How to Turn Back Our Biological Clocks

Using four factors to rejuvenate cells and prevent age-related disease

Parmin Sedigh
Predict

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Photo by Jon Tyson on Unsplash

You feel the sweet taste of the elixir of youth as your body begins to rejuvenate itself, your wrinkles disappearing, your cells becoming younger, your scars healing. You’ve finally managed to find a way to reverse aging. How did you do it?

With the help of four transcription factors, of course. Four what?

While scientists have not reached the point of finding an “elixir of youth”—and importantly, are not trying to—there is a lot of ongoing research looking into making our cells more youthful which can help prevent or ease the pain of age-related diseases.

So let’s dive deep into the field of partial reprogramming, one aspect of cellular rejuvenation, see how it all works, and separate fact from fiction.

Table of Contents
Part I: Damaged Lego Bricks — Epigenetics & Aging
Part II: Righting Wrongs — Cellular Rejuvenation
Part III: The Future — Obstacles & Hopes

Part I: Damaged Lego Bricks — Epigenetics & Aging

The Hallmarks of Aging

Before we can understand how rejuvenation works, we need a basic understanding of what aging is. And I don’t mean how you and I associate aging with sagging skin and the like.

We need to understand how scientists view aging from a research point of view since it holds the key to minimizing the risks associated with aging.

Let me set the scene: It’s late 2013. Frozen will soon come out; you will undoubtedly hear the song “Let It Go” playing everywhere you go. But there’s another important event happening. A group of scientists have just published a paper titled “The Hallmarks of Aging” which grouped “common denominators of aging” into nine categories.

A visual representation of the hallmarks of aging. Adapted from The Hallmarks of Aging. Image by author.

Some of the categories included genomic instability, telomere attrition, epigenetic alterations, and cellular senescence. Notably, the nine hallmarks have recently been criticized for incorrectly suggesting that these are causes of aging when not all of them truly are and new research has led to the addition of new hallmarks.

Regardless of these ongoing discussions, there is one concept some scientists agree on including Dr. Vittorio Sebastiano, Associate Professor of Reproductive And Stem Cell Biology at Stanford University. He and others believe that epigenetic alterations are the most important hallmark.

During a chat with Dr. Sebastiano, he further explains that, in his opinion, epigenetic alterations is hierarchically the most important and dominant hallmark of aging. In other words, if we can tackle this hallmark, it’ll have an impact on multiple factors that are controlled by epigenetics.

Importantly, it’s not quite clear whether changes in the epigenome are a cause or consequence of aging; in any case, fixing changes that occur in the epigenome with aging has been found to reduce age-related issues. But wait, what are the epigenome and epigenetic alterations?

Epigenetics: The Most Important Hallmark?

To understand what the term epigenetics means, we need to take a quick trip back to high school biology class and get a basic understanding of how protein production works. If you already understand epigenetics, skip ahead to this section.

Let’s begin with defining genome. Our genome contains recipes for every protein our body needs to produce. DNA forms the building blocks of these recipes, just like how we use the alphabet to write sentences in a real-world recipe.

These lego bricks represent our genome. Image by author.

Here’s a very simplified explanation of how our body uses our genome to produce proteins. A group of enzymes (represented below by one purple block) read the genome and then, through a complex process that’s not important to us for now, a protein is formed.

A simplified diagram of the protein production process. Image by author.

Here’s the catch: every cell in our body contains our entire genome. But not every cell needs to be making every protein all the time. As well, each section of the genome that has a specific function is called a gene.

In fact, if all genes were being used to make proteins (or what scientists call “were being expressed”), we would have a disaster on our hands. Imagine if our eye cells were producing the same proteins as our muscle cells; our vision would likely be impaired very quickly.

So we need to ensure only the necessary and appropriate genes are being expressed in cells. Meet the epigenome.

The blue blocks laid on top of the genome represent the epigenome. Image by author.

Taking a look at etymology can help us begin to understand the epigenome. Epi is a Greek word meaning above so epigenome translates directly to above the genome.

In simple terms, the epigenome controls which genes are expressed and which are not. By doing this, it ensures each cell is producing only the proteins it needs. Considering all of this, it becomes clear why the epigenome is so important to our health.

