Reprogramming: Rejuvenation Nature’s Way
In my last post, we explored two key areas in aging research aimed at keeping us healthy, active, and disease-free: dampening the molecular nutrient-sensing pathway used by all our cells, and removing accumulated senescent cells from our organs and tissues. While nutrient sensing has dominated research for decades, and eliminating senescent cells took off around a decade ago, the latest excitement revolves around something called reprogramming.
Even if you haven’t come across reprogramming, you are already familiar with its core feature: resetting cellular age to zero. We experience it with every new generation: your body’s cells are direct descendants of your parents’ cells, which were probably a few decades old at conception. Extend that back through the generations and it becomes clear that the cells that make up you go back millions — well, actually billions — of years. There is a continuous line of descent from the very first cells to those forming every part of you. Famines, floods, mass extinctions — your cells survived them all. They are, in this sense, immortal. So why aren’t we immortal? Why do our bodies grow old and die, yet we can propagate our cells in perpetuity simply by having children?
This is the crux of two concepts: the Immortal Germ Line, and the Disposable Soma Theory. Our germ (reproductive) cells persist indefinitely, but our bodies are mere vessels. Our germ cells don’t care what happens to “us.” They only care about being reproduced — copied widely and perpetually. Replication, duplication, expansion: these are the fundamental concepts of evolution, driven by our Selfish Genes. Whatever virus, or bacterium, or animal is best at making more of itself wins. At the level of evolution, that’s all life is: a replication competition.
So if the cells of our bodies must succumb to aging and death, how do reproductive cells survive forever? If they don’t age, or if they de-age, can we apply these rejuvenative molecular processes of reproduction outside the germ line? Scientists have searched for answers for decades. Some of the first real progress came with a technique called somatic cell nuclear transfer (SCNT). If you don’t recognize the name, you probably know its application: cloning. As in Dolly, the sheep. SCNT involves collecting the nucleus containing all the genetic, inherited information (the DNA) from a somatic cell (a non-reproductive cell, like the mammary gland cell in Dolly’s case) of the adult donor you want to clone. This nucleus is then physically transferred to an egg cell (oocyte) with its nucleus removed. After implanting the egg in a surrogate mother, it divides and grows into an exact genetic replica of the donor: a clone. Despite Dolly’s premature death, it now appears that cloned animals age no differently from normal animals. Old DNA can indeed create an entirely new, “day 0” organism. This perhaps marked the first hint that aging, at least at the DNA level, is not inevitable. But this wasn’t actually the major finding at the time. Instead, the core concept centered around the loss of cellular identity during SCNT. The DNA, originally specialized for mammary gland cell function, underwent a “reset.” It regained the ability to create every cell type in the mature body, just like an embryonic stem cell. In other words, mammary gland DNA was reprogrammed into an embryonic state.
To grasp the implications, we need a little biology. Firstly, remember that every cell in your body harbors the exact same DNA. Starting from the one, single cell formed upon conception, this DNA is copied repeatedly and exactly, ultimately inhabiting the trillions of cells of your body. Yet, despite this uniform genetic blueprint, our cells are not identical. Far from it, they display remarkable diversity. How then do cells with identical instructions perform such different functions?
It boils down to which instructions are read, and which are not. While no perfect analogy exists, envision your DNA as an aggregation of 20,000 instruction manuals (in genetic terms, each manual is called a gene). But, rather than explicitly guiding the construction of a house (i.e. a cell), these manuals dictate the creation of basic building blocks and tools that go into creating and maintaining the house: the nails, screws, bricks, mortar, hammers, drills, etc. There are many building blocks and tools all cells require to perform basic functions, and all cells read this same core set of instructions (these are called housekeeping genes). But the adult body is composed of specialized cells, those that perform specific and distinct functions. What makes these cells different is that they read a different set of instructions. Some are turned off, others on. With 20,000 instructions, the potential combinations give rise to a vast array of cell types: muscle cells that contract, immune cells that kill bacteria, neurons that transmit electricity, pancreatic beta-cells that secrete insulin. Each cell type has a specific, defined subset of the 20,000 genes turned on, and the rest are off. This set is what defines the cell’s function, and thus its identity.
