Today, we chronicle the progress of partial cellular reprogramming and discuss how this powerful treatment may be able to reprogram cells back into a youthful state, at least partially reversing epigenetic alterations, one of the proposed reasons we age.
The Yamanaka factors and the birth of partial cellular reprogramming
In 2006, a study by Drs. Takahashi and Yamanaka showed that it was possible to reprogram cells using just four master genes named Oct4, Sox2, Klf4, and c-Myc, or OSKM for short [1]. These four reprogramming factors are often called the Yamanaka factors.
Prior to this, it was assumed that egg cells (oocytes) would contain a complex array of factors needed to reprogram a somatic cell into becoming an embryonic cell. After all, the feat of transforming an aged egg cell and reprogramming it to make a new animal must be controlled by many factors present in the egg cell, or so they thought.
Takahashi and Yamanaka turned this idea upside down when they showed that just four the Yamanaka factors were needed to achieve this transformation. They used the Yamanaka factors to reprogram adult mouse fibroblasts (connective tissue cells) back to an embryonic state called pluripotency, a state where the cell behaves like an embryonic stem cell and can become any other cell type in the body.
This discovery paved the way for research into how these Yamanaka factors might be used for cellular rejuvenation and a potential way to combat age-related diseases.
Yamanaka factors for cellular and animal rejuvenation
In 2011, a team of French researchers, including Jean-Marc Lemaitre, first reported cellular rejuvenation using the Yamanaka factors [2]. During their life, cells express different patterns of genes, and those patterns are unique to each phase in a cell’s life from young to old; this gene expression profile makes it easy to identify an old or young cell. At the time, it was also known that aged cells such as fibroblasts have short telomeres and dysfunctional mitochondria, two of the nine reasons we age [3].
Jean-Marc Lemaitre and his colleagues tested the effects of Yamanaka factors on aged fibroblasts from normal old people and also from healthy people over 100 years old. They added two additional pluripotency genetic factors to the OSKM mix, namely NANOG and LIN28, and examined the effect that this had on the gene expression, telomeres, and mitochondria of these older people.
They discovered that together, the six factors were able to reset cells from old donors back into a pluripotent state, meaning they could become any other cell type in the body. These became known as induced pluripotent stem cells (iPSCs).
The researchers noted that the cells had a higher growth rate than the aged cells they had been reprogrammed from; they also had longer telomeres as well as mitochondria that behaved in a youthful manner and were no longer dysfunctional. In other words, reprogramming the cells reversed some of the aspects of aging and rolled the cells back to a similar state seen during development.
Yamanaka factors appear to reverse some aspects of aging
The final step for the researchers was to then guide these iPSCs to become fibroblasts again using other reprogramming factors. The result was that these reprogrammed fibroblasts no longer expressed the gene patterns associated with aged cells and had a gene expression profile indistinguishable from those of young fibroblasts.
Essentially they showed that epigenetic alterations (changes to gene expression patterns), a reason we age, were reversed. You can learn more about how epigenetic alterations contribute to aging in the topic box below.
In addition to this, they also showed that telomere length, mitochondrial function, and oxidative stress levels had all reset to those typically observed in young fibroblasts. Telomere attrition and mitochondrial dysfunction are two more reasons we are thought to age.
This was the first evidence that aged cells, even from very old individuals, could be rejuvenated, and this was followed by a flood of independent studies confirming these findings in the same and other types of cells.
Could Yamanaka Factors be used in living animals?
It was easy to isolate cells in a dish, take them back to developmental state, then guide them to become whatever cell type they wanted using Yamanaka factors. But this was obviously not practical in a living animal as cells could not have their memory erased so they reverted to a pluripotent state. Imagine if a heart cell forgot it was a heart cell while it was supposed to be helping pump blood around the body!
There was also the concern that the expression of Yamanaka factors was known to induce cancer in animals [4].
Some researchers believed that it might be possible to avoid cancer and reverse aging in old cells without completely reverting them to pluripotency. In other words they thought there was a way for us to have our cake and eat it. But no one had successfully managed to achieve this in living animals. This was all about to change in December 2016.
