In the intestine, epithelial renewal is reliant on stem cells and fueled by rapidly proliferating transit-amplifying cells (TA). Identification of intestinal stem cells is paramount to fully understanding this renewal process. The broad goals of the studies planned in this project are to isolate (ISCs) in order to elucidate the molecular features that distinguish them from their immediate descendents, the progenitor /TA cells and the more differentiated cell within the gut. One major obstacle in ISC biology has been the lack of definitive markers that identify ISCs. The recently described novel putative ISC marker DCAMKL-1 (doublecortin and CaM kinase-like-1) is predominantly observed in a unique quiescent cell population in the lower third of the intestinal crypt. Furthermore, cell surface expression of DCAMKL-1 allows for FAQs based sorting from intestinal tissues. Moreover the recent identification of another novel putative ISC marker LGR5 (leucine-rich-repeat-containing G-protein-coupled receptor) that is expressed in rapidly cycling cells within the crypt has allowed for examination of the effects of genotoxic injury on intestinal stem quiescence and proliferation in situ and in real time. The central hypothesis of the current proposal is that DCAMKL-1 marks quiescent ISCs and can be used to isolate, characterize, and distinguish quiescent stem cells from rapidly cycling stem/progenitor (LGR5+) cells. This hypothesis will be tested with the following specific aims: 1) To isolate stem cells from the normal adult mouse small intestine by FACS and develop in vitro and in vivo assays to assess clonogenicity, and lineage determination 2) To delineate the molecular signatures and signaling pathways that regulate intestinal stem cell fate and 3) To determine the molecular mechanisms that control intestinal epithelial stem quiescence and proliferation during the restitution phase following genotoxic injury. Collectively, these studies should provide new insights into the key cellular mechanisms and identify novel candidate genes and pathways that regulate the homeostatic interplay between stem and progenitor cells in the normal intestine and following genotoxic/cytotoxic injury during the primary regenerative response.

Public Health Relevance

The experimental studies proposed may have far reaching consequences in the study of ISC biology and in other gut stem cells and perhaps other solid tissues as well. These studies should help in the development of novel regenerative medicine strategies to treat inflammatory gastrointestinal diseases, and perhaps in targeting the stem cell in gut neoplasia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project--Cooperative Agreements (U01)
Project #
3U01DK085508-05S1
Application #
8699374
Study Section
Special Emphasis Panel (ZDK1-GRB-8 (O1))
Program Officer
Carrington, Jill L
Project Start
2009-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$27,399
Indirect Cost
$8,886
Name
University of Oklahoma Health Sciences Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Chandrakesan, Parthasarathy; May, Randal; Weygant, Nathaniel et al. (2016) Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury. Sci Rep 6:37667
Chandrakesan, P; Panneerselvam, J; Qu, D et al. (2016) Regulatory Roles of Dclk1 in Epithelial Mesenchymal Transition and Cancer Stem Cells. J Carcinog Mutagen 7:
Chandrakesan, Parthasarathy; May, Randal; Qu, Dongfeng et al. (2015) Dclk1+ small intestinal epithelial tuft cells display the hallmarks of quiescence and self-renewal. Oncotarget 6:30876-86
Sureban, Sripathi M; May, Randal; Qu, Dongfeng et al. (2015) Dietary Pectin Increases Intestinal Crypt Stem Cell Survival following Radiation Injury. PLoS One 10:e0135561
Qu, Dongfeng; Weygant, Nathaniel; May, Randal et al. (2015) Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis. PLoS One 10:e0134212
Qu, Dongfeng; May, Randal; Sureban, Sripathi M et al. (2014) Inhibition of Notch signaling reduces the number of surviving Dclk1+ reserve crypt epithelial stem cells following radiation injury. Am J Physiol Gastrointest Liver Physiol 306:G404-11
Chandrakesan, Parthasarathy; Weygant, Nathaniel; May, Randal et al. (2014) DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition. Oncotarget 5:9269-80
Chandrakesan, P; Roy, B; Jakkula, L U M R et al. (2014) Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigenesis. Oncogene 33:2639-54
May, Randal; Qu, Dongfeng; Weygant, Nathaniel et al. (2014) Brief report: Dclk1 deletion in tuft cells results in impaired epithelial repair after radiation injury. Stem Cells 32:822-7
Magness, Scott T; Puthoff, Brent J; Crissey, Mary Ann et al. (2013) A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 305:G542-51

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