TRANSIENT REPROGRAMMING FOR MULTIFACETED REVERSAL OF AGING PHENOTYPES A DISSERTATION SUBMITTED TO THE DEPARTMENT OF APPLIED PHYSICS AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TAPASH SARKAR MAY 2019 © 2019 by Tapash Jay Sarkar. All Rights Reserved. Re-distributed by Stanford University under license with the author. This dissertation is online at: http://purl.stanford.edu/vs728sz4833 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Vittorio Sebastiano, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Andrew Spakowitz, Co-Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Vinit Mahajan Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii iv Abstract Though aging is generally associated with tissue and organ dysfunction, these can be considered the emergent consequences of fundamental transitions in the state of cellular physiology. These transitions have multiple manifestations at different levels of cellular architecture and function but the central regulator of these transitions is the epigenome, the most upstream dynamic regulator of gene expression. Reproduction is the only general phenomena in nature where the age of (parental) cells is truly reset - to produce an embryo and ultimately an age 0 offspring - and core to this process is a dramatic reprogramming of the epigenome. Here we present a technology that captures part of this age reset mechanism but using a transient reprogramming - to drive more youthful phenotypes but without the full reset back to an embryo. This reprogramming technology is distinct from previous anti-aging/pro-longevity interventions as instead of just modulating a few identified aging pathways, reprogramming engages a global and balanced state transition, in the case of reproduction, or state perturbation, in our transient approach, which we show leads to a multifaceted age reversal effect at the DNA, metabolic, whole cell and local environmental levels. We further discuss the emergent tissue and organ level benefits when transplanted with cells undergoing this treatment. v Acknowledgments Thanks to our collaborators in the … Sebastiano Lab, Stanford University Vittorio Sebastiano Jens Durruthy-Durruthy Horvath Lab, University of California, Los Angeles Steve Horvath Bhutani Lab, Stanford University Nidhi Bhutani Shravani Mukherjee OneSkin Technologies Carolina Oliveira Alessandra Zonari Chu Lab, Stanford University Constance Chu Eleonora Migliore Erika Leonardi vi Rando Lab, Stanford University Tom Rando Marco Quarta Alex Colville Patrick Paine Linda Doan Mahajan Lab, Stanford University Vinit Mahajan Katherine Wert Palmer Lab, Stanford Univeristy Theo Palmer Aditya Asokan And to the services and resources provided by … ESI Biotechnologies Nakuchi Lab, Stanford University Girihlet Inc Human Immune Monitoring Facility, Stanford University vii As well as to our funding sources National Science Foundation Glenn Foundation for Medical Research American Federation of Aging Research viii Table of Contents 1) Introduction 1.1) Motivation 1 1.2) Background 2 1.3) Approach 5 2) Technology 2.1) Background 6 2.2) Results 8 2.3) Discussion 13 2.4) Methods 15 3) DNA Level 3.1) Background 17 3.2) Results 21 3.3) Discussion 32 3.4) Methods 34 4) Metabolic Level 4.1) Background 38 4.2) Results 42 4.3) Discussion 50 4.4) Methods 51 5) Niche Level 5.1) Background 54 5.2) Results 57 ix 5.3) Discussion 63 5.4) Methods 65 6) Metabolic Level 6.1) Background 67 6.2) Results 69 6.3) Discussion 73 6.4) Methods 75 7) Conclusion 7.1) Concepts 78 7.2) Future Directions 79 8) Supplemental Figures 82 9) References 93 x Figures 11 Figure 1 Verification of Results from In Vitro Sysnthesis of Reprogramming 12 Figure 2 Verification of identity retention and lack of transformation in treated cells and derived tissue by histology. 13 Figure 3 Verification of identity retention and lack of transformation in treated cells and derived tissue by gene expression 26 Figure 1 Combined single cell distritubtions of epigenetic, laminar and DNA damage markers. 27 Figure 2 Combined single cell distritubtions for treated and untreated cells from HGPS patients. 29 Figure 3 Transcriptomic landscape in aging subspace. 30 Figure 4 Transcriptomic landscape of treatment 32 Figure 5 Methylation Clock analysis 46 Figure 6 Combined single cell distritubtions and bulk redout of nutrient sensing, mitochondria activity and protien clearence 49 Figure 7 Combined single cell distritubtions and bulk redout of redox and ECM metabolism parameters. 