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.