Professor Juan Carlos Izpisua Belmonte and his team of researchers at the Salk Institute reported the conclusion of their study, which showed for the first time that the cells and organs of a living animal could be rejuvenated [5]. The researchers made this short video to explain this breakthrough in aging research.
For the study, the researchers used a specially engineered progeric mouse designed to age more rapidly than normal as well as an engineered normally aging mouse strain. Both types of mice were engineered to express the Yamanaka factors when they came into contact with the antibiotic doxycycline, which was given to them via their drinking water.
They allowed the Yamanaka factors to be transiently expressed by including doxycycline in the water for two days then removed it so that the OSKM genes were silenced again. The mice then had a five-day rest period before another two days of exposure to doxycycline; this cycle was repeated for the duration of the study.
Transient Yamanaka factors
After just six weeks of this treatment, which steadily reprogrammed the cells of the mice, the researchers noticed improvements in their appearance, including reduced age-related spinal curvature. Some of the mice from both experiment and control groups were also euthanized at this point so that their skin, kidneys, stomachs, and spleens could be examined. The control mice showed a range of age-related changes compared to the treated mice, which had a number of aging signs halted or even reversed, including some epigenetic alterations.
The treated mice also experienced a 50% increase in their mean survival time in comparison to untreated progeric control mice. It should be noted that not all aging signs were affected by partial cellular reprogramming, and if treatment was halted, the aging signs returned.
Perhaps most importantly, while the partial cellular reprogramming conducted in this periodic manner reset some epigenetic aging signs, it did not reset cell differentiation, which would cause the cell to revert to an embryonic state and forget what kind of cell it previously was; as you can imagine, this would be a bad thing in a living animal.
Finally, not only did the transient expression of Yamanaka factors at least partially rejuvenate cells and organs in progeric mice, but it also appeared to improve tissue regeneration in the engineered 12-month-old normally aging mouse group. The researchers observed that the partial reprogramming improved these mice’s ability to regenerate tissue in the pancreas, resulting in an increased proliferation of beta cells; additionally, there was an increase of satellite cells in skeletal muscle. Both of these types of cells typically decline during aging.
Yamanaka factors used in living animals again
In October 2020, another study took us a step close to partial cellular reprogramming reaching the clinic when researchers showed that partial cellular reprogramming improves memory in old mice. As the previous studies have shown, partial cellular reprogramming is a balancing act between epigenetically rejuvenating cells and resetting their aging clocks, without completely resetting their cell identity so they forget what kind of cell they are [6].
Previous studies have also shown us that this balancing act is possible and that by exposing cells just long enough to the reprogramming factors, rejuvenation of the cell is possible without erasing its cellular identity.
As in the previous study we talked about, mice in this study had their cells engineered to react to doxycycline, a common antibiotic used in veterinary practice, in order to express the OSKM reprogramming factors. The researchers found that giving the mice just enough exposure to Yamanaka factors improved their cognitive function without an increase in mortality during a four-month period.
Another step forward for partial cellular reprogramming
In late 2020, researchers including Dr. David Sinclair, published a study that showed that they had managed to restore lost vision to old mice, and mice with damaged retinal nerves, using partial cellular reprogramming [7].
To reduce cancer risk they opted to try partial cellular reprogramming minus one of the Yamanaka Factors. One of the study authors, Dr. Yuancheng Lu, was looking for a safer way to rejuvenate aged cells, as there were some concerns that using c-Myc could cause cancer under certain circumstances. So in the end they opted to use just Oct4, Sox2, Klf4 (OSK).
The good news was that even OSK was able to rejuvenate the damaged eye nerves in mice and restore vision. It also worked to improve age-related vision impairment in treated mice and in mice that experienced increased eye pressure, an emulation of glaucoma.
Study co-author, Dr. David Sinclair, said in an article in Nature, “We set out with a question: if epigenetic changes are a driver of ageing, can you reset the epigenome?”, or, in other words, “Can you reverse the clock?”. The answer to that appears to be a resounding yes!
