Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - Neobetacell (Neogenesis of new functional Beta Cells through Modulation of Neurogenin-3 Expression to provide Regenerative Therapy for Diabetes Patients)
Teaser
Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels and increased risk of vascular complications. Currently 415 million people have diabetes globally, and that number is expected to rise to 642 million by 2040. Several subtypes of...
Summary
Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels and increased risk of vascular complications. Currently 415 million people have diabetes globally, and that number is expected to rise to 642 million by 2040. Several subtypes of diabetes can be clinically distinguished, including polygenic/multifactorial, monogenic and secondary forms. The two most common forms of diabetes, type 1 (T1DM) and type 2 (T2DM) diabetes are polygenic and multifactorial. Reduced insulin signaling (be it due to insulin resistance or beta cell failure), results in decreased glucose uptake in target organs and increased release by the liver and kidney. The resulting chronic hyperglycemia in turn damages blood vessels and nerves, eventually leading to multi-organ damage.
The current standard of care for diabetes consists of exogenous insulin injections and/or oral glucose-modifying drugs. None of these therapies directly address the underlying beta cell deficit in most patients with diabetes. Beta-cell replacement therapies could effectively restore and protect the functional beta cell-mass. Transplantation of cadaveric human donor islets represents one promising approach for beta cell replacement. Shortage of donor islets however severely limits the more widespread clinical use of this alternative therapy. As such, the need for alternative supplies of human islet cells is evident. Insulin-producing cells can be generated by replication of pre-existing beta cells and/or by (trans)differentiation of non-beta cells.
The current project will identify the mechanisms regulating embryonic beta cell development, expand these mechanisms to in vivo mouse models for beta cell generation, generate a novel humanised mouse model to test beta cell regeneration and expand on the specific targeting of the therapy to human cells in vivo. Many of our prior studies were performed in mice. While mice remain a valuable tool for studying these processes in whole organism, and for preclinical tests of potential therapies, we aim to increasingly move studies into human tissues and ultimately into people.
The impact of the proposed model is potentially very high and can cause a paradigm shift in the field of beta cell regeneration. The current focus within the diabetes field is on generating sufficient functional beta cells that can improve cell therapeutic approaches. The current project proposes to develop a preclinical model representative for human diabetes to study a treatment that would omit the need for cell therapy and thus circumvent the problems associated with allograft rejection. If successful, this approach may lead to revolutionary therapies that alleviate the burden diabetes imposes on patients, with the highest incidence of type 1 diabetes observed in children between the age of 2 and 14 years old. Diabetes remains incurable, has a terrible impact on the quality of life and causes a massive socio-economical problem. Insulin-dependent patients, including young type 1 patients, can still only resort to daily injections of exogenous insulin to control disease progression.
Work performed
Our results revealed that a novel regulator can interact with genomic sequences located within known regulatory regions of transcription factors previously identified to regulate the primary pancreas specification domain, such as Pdx1, Onecut1/Hnf6 and Nkx6.1. The peaks identified are highly enriched compared to the input samples.
In addition, we have identified a potential mechanism by which this protein can modulate the expression of the pro-endocrine master switch transcription factor Neurog3. We found 2 highly enriched binding sites in the promoter region of Hes1. Hes1 is known to be an effector of activated Notch signaling and important to establish lateral inhibition. Lateral inhibition is a way of regulating the number of cells that can adopt an endocrine phenotype by preventing neighbouring cells from adopting a similar Neurog3+ fate. Possibly, our target protein negatively regulates Hes1 expression allowing for amplified Hes1 activation in KO mice. A second important factor regulating Neurog3 expression is Cdkn1a. Induction of Cdkn1a normally inhibits proliferation thus allowing Neurog3 expression in quiescent cells. A potential mechanism for our target is the stimulation or stabilization of Cdkn1a expression, leading to a decrease of Cdkn1a in KO mice.
We studied the mechanisms by which our target regulates Neurog3 expression and how it affects Neurog3 action during human and murine pancreas development. It appears to controls the transition from general pancreas progenitor to differentiating endocrine cell through Neurog3 modulation and acts as determining factor in beta cell differentiation in mice and men.
We identified the targets of our protein using high throughput screening, phenotypic analyses and gain-of-function/loss-of-function experiments. Insights into the transcripts that are differentially regulated hold clues to the mechanisms of action in the pancreas and allow using this knowledge to improve human beta cell regeneration.
1. Using mouse models generated by the applicant, transcriptome analysis of KO animals is compared at critical stages of mouse pancreas development (RNA sequencing analysis on E10: onset of primary transition; E13.5: onset of secondary transition; E15.5: peak of pro-endocrine differentiation). Differentially regulated genes were confirmed by qPCR. In addition, potential rescue of the knockout phenotype by introduction of selected hits is analyzed in ex vivo explant cultures of knockout pancreases.
2. We show evidence that our target protein is regulated in the pancreas by posttranslational modifications, specifically by phosphorylation of serine residues previously shown in other organs.In addition we investigated if either wildtype or mutant protein could rescue endocrine differentiation in deficient pancreatic explants.
3. A recent report has indicated that Neurogenin-3, the master switch for embryonic endocrine differentiation is regulated by posttranslational modifications. By mass spectrometry and mutated forms of Neurog3, we have also identified the described modifications (phosphorylation of serine residues 183 and 187) that affect Neurog3 function. In addition, we have identified a novel modification (acetylation of lysine 72) that appears to affect protein stability and turnover. We studied the effects of our protein on Neurog3 modification during development and assess whether Neurog3 modifications are more affected by phosphorylated or dephosphorylated target.
4. The knowledge gained from the mouse models is applied to study the effect of our protein during human pancreas development. To study human pancreas development, we use fetal human pancreas obtained from terminated pregnancies (9-16 weeks post-conception). Phenotypic analyses of developing human pancreas of various ages determined the expression pattern of our target (especially in relation to Neurog3). Fetal human pancreases were cultured ex vivo as explants in an air-liquid interfase setup. The effects of
Final results
We have found a method to efficiently isolate exosomes from human placenta. This allowed us to study the effects of exosome biology on human pancreas cells. In addition, this discovery will help to devise a delivery system to target regenerative signals to the human cells. We also developed a platform to help in performing sequencing and nucleic acid studies, as well as image analysis tools.
We anticipate to use the knowledge gathered to study regenerative stimuli on human tissues in vivo.