The epigenome can prevent some genes from being expressed. One way this happens is by blocking certain enzymes from reading the genome. Image by author.

So how does our epigenome, a critical control mechanism, get damaged as we age?

Studies have found that our epigenome can change as we age and this throws off our body’s balance. Using the Lego blocks from before, imagine if the blue epigenome blocks were moved from one location to another. Now, the enzymes would be able to produce proteins they weren’t able to before.

In our older years, different molecules may begin being produced in incorrect cells which is certainly not good.

Now, studies are exploring how we can reverse these changes and return our cells and ultimately our bodies to a more youthful and healthy state.

Part II: Righting Wrongs — Cellular Rejuvenation

Before we understand how to make our cells more youthful, we need to take one last time travelling trip back to 2006. Two scientists, Kazutoshi Takahashi and Shinya Yamanaka, have just published their seminal paper describing the process of turning adult cells back into stem cells.

Stem cells are able to make infinite copies of themselves and specialize into other cell types. Think of them like young children that still haven’t decided on which career path they’ll choose.

Stem cells can specialize into different cell types. Image by author.

What’s important for the purposes of this article, however, is the fact that reprogramming (what scientists call turning adult cells back to stem cells) is able to change back some of the epigenetic alterations that occur with cellular aging.

A Deeper Dive Into Reprogramming

After the 2006 paper, reprogramming took the biology field by storm. Epigenetic reprogramming had existed in different forms in the past but within a few years of Takahashi and Yamanaka’s groundbreaking discovery, the number of studies using reprogramming skyrocketed.

This begs the question: how does reprogramming work? How can you change one cell type into another? The answer lies with the four transcription factors mentioned at the beginning.

Transcription factors are proteins that are essential to the protein production process. They help turn genes “on” and “off”. Yes, it’s a bit confusing; we need proteins to create proteins!

But where do these transcription factors come from? They’re encoded in genes. To make things even more confusing, the genes that code for the transcription factors we’ll be talking about have the same name as the transcription factors themselves.

It’s now finally time to meet our fantastic four: Oct3/4 (Oct3 and Oct4 are slightly different genes/factors but they act very similarly), Sox2, Myc, and Klf4. Collectively, these factors are referred to as the Yamanaka factors or OSKM.

To really understand how OSKM results in reprogramming, let’s return to our Lego blocks.

A set of genes including the Oct3/4, Sox2, Myc, and Klf4 genes. (Remember the blue blocks on top represent the epigenome). Image by author.

In the diagram above, you can see a set of genes including all of the OSKM genes. Remember the group of enzymes from before? They’re back too.

The enzymes read the genes and result in the production of messenger RNA or mRNA molecules. If a gene is like a chapter in a cookbook, an mRNA molecule would be a recipe card with only the most important information written on it.

Now it’s time to execute the instructions from the recipe card (or mRNA molecule) and create the final protein. Looking at the diagram below, an mRNA molecule was created from the Oct3/4 gene and ultimately, we have an Oct3/4 transcription factor. Woohoo!

A group of enzymes (shown in purple) leads to the production of mRNA which then leads to the final transcription factor molecule. (TF = transcription fator). Image by author.

All we need to do now is repeat the process for the remaining three genes. What now? We have four transcription factors ready to perform gene regulation.

Specifically, OSKM have been shown to act as pioneer factors. That means they can bind to parts of the genome that can’t currently be accessed and change their accessibility. This can then turn genes “on” or “off.”

All of this eventually leads to our goal: reprogramming.

All four transcription factors work together to reprogram a cell; it’s collaboration on a microscopic scale. Note: Sox2, Myc and Klf4 all undergo the mRNA step as well. It’s been omitted here for simplicity. Image by author.

As if this isn’t mind-blowing enough on its own, here’s another fascinating fact: we all have OSKM in our cells’ genomes right now.

However, the four genes are expressed at very low levels in adult bodies and are instead found at high levels in early-stage embryos.

Despite the fact that our cells do contain OSKM, it’s difficult to specifically target and express them. “We don’t really have a way to activate these four specific genes” in our cells, explains Dr. Tamir Chandra, leader of the Systems Biology of Aging and Disease group at the University of Edinburgh’s Institute of Genetics and Cancer.

