Dr. Kaestner’s lab is employing modern genetic, genomic and epigenomic approaches (ChIP-Seq, RNA-Seq, gene targeting, tissue-specific and inducible gene ablation, CyTOF) to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract. Disease areas targeted by our research include diabetes and cancer.
Description of Research
Epigenomic rejuvenation of human 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. Stem cell niches provide essential signals and growth factors to sustain proliferation and self-renewal of stem cells in regenerative organs such as the intestine. Here, we identify subepithelial telocytes as an obligatory source of Wnt proteins, without which intestinal stem cells cannot be maintained. Telocytes are large but rare mesenchymal cells that are marked by Foxl1 and PDGFRα expression and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, Foxl1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional gene ablation of Porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in Foxl1+ telocytes causes cessation of Wnt signaling to intestinal crypts, loss of stem cell proliferation, and death of mutant mice within four days of tamoxifen treatment. Thus, Foxl1+ telocytes are the critical source of niche signals to intestinal stem cells..
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. We will determine which genes and gene combinations promote or repress hepatocyte repopulation following toxic liver injury using an innovative genetic approach. I 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.
Rotation Projects for 2018
1. Analysis and integration of complex genomic and epigenomic data sets
2. Molecular, histological and metabolic analysis of mouse models of diabetes and gastrointestinal cancer.
3. ChIP-Seq analysis. Chromatin immunoprecipitation using various transcription factor or modified histone antibodies. Library construction, ultra-high throughput sequencing and computational analysis of target sequences.
Amber Wang, Graduate Student
Ayano Kondo, Graduate Student
Kristy Ou, Graduate Student
Dr. Long Gao, Postdoc - Computational Biology
Dr. Yitzak Reizel, Posdoc
Dr. Michal Shoshkes, Postdoc
Dr. Yue Wang, Postdoc
Dr. Amanda Misfeldt, Postdoc
Dr. Catherine Lee, Research Associate
Dr. Adam Zahm, Research Associate
Dr. Maria Golson, Research Assistant Professor
Dr. Jonathan Schug, Technical Director, Functional Genomics Core
Dr. Shilpa Rao, Programmer/Analyst
Ashley Morgan, Research Specialist
Teguru Tembo, Research Specialist
Olga Smirnova, Research Specialist
Michal Shoshkes-Carmel, Yue J. Wang, Kirk J. Wangensteen, Beáta Tóth, Ayano Kondo, Efi E. Massasa, Shalev Itzkovitz, and Klaus H. Kaestner: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts. Nature doi: 10.1038/s41586-018-0084-4, May 2018.
Wang AW, Wangensteen KJ, Wang YJ, Zahm AM, Moss NG, Erez N, Kaestner KH: TRAP-seq identifies cystine/glutamate antiporteras a driver of recovery from liver injury. J Clin Invest Page: DOI: 10.1172/JCI95120, April 2018.
Wangensteen Kirk J, Wang Yue J, Dou Zhixun, Wang Amber W, Mosleh-Shirazi Elham, Horlbeck Max A, Gilbert Luke A, Weissman Jonathan S, Berger Shelley L, Kaestner Klaus H: Combinatorial genetics in liver repopulation and carcinogenesis with a novel in vivo CRISPR activation platform. Hepatology (Baltimore, Md.) Nov 2017.
Kim, R., Sheaffer, K.L., Choi, I., Won, K.-J., and Kaestner, K.H.: Epigenetic regulation of intestinal stem cells by Tet1-mediated DNA hydroxymethylation. Genes Dev in press, December 2016.
Bernstein, D.A., Le Lay, J.E., Ruano, E.G. and Kaestner, K.H.: TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts. J. Clin. Invest. 125(5): 1998-2006, May 2015.
Wang YJ, Schug J, Won KJ, Liu C, Naji A, Avrahami D, Golson ML, Kaestner KH: Single cell transcriptomics of the human endocrine pancreas. Diabetes 65(10): 3028-38, October 2016.
Wangensteen KJ, Zhang S, Greenbaum LE, Kaestner KH.: A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation. Genes Dev 29(9): 904-9, May 2015.
Avrahami D, Li C, Zhang J, Schug J, Avrahami R, Rao S, Stadler MB, Burger L, Schübeler D, Glaser B, Kaestner KH.: Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved β Cell Function. Cell Metabolism 22(4): 619-32, October 2015.
Sheaffer, K.L., Kim, R., Aoki, R., Elliott, E.N., Schug, J., Burger, L., Schubeler, D., and Kaestner, K.H.: DNA methylation is required for the control of stem cell differentiation in the small intestine. Genes Dev 28(6): 652-664 March 2014.
Bramswig, N., Evertt, L., Schug, J., Dorrell, C., Liu, C., Luo, Y., Streeter, P., Naji, A., Grompe, M., And Kaestner, K.H.: Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J. Clin. Invest. 123(3): 1275-84, March 2013.
Li, Z, Gadue, P, Chen, K, Jiao, Y, Tuteja, G, Schug, J, Li, W, and Kaestner, KH: Foxa2 and H2A.Z Mediate Nucleosome Depletion during Embryonic Stem Cell Differentiation. Cell 151(7): 1608-16, December 2012.
Li, Z., Tuteja, G., Schug, J. and Kaestner, K.H.: Foxa1 and Foxa2 are Essential for Sexual Dimorphism in Liver Cancer Cell 148: 72-83, January 2012.
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Last updated: 05/06/2018
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