The Kaestner lab employs modern mouse genetic approaches, such as gene targeting, tissue-specific and inducible gene ablation, to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract. We also use next-generation sequencing to look at differences between the transcriptome and epigenome of normal vs diseased tissues.
Some of our current research includes:
Epigenomic rejuvenation of pancreatic beta-cells.
The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide, and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. No treatments have been devised that increase beta-cell mass in vivo in humans, and transplantation of beta-cells is extremely limited due to lack of appropriate donors. For these reasons, increasing functional beta-cell mass in vitro, or in vivo prior to or after transplantation, has become a “Holy Grail” of diabetes research. Our previous studies clearly show that adult human beta-cells can be induced to replicate, and – importantly - that cells can maintain normal glucose responsiveness after cell division. However, the replication rate achieved was still low, likely due in part to the known age-related decline in the ability of the beta-cell to replicate. We propose to build on our previous findings and to develop more efficacious methods to increase functional beta-cell mass by inducing replication of adult beta-cells, and by restoring juvenile functional properties to aged beta-cells. We will focus on mechanisms derived from studies of non-neoplastic human disease as well as age-related phenotypic changes in human beta-cells.
In Aim 1, we will target the genes altered in patients with marked beta-cell hyperplasia, such as those suffering from Beckwith-Wiedemann Syndrome or Multiple Endocrine Neoplasia. Expression of these genes will be altered in human beta-cells via shRNA-mediated gene suppression and locus-specific epigenetic targeting. Success will be assessed in transplanted human islets by determination of beta-cell replication rate and retention of function. In Aim 2, we will determine the mechanisms of age-related decline in beta-cell function and replicative capacity, by mapping the changes in the beta-cell epigenome that occur with age. Selected genes will then be targeted as in Aim 1 to improve human beta-cell function, as assessed by glucose responsiveness. To accomplish these aims, we will use cutting-edge and emerging technologies that are already established or are being developed in our laboratories. The research team combines clinical experience with expertise in molecular biology and extensive experience in genomic modification aimed at enhancing beta-cell replication. By basing interventions on changes found in human disease and normal aging, this approach will increase the chances that discoveries made can be translated more rapidly into clinically relevant protocols.
Regulatory cascades in differentiation and proliferation of the gastrointestinal epithelium.
The mammalian gut epithelium is a highly organized and dynamic system which requires continuous controlled proliferation and differentiation throughout life. Proliferation, cell migration and cell adhesion all must be tightly controlled in order to prevent either inflammatory diseases or epithelial cancers. As with many other vertebrate organs, the digestive tract develops from heterogeneous embryonic origins. While the musculature and the connective tissue are derived from lateral plate mesoderm, the epithelium is derived from the endoderm. We have identified a novel member of the winged helix gene family termed Foxl1 which is expressed in the gut mesoderm and have begun its functional analysis in vivo through targeted mutagenesis in mice. Null mutations in the mesodermal transcription factor Foxl1 result in dramatic alterations in endoderm development, including epithelial hyperproliferation. We have now identified APC/Min and GKLF as downstream targets of Foxl1 and have begun the analysis of these genes in gastrointestinal differentiation by tissue-specific gene ablation.
Innovative genetic approaches for hepatic repopulation.
A better understanding of the liver’s response to toxic injury, which includes hepatocyte proliferation, activation and differentiation of facultative hepatic stem cells (“oval cells”), and – unfortunately – an increased risk for hepatocellular carcinoma, is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Likewise, cell replacement therapy, either through direct hepatocyte transplantation or in bio-artificial liver devices, needs to be improved in order to become a reliable alternative to liver transplantation. To date, investigations of hepatocyte proliferation have frequently focused on the partial hepatectomy paradigm, a “non¬injury” model that is not reflective of liver injury in humans and which has therefore failed to identify specific targets for either improved regeneration following toxic injury or for limiting proliferation in HCC in humans.
In Specific Aim 1, we will determine which genes and gene combinations promote or repress hepatocyte repopulation following toxic liver injury using an innovative genetic approach. In Specific Aim 2, we will employ expression of key hepatic transcription factors to improve the differentiation of hepatic progenitor cells to functional hepatocytes. Together, these approaches will provide an improved understanding of the liver’s response to toxic injury, and facilitate the discovery of new cell replacement therapies to treat chronic liver disease and liver failure.
- Redox regulation during liver repopulation
- Mapping liver tumor signaling pathways in vivo
- In vivo CRISPR-Cas9 activation and inhibition screening during liver repopulation
- Hepatocyte polyploidy and its association with human liver disease
- Pairwise microRNA inhibition in the regenerating mouse liver
- Mitochondrial genome editing to alleviate a nuclear gene deficiency
- Single cell applications in liver triomics
- Liver regeneration to stimulate epigenetic rejuvenation of hepatocytes
- FoxA1/2 regulation of hepatocyte-specific DNA methylation patterns
Regulation and function of the Meg3 locus in beta cells.
Maintenance of glucose homeostasis is central to our health, and its failure results in severe debilitating diseases including diabetes and familial hyperinsulinism. Diabetes mellitus is a metabolic disorder that affects over 285 million people worldwide and is a leading cause of death in many countries. The disease is characterized by either absolute insulin deficiency due to the autoimmune destruction of pancreatic insulin-producing beta-cells [Type 1 diabetes mellitus (T1DM)], or relative insulin deficiency due to defective insulin secretion or insulin sensitivity [Type 2 diabetes mellitus (T2DM)]. The resulting elevated blood glucose levels eventually lead to an impairment of the microvasculature followed by kidney failure, blindness, neuropathy and heart disease. Consequently, diabetes is currently the sixth leading cause of death in the United States (CDC). During the past grant cycle, we have made the exciting discovery that the imprinted MEG3 locus is strongly down-regulated in islets from type 2 diabetics. The MEG3 locus is of particular interest in that it encodes a cluster of 54 microRNAs, which we have found to target anti-apoptotic genes, suggesting that dysregulation of this locus contributes to beta-cell failure in type 2 diabetes. Here, we propose to investigate both epigenetic regulation and biological function of this locus in mouse and human beta-cells. In Aim 1, we will investigate the molecular mechanism that causes dysregulation of the MEG3 locus in diabetes. Specifically, we will test the hypothesis that hyper-methylation of a differentially methylated region (DMR) near the promoter of the MEG3 gene causes loss of binding of beta-cell specific transcription factors in an enhancer in the MEG3 gene. In Aim 2, we will determine the specific function of the MEG3 locus in beta-cell physiology and survival using both mouse genetic and innovative human epigenetic inactivation. Multiple biochemical and molecular assays will be performed on MEG3 deficient beta-cells. Together, these experiments will provide important new insights into the molecular etiology of beta-cell failure in type 2 diabetes.
Penn integrated human pancreas procurement and analysis program.
The past decades have seen a dramatic improvement in our ability to phenotype and molecularly profile human tissues relevant to the etiology of Type 1 diabetes with unprecedented resolution, at the genomic, epigenomic, protein, and functional levels. Here, in collaboration with Dr. Ali Naji's group and others, we will employ state-of-the-art technologies to determine all aspects of pancreas biology as it pertains to type 1 diabetes, juvenile organ donors, and other cases of beta- cell dysfunction. We will profile both the endocrine and immune systems with multiple modalities, and make the vast data accumulated available through the highly accessible PANC-DB to be developed here. This comprehensive profiling of the natural history of Type 1 diabetes will pave the way for future discoveries of new treatment modalities for diabetes.