To overcome this, scientists insert a genetic construct into the cells they are studying in laboratories. That genetic construct allows for the use of chemicals, like the antibiotic doxycycline, to activate the expression of these genes.

Put simply, the “gatekeeper” controlling whether a gene can be read or not normally does not allow for the reading of the OSKM genes. However, this “gate” can be opened when the cells are exposed to doxycycline.

The Microscopic Dilemma

This finding that reprogramming can return the epigenome to a more youthful state was incredibly exciting to scientists and the general public alike. There was only one problem: we can’t fully reprogram all of the cells in our bodies. The result would be disastrous and tumors would begin to form in our bodies. Why?

A certain group of stem cells called adult stem cells are critical for our normal bodily functions. But full reprogramming doesn’t lead to adult stem cells—stem cells that can only turn into a few cell types.

Instead, it results in pluripotent stem cells which can turn into all body cells. That means a pluripotent stem cell located in our stomach could eventually give rise to bone cells. We clearly don’t want that!

So, how do we harness the power of reprogramming for treating aging safely?

Enter Partial Reprogramming

In 2010, Prim B. Singh & Fred Zacouto proposed an idea that would forever change the aging field: what if we could epigenetically rejuvenate cells using reprogramming? This is what we now call partial reprogramming.

Instead of expressing the four transcription factors in a way that results in a complete return to stemness, we can express the factors for a shorter period of time and erase the altered epigenetic markings without the cell losing its identity.

A few months earlier, in 2009, Andras Nagy & Kristina Nagy suggested an interesting way to think about partial reprogramming—though they themselves were perhaps not aware of partial reprogramming yet.

They explained how there are two points of importance on a timeline showing a specialized cell’s journey back to becoming a stem cell: the point of no return and the commitment point to pluripotency.

If we stop providing a specialized cell with the reprogramming factors before the point of no return, it will revert back to a specialized cell. If we stop the reprogramming factors after the commitment point to pluripotency, the cell will go on to become a pluripotent stem cell.

A cell’s reprogramming journey. Adapted from Nagy & Nagy. Image by author.

But there’s an interesting area in between the two points which the authors named “Area 51.” During this point, stopping the factors wouldn’t lead back to a specialized cell but neither would the cell become pluripotent. This is exactly what partial reprogramming is!

Partial Reprogramming & Aging

Now that we have a deep understanding of what partial reprogramming is, it’s time to see what it can do in action. How can it truly help rejuvenate cells?

Let’s begin with the paper that kicked off the partial reprogramming field in earnest. Though previous papers, such as the one by Singh & Zacouto conceptualized partial reprogramming, robust experimental data supporting it wasn’t published until 2016 in a paper by Ocampo et al.

They began by using doxycycline-inducible genes, just like we talked about. They then treated fibroblasts—connective tissue cells—from the tips of the mice’s tails with doxycycline for two or four days. Thankfully, the cells didn’t lose their identity, meaning they didn’t turn into pluripotent stem cells.

Up next, they tested partial reprogramming on mice (with doxycycline-inducible genes). Interestingly, when they continuously gave mice water with doxycycline to induce the four reprogramming factors, many died and experienced extreme weight loss likely because their cells began to dedifferentiate.

But, when giving the mice doxycycline cyclically (meaning two days of doxycycline, followed by five days of no doxycycline), weight loss, mortality, or cancer wasn’t seen even after 35 cycles (or 35 weeks).

The continuous vs. cyclical doxycycline groups. Image by author.

Even more interesting results emerged when the four reprogramming factors were induced in progeria mice—a disease that causes rapid aging. These mice had a significantly longer lifespan compared to progeria mice in which the factors weren’t induced.

Another important study that established the use of partial reprogramming was published in 2020 by Sarkar et al. Though the Ocampo et al. paper also studied partial reprogramming in human cells, this 2020 study made human cells their focus. They tested partial reprogramming on young and aged fibroblasts and endothelial cells, using mRNA.

