Characterization of dormant intestinal stem cells () by a novel Optical Stem Cell Activity Reporter (OSCAR)

• Applicant: Dr. Christoph Kaether, since 8/2023
• Subject Area: Cell Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 438779085

Aging is characterized by loss of homeostatic tissue maintenance. Adult stem cells serve as the main source of new cells during tissue repair, regeneration and maintenance, thus raising the possibility that aging is in fact caused by a decline in adult stem cell function. Most adult stem cells are in state of relative dormancy, which protects them from genetic and environmental damage. However, the exact mechanism of induction and maintenance of this dormant state is currently unknown. Despite this protective mechanism, dormant stem cells accumulate molecular damages (genetic mutations as well as epigenetic and metabolic alterations) over time, which have been shown to be drivers of most of the aging-associated human organ dysfunctions and diseases (Ermolaeva, Neri et al, 2018). Aging-related epigenetic alterations in stem cells represent a valuable therapeutics target because of their reversibility, but they are still not fully characterized in many adult tissues.It was previously found that dormant adult stem cells of at least seven different lineages display a down regulation of productive mRNA transcription, characterized by absence of the active Serine 2-phosphorylated form of RNA polymerase II (Freter et al, 2010). We have now developed a fluorescent, genetically encoded reporter of RNApII-pSer2 kinase activity, termed Optical Stem Cell Activity Reporter (OSCAR). We used OSCAR for generation of a new transgenic mouse line, showing OSCAR fluorescence in all tissues. Following our preliminary results and pilot experiments we performed, we found that the newly established OSCAR system and the OSCAR mouse line clearly label dormant stem cells in the intestine and other tissues. We propose to use young and old OSCAR mice to investigate the impact of aging on adult stem cell dormancy. We will isolate OSCARhigh (marking dormant stem cells) cells from the small intestine, a well-described stem cell compartment. Characterization of these cells in terms of RNA-Seq, epigenetic analysis and in vitro assays will determine if aging results in activation and loss of dormant stem cells and highlight pathways and factors contributing to healthy stem cell aging. This novel tool represents a unique opportunity to validate our hypothesis of dormant stem cell activation and exhaustion in the aging intestine and to characterize epigenetic alterations driving this mechanism. Understanding the epigenetic basis of the stem cell aging is fundamental for the development of therapeutic / preventive approaches aimed to contrast aging-associated dysfunctions and diseases including cancer, loss of intestinal barrier and reduced regenerative capacity. Our group will provide the research infrastructure, mouse lines husbandry and bioinformatics expertise for the performing of the study. The DFG funding will be necessary to cover personnel and part of the consumable costs for this project.

• DFG Programme: Research Grants
• Ehemalige Antragsteller: Francesco Neri, Ph.D., until 6/2021; Privatdozent Dr. Alessandro Ori, from 7/2021 until 7/2023