Project Investigators:

Gordon Keller

Philip Streeter

Lay Abstract:

Stem/progenitor cells offer the promise that transplantable beta cells may be generated in the laboratory. For this promise to be realized, these cells will need to be isolated and cultured, they will need to be differentiated to become beta cells, and the differentiated cells may need to be isolated from complex cell mixtures following cell culture. In this work, we propose to further characterize novel reagents developed during the first year of this project that are directed against endoderm, and pancreatic and hepatic lineage cells, and to develop additional reagents directed against cell surface molecules expressed by stem cell-derived insulin expressing cells. These reagents will be used to accelerate progress in the development of methods for generation of transplantable beta cells.

Project Investigators:

Maike Sander

Anil Bhushan

Klaus Kaestner

Lori Sussel

Lay Abstract:

Significant research efforts are currently underway to develop treatments or even a cure for diabetes. In particular, many researchers are attempting to exploit embryonic stem (ES), induced pluripotent stem (iPS) and/or adult progenitor cell populations as alternative sources of insulin-producing beta-cells. Thus far, however, the efficient production of large numbers of fully functional and glucose-responsive beta-cells has not yet been achieved. Each cell type within the body contains characteristic chemical modifications in the DNA that are determined by the cell's origin, genetic identity and environment. Furthermore, the characteristic DNA modifications within a particular cell influence its ability to differentiate into specific cell types. Therefore, the failure to transform non-pancreatic cells into beta-cells may be due to the presence of underlying DNA modifications that are resistant to, or provide a barrier against, the activation of beta-cell-specific genes.
DNA methylation is a DNA chemical modification that is believed to be essential for programming different cell types. Most DNA methylation patterns are established during fetal development through highly regulated cell-specific processes that are not well understood. Therefore, we hypothesize that it will be necessary to manipulate the DNA methylation state of a non-pancreatic cell, regardless of its origin, to render it competent to become a functional insulin-producing beta-cell. This proposal aims to understand how gene-specific DNA methylation is established to initiate and/or maintain islet alpha- and beta-cell fates and gene expression patterns. This knowledge will facilitate the differentiation of beta-cells from alternative cell sources. It will also provide insight into the mechanisms that ensure the beta-cell fate is maintained in a transplant setting.

Project Investigators:

Markus Grompe

Klaus Kaestner

Christian Stoeckert

Lay Abstract:

In order to produce functional insulin producing β-cells for the cure of diabetes it is necessary to know all of the genes expressed in these cells inside a normal human pancreas. It is also important to know how the other pancreatic cell types (other hormone producing cells and cells making digestive enzymes) are regulated, because proper β-cells should not activate these other genes. In this work we will produce the first comprehensive catalog of all genes active or inactive in seven major human pancreatic cell types, including β-cells.

Project Investigators:

Ray MacDonald

Anne Grapin-Botton

Mark Magnuson

Palle Serup

Kenneth Zaret

Lay Abstract:

The availability of effective sentinels for developmentally important signaling pathways would facilitate the progress towards understanding how cells are directed toward pancreatic fate, specified for endocrine versus exocrine development, and committed to beta-cell differentiation. A detailed temporal, cell-centric understanding of signaling events is needed for further critical advances. It will be of great value to be able to follow the signaling status of embryonic pancreatic progenitor cells and their progeny dynamically, in order to decipher the program of beta-cell differentiation.
The objective of this Collaborative Bridging Project is to provide genetically modified ES-cells and mice to the BCBC for monitoring signaling events during pancreatic beta-cell development and regeneration, in vivo and in vitro. We propose to create ES-cells and mice bearing sentinel genes that report the receipt of intercellular signals for the major pathways directing development and regeneration: DSL-Notch, Wnt, Hedgehog, retinoic acid (RA), FGF, BMP, and TGFb/nodal/activin. In this phase of the project, sentinels for most pathways (DSL-Notch, Wnt, Hedgehog, retinoic acid (RA), BMP, and TGFb/nodal/activin ) have been tested in transient transfection, transient transgenics and/or chick electroporation. For most of them, effective reporter constructs have been inserted into the Rosa26 locus after deletion of part of the Rosa26 promoter to provide a neutral environment of opened chromatin. The preliminary experiments with the RA reporter show that the strategy is bearing fruits and will allow us to monitor the activity of the pathways in a dynamic manner, coordinating specific events of beta cell formation and function with changes in signaling activities. It is now our goal to transform the knowledge generated in this project into validated tools and to share them with the BCBC community.
The following Specific Aims outline the strategies to create genetically modified ES-cell lines and mice bearing signal-activated sentinels, test these for the veracity of the sentinels to report activation of individual pathways, and provide cell and mouse lines we generate to BCBC investigators. We emphasize the novel use of a modified Rosa26 locus and the creation and distribution of sentinel mice.

