Novel Integrated Platform to Quantitate Rejuvenation in vivo

Partial reprogramming in vivo is an exciting emerging field based on observation that short-term expression of Yamanaka factors (Oct4, Sox3, Klf4, cMyc, abbreviated as OSKM) in vivo rejuvenate diverse functional aspects of several tissues. However, prolonged expression of OSKM factors in vivo leads to teratomas, which emphasizes the need for tightly controlled delivery and accurate detection of the epigenetic changes as a means to monitor reprogramming. Given the astounding therapeutic potential of this approach and an exponentially growing interest both from academia and in the industry, the need for accurate quantitation of epigenetic changes in vivo, including at the single-cell level, is pressing. We propose a single cell method that is robust, scalable, and cost-effective assay to quantitate epigenetic changes following partial reprogramming - microscopic imaging of Epigenetic Age (miEpiAge). The Terskikh laboratory has developed a novel technique rooted in the analysis of epigenome topography at the single-cell level to quantitate change in chromatin landscape. We capture patterns of nuclear staining of epigenetic marks (e.g. acetylated and methylated histones) and employ automated microscopy and machine learning to determine multiparametric signature of cellular state. Application of this technique to aging, miEpiAge, demonstrated robust separation of young and old mouse and human cells. miEpiAge signatures of PBMC/CD3+ cells correlate (R2=0.86) with chronological age without linear regression. Doxorubicin (DOX) and Caloric Restriction (CR) interventions indicate that miEpiAge successfully detects the acceleration of aging after chemotherapy treatment and the slowdown of aging after CR diet in chronologically matched animals. Critically, miEpiAge successfully detects the rejuvenating effect of partial reprogramming in whole livers, revealing, for the first time, single-cell heterogeneity. We will expand these studies to validate miEpiAge technique in different tissues and directly compare it with whole genome analysis of chromatin state. To obtain orthogonal validation, we will interrogate the same samples using ATAC-seq and Cut&Tag to map and correlate miEpiAge multiparametric signatures with overall chromatin accessibility (ATAC-seq) and genome-wide distribution of specific epigenetic marks (Cut&Tag). Phase I will provide proof of concept and will yield a established correlation of miEpiAge to the orthogonal whole genome chromatin states thereby establishing miEpiAge as a high information content analysis tool. These experiments will demonstrate the broad utility of miEpiAge as a robust and cost-effective platform for evaluating multi-tissue rejuvenation in vivo.