Think back to our Lego model: enzymes turn genes into mRNA which eventually become proteins (specifically transcription factors in the case of OSKM). In this case, scientists skipped the gene part, directly putting mRNA from OSKM genes into the cell. (Technically, in this case, scientists used two additional genes too, creating a combination called OSKMLN. We’ll discuss this further below).

Why did they use mRNA? According to Dr. Sebastiano, reprogramming using mRNA is safer because there is no possibility of mutations caused by new genes being inserted into the genome. But unfortunately, mRNA reprogramming may not always be the best method of reprogramming.

Dr. Ryan Lu, a Postdoctoral Researcher at the Whitehead Institute and MIT who previously worked in Dr. David Sinclair’s lab, explains that the choice of reprogramming method mostly comes down to which tissue the reprogramming is being done in.

In the eye, for example, you need to express the reprogramming factors for four weeks. This would make using mRNA very difficult since it usually lasts about 48 hours in the body. That’s when methods like doxycycline-inducible genes are often used.

Reprogramming & the Heart

Scientists have now tried partial reprogramming in many organs but one that stands out is the heart — specifically cardiomyocytes or heart muscle cells.

Adult cardiomyocytes are not able to regenerate nearly as well as fetal cardiomyocytes. So researchers wondered whether partial reprogramming could return some regenerative capabilities to adult cardiomyocytes.

Regeneration of fetal vs. adult cardiomyocytes. Image by author.

Scientists created mice with doxycycline-inducible genes that specifically turn on OSKM in cardiomyocytes. After performing some experiments, they found that 6 days was the sweet spot for expressing OSKM — beyond that, some cells became abnormal.

Next up, researchers tested whether OSKM expression had an effect on how well cardiomyocytes could regenerate following a heart attack. There were several groups of mice that were treated with OSKM at various time points and for different lengths of time (some were before the heart attack, some right after for a day, some 6 days after the heart attack).

The fascinating finding was that all treated mice had decreased scar size compared to controls after the heart attack.

A representation of the difference in scar size between the control and treatment groups (not to scale). Heart image adapted from Freepik. Image by author.

It’s important to note here that the rates at which mice died because of the heart attacks were not different between the OSKM-treated groups and the control groups. It was the scar size specifically that was affected. Regardless, this finding holds a lot of promise.

Do We Need All the Factors?

As you may have noticed above, different research groups have used different combinations of reprogramming factors to achieve partial reprogramming.

Many have used the traditional OSKM but others have used additional factors and some have used fewer than four and eliminated c-Myc. The elimination of c-Myc is particularly attractive to scientists because this gene is a potent oncogene, meaning it can cause cancer.

So what combination of factors works best? The answer is: it depends. Dr. Sebastiano explains that, for some cell types, we may be able to take away some factors and achieve the same results. In other cases, we may need extra factors to optimize reprogramming. It all comes down to the specific instance.

One recent paper found that no one factor was necessary for partial reprogramming. In fact, most combinations of three factors had very similar effects as the full set of four. The researchers hypothesized that the three factors could be causing the activation of the other. But, the paper looked at only two cell types so other cells may act differently.

Part III: The Future — Obstacles & Hopes

There’s no doubt that the future of partial reprogramming is bright. There are many companies that are now investing both money and incredible talent into creating a future of more youthful cells.

But some obstacles remain.

Dr. Sebastiano believes the next big barrier in the partial reprogramming field is finding an efficient way of targeting specific cells in vivo (within an organism) using mRNA reprogramming.

Dr. Lu adds on that he thinks obtaining regulatory approval will be very difficult if c-Myc is used as a factor in partial reprogramming. It may be necessary for scientists to use reprogramming factor combinations that exclude c-Myc to bring partial reprogramming to patients.

Some of the upcoming obstacles for the partial reprogramming field. Image by author.

Despite these obstacles, there is a lot to be excited about. Dr. Lu thinks that there will be therapies using OSK (not including c-Myc) within the next 10 years. Dr. Sebastiano believes that, in the future, we’ll be able to reverse the aging process in a tissue-specific manner.

I can nearly feel the sweet taste of the elixir of youth. And now it feels even sweeter. Because it’s backed by science.

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Parmin Sedigh
Predict

Science communicator trying to learn something new everyday | Published in Start It Up, Predict & The Writing Cooperative