During reprogramming, when converting Dolly’s mammary gland cell DNA into the DNA of an embryonic stem cell, the set of genes that are on versus off is changed. Mammary gland-specific genes are turned off, while other genes are turned on. Although numerous complex mechanisms regulate gene activation, one extensively studied process is DNA methylation, in which a small chemical modification is added to the DNA. This methyl modification is typically repressive — that is, it blocks access to the on-switch of each gene. Mature cells, like adult mammary gland cells, exhibit highly methylated DNA — on-switches to many genes are blocked leaving only housekeeping genes and mammary gland cell-specific genes on. During reprogramming to an embryonic cell, similar to what occurs after fertilization during early embryo development, DNA undergoes extensive demethylation. The repressive chemical modifications are removed, priming the cell with the ability to activate almost any of the 20,000+ genes. This demethylation, and other associated molecular changes, is thought to underpin both the loss of cell identity (conversion to an embryonic stem cell) and the rejuvenation (resetting to age zero).
But why exactly does transferring a mature nucleus into an egg cell reprogram the DNA? What is inside this oocyte that triggers this process? A clue came in the early 2000s with the discovery that reprogramming also occurs when a mature cell fuses with an embryonic stem cell (ESC). This suggested that something inside the ESC spurs reprogramming, similar to the oocyte. The major breakthrough — and one that won a Nobel Prize — came in 2006, after researchers discovered four specific genes (abbreviated “OSKM”, also known as the “Yamanaka factors”) that, when activated simultaneously in a fully specialized cell, could reprogram it into an embryonic-like state capable of forming any specialized cell type — a state termed pluripotent. This discovery of induced pluripotent stem cells (iPSCs) marked a new era in biomedical research. With iPSCs came the promise of creating any cell type at will, opening endless possibilities for new treatments — a feat possible before only with ethically fraught and sparsely available embryonic stem cells. Using a patient’s own skin cells, for example, one could conceivably convert them into dopaminergic neurons and transplant them into Parkinson’s patients, all without the risk of immune rejection.
And despite the staggering potential for iPSCs in medicine (more on that next time when we discuss replacing aged organs and cells), only a few recognized the arguably even bigger potential — the possibility of rejuvenating the body’s trillions of cells via reprogramming. With the newfound ability to reprogram cells at will, could we thus rejuvenate any mature cell? Could this be a universal age-reversal therapy? It wasn’t immediately obvious this could work. After all, reprogramming converts mature cells into highly replicative embryonic-like stem cells, the same type known to lead to horrendous tumors composed of different tissues and cell types called teratomas. However, in 2016, researchers tackled this challenge by employing what they termed “partial reprogramming,” a scheme by which the four iPSC genes are turned on and off cyclically every few days. Remarkably, this appeared to induce rejuvenation without the loss of cell identity that leads to teratomas, and the authors reported lifespan extension in a genetic mouse model of premature aging.
This marked a seminal moment in the aging field, triggering an explosion of research and investment, both in academic circles and for-profit biotechnologies companies seeking to capitalize on new therapies for humans. Making headlines in 2021, Altos Labs, replacing the fledgling philanthropic Milky Way Research Foundation, emerged as perhaps the best-funded biotechnology company ever established. Following a flurry of recruiting top talent and renowned figures in reprogramming and the closely associated field of DNA methylation aging clocks, Altos now encompasses over 20 PI-led laboratories across three sites: San Diego, San Francisco, and Cambridge, UK.
While the excitement is perhaps warranted — as reprogramming stems from what is arguably the most fundamental way to elicit cellular age-reversal found in nature — the field remains nascent, with dozens of fundamental questions yet unanswered. What features of aging does reprogramming genuinely reverse? Is that reversal permanent, or will cells revert to their aged state? Are other features of aging missed that make reprogramming moot? How often must one perform reprogramming to maintain youthful function? Can reprogramming ever be deemed safe? Does the technology even exist to perform reprogramming in humans, or to deliver the factors to the right organs and cell types? Do different cell types require different factors? Are there factors superior to the Yamanaka factors? Can we separate rejuvenation from the loss of cell identity?
The sprint for answers is on, with potentially billions of dollars at stake. At the heart of this race is safety — the pursuit for novel reprogramming methods that avoid erasing cellular identity while preserving rejuvenation. Strategies fall into a few categories, the first of which we’ve already visited: cyclic bursts or transient induction of the Yamanaka factors. For example, Turn Biosciences delivers messenger RNA (mRNA) via lipid nanoparticles (like some Covid vaccines) to instruct cells to produce the four Yamanaka factors (and, at Turn, two additional mRNAs for Lin28 and Nanog). But, as mRNAs degrade relatively quickly, the effect is a brief burst of reprogramming.