Aim 1. Demonstrate that engineering of reporters in the Rosa locus is more powerful than random transgenesis and validate mouse RA- and Wnt reporter lines for release to the community.

Aim 2. Establish whether signal-activated promoters constructed of integrated ‘response modules' are more sensitive for monitoring and characterize a mouse Notch reporter line for release to the community.

Aim 3. Generate sentinels for complex signaling pathways (FGF, BMP, and TGFβ) using composite fragments from natural promoters.

Project Investigators:

Alvin Powers

Yuval Dor

Dale Greiner

Leonard Shultz

Lay Abstract:

Implementation of regenerative therapies for type 1 diabetes will require inhibition of patient's immune system, to halt autoimmunity and/or graft rejection. Recent studies in mice have shown that beta cells have a surprising capacity for regeneration, suggesting that regenerative therapy for diabetes is possible in principle if the immune attack is halted. However, these studies also showed that currently used immune suppression drugs, such as those used in the Edmonton protocol for islet transplantation, are highly toxic to beta cell replication and regeneration, providing a potential explanation for the failure of islet grafts over time. We propose to utilize several advanced experimental models to test the impact of select immunosuppressive agents on mouse and human beta cell regeneration. The results of these experiments will provide important information for the design of clinical trials and the design for future immunosuppressive agents for the treatment of type 1 diabetes.

Project Investigators:

Kenneth Zaret

Ondine Cleaver

Gerard Gradwohl

Harry Heimberg

Lay Abstract:

The pancreatic islet is an endocrine gland; thus, the developing beta cells must coordinate their specification and differentiation with the vasculature so that the final architecture of the islet and pancreas enables rapid and efficient sensing of circulating glucose and secretion of insulin into the bloodstream. Although certain organisms such as zebrafish can survive early development without a vasculature, genetic and functional studies have shown that amniotes, including mammals, require proper vascular development and direct interactions with vascular endothelial cells for beta cell development and islet function. Research from our laboratories and others has shown that endothelial cells help promote endocrine and beta cell development at multiple discrete steps, including the initial differentiation of Ptf1a gene-positive pancreatic progenitors from an initial pool of Pdx-1 gene positive endoderm progenitors; the induction of insulin gene expression from a later pool of Ngn3 gene positive endocrine progenitors; and the normal function of the islet in adult animals. Given the known complexity of beta cell development and the difficulty in reconstituting proper glucose-sensing, insulin secreting cells from stem cells, additional steps of beta cell development may require endothelial cell interactions. To address these issues, in preliminary studies we showed that the Ptf1a induction step of the Pdx-1 positive endoderm, where endothelial cell interactions are crucial, could be reconstituted with secreted endothelial cell proteins in an in vitro explant assay with tissues from flk-1-/- embryos, which lack endothelial cells. Further reiterative cycles of biochemical fractionation and explant screening, followed by proteomic analysis, identified netrin-4 as a secreted endothelial factor that is sufficient to completely replace the effect of endothelial cells in co-cultures. Subsequent analyses of netrin-4 expression in embryogenesis show that the protein is normally induced just prior to the time of Ptf1a induction and is necessary, in the explant assay, for coordinate induction of various early pancreatic regulatory genes. Others in the BCBC have found that netrin-4 promotes endocrine and later insulin expression in mouse embryonic stem cells. Given these successes, we propose to repeat the above screening and proteomic approach to discover endothelial secreted molecules that could: 1) induce later endocrine and insulin expression in embryo pancreatic explant cultures; 2) promote the differentiation of Pdx-1 positive beta cell progenitors that arise after pancreatic duct ligation in adult animals.

Project Investigators:

Yuval Dor

Howard Cedar

Doug Melton

Lay Abstract:

Differentiation of embryonic stem cells to beta cells will be key for the development of cell therapy for diabetes. Progress in this field so far includes the generation of various progenitor cell types from cultured ES cells, which resemble the native, embryonic progenitor cells in terms of marker gene expression. However the derivation of functional beta cells from these progenitors has proven challenging, for unknown reasons.
Whether in vitro generated progenitors indeed have the full potential to become functional beta cells, or are restricted in their potential, is a problem of great importance that is difficult to addess. We propose to study in pancreatic progenitor cells as well as in ES cell-derived pancreatic progenitors the chemical modifications of DNA known as epigenetic marks. These marks, specifically methylation of DNA, are known as important determinants of the fate of cells during embryonic development. However their significance in directed differentiation of stem cells to beta cells has not been examined so far. We will characterize the pattern of DNA methylation as a novel and sensitive tool to ask if seemingly homogenous populations of progenitor cells are already biased to a particular fate.
Our experiments are expected to provide information on questions such as: are ES cell-derived pancreatic progenitors equal to native pancreatic progenitors from embryos? When does commitment of pancreatic progenitors to specific fates take place? When are these cells still amenable for manipulations that will direct them to become beta cells? Answers to these fundamental questions may have important implications for the design of ES cell-based therapy for diabetes. Moreover, DNA methylation profiling may provide a new quality control tool for the assessment of intermediate steps in the process of directed differentiation of beta cells.