59 Figure 8 Evaluation of senescence parameters in aged monolayer endothelial cells and fibroblast-keratinocyte constructs 61 Figure 9 Evaluation of senescence parameters in fat pat derived mesenchymal stem cells. xi 62 Figure 10 Intracellular expression and extracellular secretion of pro-inflammatory factors. 71 Figure 11 First injury response after transplanting in treated, aged and young mouse MuSC 72 Figure 12 Second injury (mouse) and human MuSC transplant results. 82 Supplementary Figure 1 Demonstration of in vivo, in situ transfection 83 Supplementary Figure 2 Comparison of Shorter vs Extended Treamtment (Fibroblast) 85 Supplementary Figure 3 Comparison of Shorter vs Extended Treamtment (Endothelial Cell) 86 Supplementary Figure 4 Effects of control transfections (Fibroblasts). 88 Supplementary Figure 5 Effects of control transfections (Endothelial Cells). 89 Supplementary Figure 6 Time course study of retentiation of rejuvenative benefits (Fibroblasts). 91 Supplementary Figure 7 Time course study of retentiation of rejuvenative benefits (Endothelial Cells). 92 Supplementary Figure 8 Example single patient distribution comparison 1 1) Introduction 1.1) Motivation Progression into middle and old age is marred by multitude of diseases and dysfunctions like arthritis, cardiovascular disease, muscle atrophy and skin aging [Jaul and Barron, 2017]. Research to combat these conditions has been largely specialized to develop solutions for each specific “downstream” conditions. However, intervention into physiological process of aging itself, as the “upstream” driver of all these conditions, has remained relatively unexplored compared to the dramatic advances in medicine over the last few decades [Finkel, 2005]. Though admittedly the lack of scientific consensus on the defining biomarker of aging presents a key obstacle to this day, a more general impediment was the long-standing dogma on the irreversibility of age, irreversible like time itself as an almost entropic concept [Newman et al., 2016]. Though there existed previous work studying interventions to slow or delay aging, the first demonstration of age reversal, or rejuvenation, came around the turn of the new millennium [Mahmoudi and Brunet, 2012]. In their landmark heterochronic parabiosis study, merging the vasculature of mice of different ages together (2-3 month young with 19-26 month old), the Rando and Weismann labs at Stanford demonstrated that the youthful blood from the young parabiont was sufficient to reverse multitude of aging phenotypes in the liver and muscle of the old parabiont [Conboy et al., 2005]. This approach demonstrated that extracellular factors, cytokines, growth factors, exosomes etc., were key to establishing and progressing aging and likewise youth. Since then the list of tissues that have been shown to exhibit a similar youthful reversal by this approach has grown to also include brain, bone, pancreas and heart [Castellano et al., 2015]. This work introduced a greater 2 aspiration for the field of ageing from a just mitigating age related dysfunction and extending lifespan, to the possibility of rejuvenation and health restoration. Since then, another contender has also risen in senolytic treatments, which we will discuss in further detail in later chapters but essentially presents another way of promoting a youthful environment by clearing the cells which most emphatically secrete aging paracrine factors [Jeon et al., 2017]. The work of this thesis highlights a third possible contender that is in many ways the complement of these approaches as it introduces a method to reverse aging in a cell autonomous fashion rather than by affecting the extracellular environment. It is also, as we will see, the one proposed rejuvenation method that is based on a rejuvenation mechanism already routinely employed in nature. 1.2) Background When considering exceptions to one-way progression of aging in nature, one is typically predisposed to think of the obscure corner cases, specifically organisms considered to be “biologically immortal”. The select organisms, specific species like the jellyfish Turritopsis dohrnii or the general hydra genus, have evolved the ability to reset the age related dysfunction of their cells by altering their epigenetic identity, through differentiation of stem cells to