The Importance of REST for Development and Function of Beta Cells.

Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST is expressed in the progenitors of both neurons and beta cells during development, but it is down-regulated as the cells differentiate. Although REST mutations and deregulation have yet to be connected to diabetes in humans, REST activation during both development and in adult beta cells leads to diabetes in mice

Endoderm specification

In this chapter I focus on the emergence of endoderm, the origin of these cells and their organization in space. I also discuss the molecular events that lead to endoderm formation and how endoderm can be molecularly defined. Although the molecular control of endoderm formation has initially been deciphered using Xenopus, Zebrafish, sea urchin and several other species many molecular switches have been confirmed in mice. This article preferentially cites references in the mouse model system but data from other model organisms are used when they provide important information missing in mice. Extensive references to other species can be found in Grapin-Botton; Constam, 2007 and Stainier, 2002. This article presents endoderm engineering from ES cells and provides molecular triggers and landmarks that may be used for optimized engineering based on normal development. Due to the similarity of markers between definitive and extraembryonic endoderm and the recent discovery that visceral endoderm can contribute to the digestrive tract, the generation of these lineages is also discussed (Kwon et al., 2008). Although endoderm stem cells, that is stem cells endowed with the ability to give rise to all endodermal derivatives but not ectoderm or mesoderm, have not been reported yet, there are stem cells in specific endodermal organs which will be discussed in the following chapters

Concise reviews: in vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors.

Three-dimensional models of organ biogenesis have recently flourished. They promote a balance between stem/progenitor cell expansion and differentiation without the constraints of flat tissue culture vessels, allowing for autonomous self-organization of cells. Such models allow the formation of miniature organs in a dish and are emerging for the pancreas, starting from embryonic progenitors and adult cells. This review focuses on the currently available systems and how these allow new types of questions to be addressed. We discuss the expected advancements including their potential to study human pancreas development and function as well as to develop diabetes models and therapeutic cells

Retinoic Acid Signaling Organizes Endodermal Organ Specification along the Entire Antero-Posterior Axis

Background: Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer. Methodology/Principal Findings: RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA + cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically wit

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. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program

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. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle-dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy

Aims/hypothesis: Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. Methods: Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. Results: xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. Conclusions/interpretation: Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplant

Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells

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

The pancreas arises from a small population of cells but how individual cells contribute to organ formation is unclear. Here, the authors deconstruct pancreas organogenesis into clonal units, showing that single progenitors give rise to heterogeneous multi-lineage and endocrinogenic single-lineage clones

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. Moreover, through a screening of a polymer library we identify a polymer that can replace fibronectin. Our experiments, comparing expanded progenitors to directly differentiated progenitors, show that the expanded progenitors differentiate more efficiently into glucose-responsive β cells and produce fewer glucagon-expressing cells. The ability to expand progenitors under defined conditions and cryopreserve them will provide flexibility in research and therapeutic production