Dr. Lu also joined us for the Journal Club December 2020 to talk us through the study.
Refining the partial cellular reprogramming method
In January 2021 researchers showed that partial reprogramming rejuvenates human cells by 30 years, making old worn out cells function like the cells of a person around 25 years old. The researchers of this study used an approach that exposed cells to enough reprogramming factors to push them beyond the limit at which they were considered somatic rather than stem cells – but only just beyond [8].
The fibroblasts that were reprogrammed in this way retained enough of their epigenetic cellular memories to return to being fibroblasts once again. Exposing these cells to the OSKM factors was performed with a doxycycline-activated lentiviral package as previous animal studies had also done.
Perhaps most interesting, according to Horvath’s 2013 multi-tissue clock, sample cells that were just under 60 years old became epigenetically equivalent to cells that were approximately 25 years old after 13 days of partial cellular reprogramming, and the Horvath 2018 skin and blood clock showed that cells that were approximately 40 years old were also epigenetically returned to those of a 25-year-old. It seems that the cells revert an epigenetic age of 25 or so suggesting this is a peak of cellular prime or the optimal functional age for cells.
The future potential and challenges ahead for partial cellular reprogramming
By far, the biggest hurdle to translating partial cellular reprogramming to people is the need to find a way to activate the Yamanaka factors in our cells without needing to engineer our bodies to react to a drug such as doxycycline. Doing this may require us to develop drugs capable of activating OSKM, editing every cell in our body to respond to a particular compound like doxycycline, which would be extremely challenging though plausible.
Another possibility is editing the germline so that our children are born with such a modification to respond to a chosen compound, an idea that is currently an ethical nightmare to even consider not to mention the technical challenges of doing so successfully. Whatever the solution is, it needs to be practical.
The other major hurdle is to find a method suitable for the long term that does not require constant upkeep, lest the aging signs return rapidly, as they did in mice when treatment was interrupted. While there is some reason to believe that these signs would not return as rapidly in people given the differences between mouse and human metabolisms and our superior repair systems, it would likely return in due course. So, finding a cost-effective way to keep the cyclic treatment going is paramount; this could potentially be achieved using drugs or transient gene therapy.
Conclusion
Assuming that these barriers can be overcome, and the rapid advances in biotechnology offer a reason to think that they will, then partial cellular reprogramming could feasibly hold a great deal of potential for preventing or even curing the diseases of aging.
One might envision the early, first-pass use of this approach in a preventative way: older people at risk of age-related diseases could be given partial reprogramming in order to halt or at the least significantly slow down this aspect of aging and thus reduce their risk of developing age-related diseases.
More refined stages may see it being used in a more focused manner to repair a certain organ or tissue damaged by injury or disease. In another, more advanced, scenario, the gradual whole-body rejuvenation of older people might be attempted in order to totally prevent age-related diseases and keep them healthy, active and able to continue enjoying life.
Companies such as Google Calico are also currently investigating alternative ways to achieve partial cellular reprogramming without using Yamanaka factors. This is another direction of research that may prove more practical and safer than using Yamanaka factors.
The rapid progress of medical technology could potentially mean that such partial cellular reprogramming therapies may become available in the not too distant future. We certainly hope so.
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Literature
[1] Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell, 126(4), 663-676.
[2] Lapasset, L., Milhavet, O., Prieur, A., Besnard, E., Babled, A., Aït-Hamou, N., … & Lehmann, S. (2011). Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes & development, 25(21), 2248-2253.
[3] López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194-1217.
[4] Abad, M., Mosteiro, L., Pantoja, C., Canamero, M., Rayon, T., Ors, I., … & Manzanares, M. (2013). Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature, 502(7471), 340.
[5] Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, Li M, Lam D, Kurita M, Beyret E, Araoka T, Vazquez-Ferrer E, Donoso D, Roman JLXJ, Rodriguez-Esteban C, Nuñez G, Nuñez Delicado E, Campistol JM, Guillen I, Guillen P, Izpisua Belmonte JC. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell. 2016;167:1719–33.
