Integrating single-cell imaging and RNA sequencing datasets links differentiation and morphogenetic dynamics of human pancreatic endocrine progenitors.

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet information on their dynamics is limited, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. By monitoring both NEUROG3 gene expression and protein in single cells using a knockin dual reporter in 2D and 3D models of human pancreas development, we show an approximately 2-fold slower expression of human NEUROG3 than that of the mouse.

Pancreas organoid models of development and regeneration.

Organoids have become one of the fastest progressing and applied models in biological and medical research, and various organoids have now been developed for most of the organs of the body. Here, we review the methods developed to generate pancreas organoids in vitro from embryonic, fetal and adult cells, as well as pluripotent stem cells. We discuss how these systems have been used to learn new aspects of pancreas development, regeneration and disease, as well as their limitations and potential for future discoveries.

Organoid Imaging: Seeing Development and Function.

Organoids are miniaturized and simplified versions of an organ produced in vitro from stem or progenitor cells. They are used as a model system consisting of multiple cell types forming an architecture relevant to the organ and carrying out the function of the organ. They are a useful tool to study development, homeostasis, regeneration, and disease.

Long-term feeder-free culture of human pancreatic progenitors on fibronectin or matrix-free polymer potentiates β cell differentiation.

With the aim of producing β cells for replacement therapies to treat diabetes, several protocols have been developed to differentiate human pluripotent stem cells to β cells via pancreatic progenitors. While in vivo pancreatic progenitors expand throughout development, the in vitro protocols have been designed to make these cells progress as fast as possible to β cells. Here, we report on a protocol enabling a long-term expansion of human pancreatic progenitors in a defined medium on fibronectin, in the absence of feeder layers.

A 3D system to model human pancreas development and its reference single-cell transcriptome atlas identify signaling pathways required for progenitor expansion.

Human organogenesis remains relatively unexplored for ethical and practical reasons. Here, we report the establishment of a single-cell transcriptome atlas of the human fetal pancreas between 7 and 10 post-conceptional weeks of development. To interrogate cell-cell interactions, we describe InterCom, an R-Package we developed for identifying receptor-ligand pairs and their downstream effects.

Self-organization of organoids from endoderm-derived cells.

Organoids constitute biological systems which are used to model organ development, homeostasis, regeneration, and disease in vitro and hold promise for use in therapy. Reflecting in vivo development, organoids form from tissue cells or pluripotent stem cells. Cues provided from the media and individual cells promote self-organization of these uniform starting cells into a structure, with emergent differentiated cells, morphology, and often functionality that resemble the tissue of origin.

Recapitulating and Deciphering Human Pancreas Development From Human Pluripotent Stem Cells in a Dish.

Here, we review how human pluripotent stem cell models of pancreas development have emerged and became an important tool to study human development and disease. Initially developed toward the production of β cells for diabetes therapy, the protocols have been refined based on knowledge of pancreas development in model organisms. While the cells produced are closer and closer to the end goal of a functional β cell, these models have also been used to carry out functional experiments addressing gene function and expression as well as regulatory and epigenetic landscape changes during human pancreas development.

Stochastic priming and spatial cues orchestrate heterogeneous clonal contribution to mouse pancreas organogenesis.

Spatiotemporal balancing of cellular proliferation and differentiation is crucial for postnatal tissue homoeostasis and organogenesis. During embryonic development, pancreatic progenitors simultaneously proliferate and differentiate into the endocrine, ductal and acinar lineages. Using in vivo clonal analysis in the founder population of the pancreas here we reveal highly heterogeneous contribution of single progenitors to organ formation.

REST represses a subset of the pancreatic endocrine differentiation program.

To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate.

Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas.

Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells.

Bicaudal C1 promotes pancreatic NEUROG3+ endocrine progenitor differentiation and ductal morphogenesis.

In human, mutations in bicaudal C1 (BICC1), an RNA binding protein, have been identified in patients with kidney dysplasia. Deletion of Bicc1 in mouse leads to left-right asymmetry randomization and renal cysts. Here, we show that BICC1 is also expressed in both the pancreatic progenitor cells that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells.

Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals.

Coordinated arterial-venous differentiation is crucial for vascular development and function. The origin of the cardinal vein (CV) in mammals is unknown, while conflicting theories have been reported in chick and zebrafish. Here, we provide the first molecular characterization of endothelial cells (ECs) expressing venous molecular markers, or venous-fated ECs, within the emergent dorsal aorta (DA).

Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development.

During development, pancreatic endocrine cells are specified within the pancreatic epithelium. They subsequently delaminate out of the epithelium and cluster in the mesenchyme to form the islets of Langerhans. Neurogenin3 (Ngn3) is a transcription factor required for the differentiation of all endocrine cells and we investigated its role in their delamination.

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis.

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins.

Cell-autonomous requirement for beta1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice.

beta1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of beta1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1(-/-) embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day (e) 8.

Endothelial FAK is essential for vascular network stability, cell survival, and lamellipodial formation.

Morphogenesis of a vascular network requires dynamic vessel growth and regression. To investigate the cellular mechanism underlying this process, we deleted focal adhesion kinase (FAK), a key signaling mediator, in endothelial cells (ECs) using Tie2-Cre mice. Targeted FAK depletion occurred efficiently early in development, where mutants exhibited a distinctive and irregular vasculature, resulting in hemorrhage and lethality between embryonic day (e) 10.

Deamidation, but not truncation, decreases the urea stability of a lens structural protein, betaB1-crystallin.

Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation.