duodenal outlet.
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Live imaging Method
We make movies of pancreas explants or organoids to observe how endocrine cells form from progenitors, how the pancreas regenerates, how cells interact and move or any kind of dynamic process.
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Mechanics Questions
Organoids developing in 3D culture in gels depend on the stiffness of the gels. We use organoids to better understand how mechanics control organ development.
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Mouse pancreas Models and samples
Studying how the pancreas forms in the mouse is our reference (review). We can use genetics to study the effect of gene perturbations or reporters to study where and when genes are expressed.
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Organoids Models and samples
We have developed systems to expand mouse pancreas progenitors from a few cells or single cells in 3D in vitro (protocol). This leads to the formation of organoids similar to a pancreas in vitro. We use these in vitro models to study how a few cells self-organize into an organ. We are currently developing human pancreas organoids.
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Planar polarity Questions
A set of molecules forming the planar cell polarity pathway controls how cells organize and coordinate patterns in sheets of cells. In the embryonic pancreas, this pathway is active in ducts where it controls duct diameter and the production of endocrine cells from ducts. This pathway may be important to understand the etiology of diseases that associate cysts in multiple organs (pancreas, liver, kidney) and diabetes.
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Progenitor proliferation Questions
We use organoids and screens to study how the human pancreas grows.
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Robustness of development Questions
We perform ablations of cells during development by genetic means or laser targeting to perturb the system and evaluate how it regenerates. This is a way of testing developmental robustness. 
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Self-organization rules Questions
Pancreas organoids form complex structures in 3D, starting from a few cells. These cells establish a community where cells change with time (differentiate), exchange signals influencing each other, and organize in space to form a structure similar to a pancreas. We are investigating the rules of this self-organization (or emergence). We found that the number of cells that are in close proximity matters, resulting in a community effect.
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Single-cell transcriptome Method
We used single-cell PCR and more recently whole transcriptome to characterize the molecular "state" of individual cells. Using bioinformatics we can then identify sub-populations and compare cells at different times or after perturbations. We could show that the mouse pancreatic primordium is made of about 500 cells, half of which are committed to endocrine differentiation. We used it to show that endocrine cell formation from human pluripotent stem cells follow largely similar differentiation paths as the mouse and human cells in the body but retain a hybrid state expressing multiple hormone transcripts.
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Stem cells Models and samples
We use human pluripotent stem cell culture to attempt to model human development and compare it with the mouse where processes can be observed in vivo. Human pluripotent stem cell models of pancreas development recapitulate many aspects of development in the body but when carefully compared, also exhibit some differences. For examples, all endocrine cells produced in vitro continue to transcriptionally express multiple hormones, unlike in vivo, in several commonly used differentiation protocols.
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Structure of ducts Questions
We are interested in the beautiful tree-structure the pancreatic ducts form. This structure is important in the adult to deliver digestive enzymes to the duodenum. We showed that secretion test-runs  by exocrine cells from E12.5 likely creates a flow running towards  the duodenal outlet, thereby remodelling the redundant ductal network from a mesh to an optimized hierarchical tree. Moreover the ductal organization, including the planar polarization of their cells, is important for the generation of endocrine cells that form in the ducts. Mutants with duct enlargements exhibit perturbed endocrine differentiation.
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Source Link Target Date
Beta cell formation Clonal analysis
Beta cell formation Live imaging
Cell ablations Mouse pancreas
Clonal analysis Mouse pancreas
Control by medium Organoids
Cysts and diabetes Diabetes
Cysts and diabetes Genetic perturbations
Cysts and diabetes Mouse pancreas
Cysts and diabetes Organoids
Cysts and diabetes Planar polarity
Diabetes Human pancreas
Diabetes Mouse pancreas
Diabetes Organoids
Diabetes Stem cells
Differentiation paths Human pancreas
Differentiation paths Single-cell transcriptome
Differentiation paths Stem cells
Genetic perturbations Mouse pancreas
In silico modelling Clonal analysis
In silico modelling Mouse pancreas
In silico modelling Organoids
Live imaging Mouse pancreas
Live imaging Organoids
Mechanics Organoids
Planar polarity Genetic perturbations
Planar polarity Mouse pancreas
Progenitor proliferation Organoids
Robustness of development Cell ablations
Robustness of development Mouse pancreas
Self-organization rules In silico modelling
Self-organization rules Organoids
Single-cell transcriptome Human pancreas
Single-cell transcriptome Mouse pancreas
Single-cell transcriptome Organoids
Single-cell transcriptome Stem cells
Structure of ducts In silico modelling
Structure of ducts Mouse pancreas
Structure of ducts Organoids
Human pancreas
Mouse pancreas
Organoids
Stem cells