[6] Rodríguez-Matellán, A., Alcazar, N., Hernández, F., Serrano, M., & Ávila, J. (2020). In Vivo Reprogramming Ameliorates Aging Features in Dentate Gyrus Cells and Improves Memory in Mice. Stem cell reports, 15(5), 1056-1066.
[7] Lu, Y., Brommer, B., Tian, X., Krishnan, A., Meer, M., Wang, C., … & Sinclair, D. A. (2020). Reprogramming to recover youthful epigenetic information and restore vision. Nature, 588(7836), 124-129.
[8] Gill, D., Parry, A., Santos, F., Hernando-Herraez, I., Stubbs, T. M., Milagre, I., & Reik, W. (2021). Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. bioRxiv.
Cellular rejuvenation therapy safely reverses signs of aging in mice [Salk Institute]
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It is well known that adding a mixture of four reprogramming molecules (Oct4, Sox2, Klf4 and c-Myc) also known as “Yamanaka factors” to cells can reset epigenetic marks to their original patterns. Partial reprogramming, by expression of reprogramming factors for short periods of time, restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown.
Now, researchers show that long-term partial reprogramming leads to rejuvenating effects in different tissues of mice and at the organismal level. In addition, they show that duration of the treatment determined the extent of the beneficial effects.
“We are elated that we can use this approach across the life span to slow down aging in normal animals. The technique is both safe and effective in mice,” says Juan Carlos Izpisua Belmonte, PhD, professor of the Gene Expression Laboratory at the Salk Institute for Biological Studies and an Institute Director at Altos Labs. “In addition to tackling age-related diseases, this approach may provide the biomedical community with a new tool to restore tissue and organismal health by improving cell function and resilience in different disease situations, such as neurodegenerative diseases.”
Juan Carlos Izpisua Belmonte, PhD, and Pradeep Reddy, PhD [Salk Institute]
In 2016, Izpisua Belmonte’s lab reported for the first time that they could use the Yamanaka factors to counter the signs of aging and increase life span in mice with a premature aging disease. More recently, the team found that, even in young mice, the Yamanaka factors can accelerate muscle regeneration. Following these initial observations, other scientists have used the same approach to improve the function of other tissues like the heart, brain and optic nerve, which is involved in vision.
In this work, Belmonte and colleagues performed various long-term partial reprogramming regimens in healthy animals, including different onset timings, during physiological aging.
“What we really wanted to establish was that using this approach for a longer time span is safe,” says Pradeep Reddy, PhD, a staff scientist at the Salk. “Indeed, we did not see any negative effects on the health, behavior or body weight of these animals.”
One group of mice received regular doses of the Yamanaka factors from the time they were 15 months old until 22 months, approximately equivalent to age 50 through 70 in humans. Another group was treated from 12 through 22 months, approximately age 35 to 70 in humans. And a third group was treated for just one month at age 25 months, similar to age 80 in humans.
Compared to control animals, there were no blood cell alterations or neurological changes in the mice that had received the Yamanaka factors. Moreover, the team found no cancers in any of the groups of animals.
The rejuvenating effects, the authors note, were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways.
When injured, the skin cells of treated animals had a greater ability to proliferate and were less likely to form permanent scars—older animals usually show less skin cell proliferation and more scarring. Moreover, metabolic molecules in the blood of treated animals did not show normal age-related changes.
This youthfulness was observed in the animals treated for seven or 10 months with the Yamanaka factors, but not the animals treated for just one month. What’s more, when the treated animals were analyzed midway through their treatment, the effects were not yet as evident. The authors suggest that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes.
The team is now planning future research to analyze how specific molecules and genes are changed by long-term treatment with the Yamanaka factors. They are also developing new ways of delivering the factors.
“At the end of the day, we want to bring resilience and function back to older cells so that they are more resistant to stress, injury and disease,” says Reddy. “This study shows that, at least in mice, there’s a path forward to achieving that.”
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