Chemically induced reprogramming to reverse cellular aging

Introduction

All life depends on the storage and preservation of information. In eukaryotes, there are two main repositories of information: the genome and the epigenome. Though these information repositories work interdependently to coordinate the production and operation of life’s molecular machinery, they are different in fundamental ways. Genetic information is digital and largely consistent across all cells in the body throughout an individual’s lifespan. In contrast, epigenetic information is encoded by a less stable digital-analog system, varying between cells and changing in response to the environment and over time.

At least a dozen “hallmarks of aging” are known to contribute to the deterioration and dysfunction of cells as they age [1, 2]. We and other researchers have gathered compelling evidence, from yeast to mammals, supporting the idea that a loss of epigenetic information, resulting in changes in gene expression, leads to the loss of cellular identity [3-7]. These findings are consistent with the Information Theory of Aging, which proposes that a decline in information, specifically epigenetic information, triggers a cascade of events, including mitochondrial dysfunction, inflammation, and cellular senescence [5, 7-9], leading to a progressive decline in cell and tissue function, manifesting as aging and age-related diseases. We have previously shown in mice that cell injuries, such as DNA double-strand breaks and cell crushing, promote epigenetic information loss, which can lead to what appears to be an acceleration of aging and age-related disease [7, 9].

Cellular senescence is a state of permanent cell cycle arrest that facilitates wound repair, tissue remodeling, and avoidance of cancer by halting proliferation in aged and damaged cells [10, 11]. Senescence is associated with alterations in cell morphology, chromatin architecture, and the release of inflammatory factors in a process referred to as the senescence-associated secretory phenotype (SASP). The transition to cellular senescence can be initiated by a loss of epigenetic information, as well as telomere shortening, irreparable DNA damage, and cytoplasmic DNA [7, 10-12]. The accumulation of senescent cells with age promotes inflammation and generates additional reactive oxygen species (ROS), both locally and across the organism, contributing to a broad range of age-related diseases, from macular degeneration, to increased blood pressure, to metabolic dysregulation [13].

Starting in 1962, Gurdon and others demonstrated that nuclei contain the necessary information to generate new individuals with normal lifespans [14-16]. In 2006, Takahashi and Yamanaka demonstrated that the expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC (collectively known as “OSKM”), reprograms the developmental potential of adult cells, enabling them to be converted into various cell types [17, 18]. These findings initiated the field of cell reprogramming, with a string of publications in the 2000s showing that the identity of many different types of adult cells from different species could be erased to become induced pluripotent stem cells, commonly known as “iPSCs” [17, 19-21].

The ability of the Yamanaka factors to erase cellular identity raised a key question: is it possible to reverse cellular aging in vivo without causing uncontrolled cell growth and tumorigenesis? Initially, it didn’t seem so, as mice died within two days of expressing OSKM. But work by the Belmonte lab, our lab, and others have confirmed that it is possible to safely improve the function of tissues in vivo by pulsing OSKM expression [22, 23] or by continuously expressing only OSK, leaving out the oncogene c-MYC [7, 8]. In the optic nerve, for example, expression of a three Yamanaka factor combination safely resets DNA methylomes and gene expression patterns, improving vision in old and glaucomatous mice via a largely obscure mechanism that requires TET DNA demethylases [8]. Numerous tissues, including brain tissue, kidney, and muscle, have now been reprogrammed without causing cancer [7, 8, 22, 24, 25]. In fact, expression of OSK throughout the entire body of mice extends their lifespan [26]. Together, these results are consistent with the existence of a “back-up copy” of a youthful epigenome, one that can be reset via partial reprogramming to regain tissue function, without erasing cellular identity or causing tumorigenesis [7-9].

Currently, translational applications that aim to reverse aging, treat injuries, and cure age-related diseases, rely on the delivery of genetic material to target tissues. This is achieved through methods like adeno-associated viral (AAV) delivery of DNA and lipid nanoparticle-mediated delivery of RNA [7, 8, 27]. These approaches face potential barriers to them being used widely, including high costs and safety concerns associated with the introduction of genetic material into the body. Developing a chemical alternative to mimic OSK’s rejuvenating effects could lower costs and shorten timelines in regenerative medicine development [26, 28-31]. This advancement might enable the treatment of various medical conditions and potentially even facilitate whole-body rejuvenation [32, 33].

In this study, we developed and utilized novel screening methods including a quantitative nucleocytoplasmic compartmentalization assay (NCC) that can readily distinguish between young, old, and senescent cells [34, 35]. We identify a variety of novel chemical cocktails capable of rejuvenating cells and reversing transcriptomic age to a similar extent as OSK overexpression. Thus, it is possible to reverse aspects of aging without erasing cell identity using chemical rather than genetic means.