Others attempt to reduce teratoma risk with a subset of Yamanaka factors, commonly OSK rather than OSKM. These include Life Biosciences, a company advancing research that demonstrated regeneration of the optic nerve with OSK, and Rejuvenate Bio, which recently showed lifespan extension after OSK delivery via adeno-associated viruses (AAVs) to mice of very advanced age.
Several companies are integrating research on established Yamanaka factors with platforms to discover novel factors. YouthBio Therapeutics has undertaken this approach, conducting tests in mouse models of both progeria (an “accelerated aging” genetic disease) and Alzheimer’s by targeting specific cell types like muscle cells and neurons. Others also avoid pan-reprogramming, opting instead to target specific cells. Retro Biosciences, for instance, investigates both novel and Yamakaka factors for reprogramming hepatocytes (the main liver cell type), T cells (an immune cell with many therapeutic applications), and hematopoietic stem cells (the fundamental stem cell that gives rise to all immune cells). NewLimit is among the few companies wholly dedicated to reprogramming, targeting both hepatocytes and T cells using high-throughput screening combined with machine learning (ML) to test and predict countless combinations of novel factors.
In fact, this approach is no longer the exception, but the norm, with many biotech companies combining novel factor screening, ML, and readouts like DNA methylation “aging clocks” to assess reprogramming success and safety. For example, Shift Bioscience has developed an accurate transcriptome clock for single cells based on the abundance of thousands of different mRNAs, with plans to iteratively screen and predict novel reprogramming factors with an ML ‘cell simulation’. Following this theme, the newly launched Junevity similarly utilizes non-Yamanaka factors for reprogramming. Finally, the just-announced Moonwalk Biosciences features a fundamentally different DNA methylation-centric approach, using tools to modify methylation more directly, rather than supplying factors that lead to downstream methylation changes.
DNA methylation, remember, is critical for governing which DNA instructions (genes) are read and thus which proteins the cell produces, which in turn governs cellular function and identity. And methylation is measurable, giving insight not only into the success of reprogramming, but also indicating the “age” of the cell. Since the inception of the first DNA methylation “clocks,” this method has been adapted by dozens of direct-to-consumer companies. Through a blood sample, they claim to determine if an individual is younger or older than their chronological age, with repeated measurements potentially uncovering your “rate” of aging. Some companies pair DNA methylation reports with supplements, exercise regimens, and special diets. However, the true value of this information and whether one can or should act on the results remains somewhat dubious, as blood tests measuring DNA methylation typically mix different cell types, making it unclear if the DNA methylation profile is a result of aging, changes in cell type proportions, or both. Fortunately, research is progressing rapidly, with single-cell DNA methylation clocks on the way.
Despite these considerations, reprogramming and DNA methylation remain hot topics, with the field poised for exponential growth. Whether this represents our best bet for the next generation of medicine remains to be seen. Undoubtedly, reprogramming stands out as one of the few methods capable of eliciting a true reversal of at least some aspects of molecular aging, not just a slowing of progression. By mimicking the immortal germ line — nature’s most robust method of rejuvenation — through targeting the driving hallmark of aging that is epigenetic alterations, reprogramming may indeed hold the potential to significantly improve the consequences of aging at the molecular, functional, and organismal level.
But what if reprogramming falls short? What if some hallmarks of aging aren’t reversed? What if traditional approaches are just too simple for the immense complexity of aging? An increasing number of scientists argue this is likely — that reversing the extremely complex phenomenon of aging is just too great a challenge. So why not avoid this altogether? Why not avoid wasting billions of dollars on drug development and the manipulation of individual molecular targets, and instead replace the aged components altogether? If your cells get old, don’t try to make them young, just replace them! We already do this in the clinic. Organ transplants and immune cell transplants are common. If we could just do this for more of our parts, wouldn’t that be the most efficient way to prevent aging and the horrible diseases it instigates? While the field grapples with ethical and technological hurdles, substantial progress is underway in more ways than you probably imagine, with implications not only for aging but for the thousands relegated to transplant wait lists. Next time, we delve into what will seem to many like science fiction: the world of replacement.
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