Project Investigators:

Pedro Herrera

Maike Sander

Lay Abstract:

"NICER" CBP: Nkx6.1-Induced β-cell Conversion & Enhanced β-cell Regeneration

The overall goal is to identify means by which to improve β-cell regeneration in the adult pancreas by promoting β-cell replication and/or α-cell reprogramming.
The working hypothesis is that the β-cell-specific transcription factor Nkx6.1 can promote β-cell regeneration by two different mechanisms: 1. Inducing the reprogramming of α-cells to β-cells, and 2. Stimulating β-cell proliferation.
Background. To study the intrinsic regenerative capacity of the adult pancreas in the absence of continuous destruction of the β-cells due to autoimmune mechanisms, Pedro Herrera (P.H.) created, with the help of the BCBC, a transgenic model of inducible, rapid, selective total (>99%) or partial (50%) β-cell ablation by administration of diphtheria toxin (DT). In these mice (termed RIP-DTR), there is significant β-cell regeneration over time. Tracking of the cells in mice has shown that after total β-cell ablation, approximately 50% of the new β-cells derive glucagon-producing α-cells (P.H., manuscript submitted). Importantly, in the non-injured pancreas α-cells normally do not convert into β-cells. P.H. further observed that a fraction (10-15%) of the α-cells start to express the β-cell-specific factor Nkx6.1 after total β-cell ablation, which suggests a possible role for Nkx6.1 in the reprogramming of α-cells into β-cells.
By overexpressing Nkx6.1 in isolated islets, a study by C. Newgard has recently suggested that Nkx6.1 promotes β-cell replication. To determine whether Nkx6.1 has similar effects in vivo, Maike Sander (M.S.) generated transgenic mice (termed Nkx6.1OE), in which Nkx6.1 can be specifically induced in any cell type, including β-cells.
Rationale. These preliminary findings suggest that Nkx6.1 could have 2 different context-dependent roles in promoting β-cell regeneration. After near complete β-cell ablation, induction of Nkx6.1 in α-cells could promote conversion of α-cells into β-cells. However, after partial β-cell loss, when β-cells regenerate largely by replication of preexisting β-cells, Nkx6.1 could function to stimulate β-cell expansion.
Using the unique genetic models generated by P.H. and M.S., this CBP will directly test whether Nkx6.1 can promote β-cell regeneration by these 2 distinct mechanisms in vivo in mice.
Approach. We propose to study the potential stimulating effect of Nkx6.1 on:
1. The observed α-to-β-cell conversion/reprogramming (inducible misexpression of Nkx6.1 in α-cells) after total β-cell ablation induced by DT administration (performed in P.H.'s lab)
2. β-cell replication (inducible overexpression of Nkx6.1 in remaining β-cells) after DT-mediated ablation of 50% of the β-cell mass (performed in M.S.'s lab)

Project Investigators:

Anne Grapin-Botton

Yuval Dor

Harry Heimberg

Palle Serup

Lay Abstract:

The objective of this Collaborative Bridging Project is to test the hypothesis that the secreted members of the BMP (Bone Morphogenic Protein) family are involved in replication and regeneration of adult beta cells. The hypothesis derives from our preliminary observation that during development, BMPs, possibly originating from blood vessels, promote beta cell proliferation. The Dor lab will provide expertise in beta cell proliferation and regeneration as well as the tools developed to manipulate blood vessel density. The Dor group has shown in the context of the BRAVE CBP that blood vessels promote beta cell proliferation. The Grapin-Botton lab will provide tools and expertise to manipulate the BMP pathway. The Serup lab will provide chemical tools to manipulate the BMP pathway and expertise with phospho-smad1 monitoring. The Heimberg lab will provide the models to study the BMP pathway during (re)generation of the beta cell mass in adult mouse pancreas.

Our aims in the frame of a one-year project are the following:

- AIM1: To evaluate BMP signaling activity in adult and regenerating pancreas (endocrine cells, ducts, blood vessels). We propose to monitor the activity of the pathway using BRE(BMP Response Element)-GFP reporter mice previously analyzed in the context of the CBP SASSY and phospho-Smad1 immunohistochemistry in normal mice at different times postnatally, in pregnant mice, in mice in which beta cell proliferation is triggered or blocked and in mice with injured pancreas and (re)generating beta cell mass. BMP activity will be correlated with proliferation.

- AIM2: To evaluate whether impairment of BMP signaling limits regeneration. We will conduct a few targeted experiments in which we will follow beta cell proliferation and total mass when the BMP pathway is inactivated either genetically or chemically in vivo.

The results of these experiments may inform efforts to boost beta cell regeneration in vivo.