Yamanaka Factors and Partial Cellular Reprogramming
Partial cellular reprogramming is powerful technique that may be able to reprogram cells back into a youthful state, at least partially reversing epigenetic alterations, one of the proposed reasons we age.
Yamanaka factors and the birth of partial cellular reprogramming
In 2006, a study by Drs. Kazutoshi and Shinya Yamanaka showed that it is possible to reprogram cells using just four master genes: Oct4, Sox2, Klf4, and c-Myc (OSKM) . These four reprogramming factors are often called the Yamanaka factors after one of their discoverers.
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 the research community thought. Takahashi and Yamanaka turned this idea upside down when they showed that just four of the Yamanaka factors were needed to achieve this transformation.
They used the Yamanaka factors to reprogram adult mouse fibroblasts (connective tissue cells) back to a state called pluripotency, in which 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 . 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 original Hallmarks of Aging .
Jean-Marc Lemaitre and his colleagues tested the effects of Yamanaka factors on aged fibroblasts from normal old people and from healthy centenarians: 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 that 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 that 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 as during development.
Yamanaka factors appear to reverse epigenetic 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), one of the hallmarks of aging, were reversed.
In addition to this, they also showed that telomere length, mitochondrial function, and oxidative stress levels had all been reset to those typically observed in young fibroblasts. Telomere attrition and mitochondrial dysfunction are two more key aspects of aging.
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.
Can Yamanaka factors be used in living animals?
It was easy to isolate cells in a dish, revert them to a developmental state, then make them become whatever cell type they wanted using Yamanaka factors. However, this was not practical in a living animal as cells could not have their identity erased as they reverted back. 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. This was the case in a 2013 experiment, the first to attempt partial reprogramming. Here, a failure to achieve the right balance and timing of reprogramming factors resulted in the development of teratomas: bizarre tumors that are often found to contain hair, teeth, eyes, and other partially formed organs .
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. However, no one had successfully managed to achieve this in living animals until December 2016, when Professor Juan Carlos Izpisua Belmonte and his team at the Salk Institute reported the conclusion of their study, which showed rejuvenation of the cells and organs of a living animal .
In that 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.
Partial cellular reprogramming
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 the experimental and control groups were also euthanized at this point so that their skin, kidneys, stomachs, and spleens could be examined. The control mice showed age-related changes that the treated mice did not; instead, those mice had several aging signs halted or even reversed, including some epigenetic alterations.
The treated mice also experienced a 50% increase in their mean survival time compared to untreated progeric control mice. Not all aging signs were affected by partial cellular reprogramming, and when 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, which 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, it also appeared to improve tissue regeneration in engineered, 12-month-old, normally aging mice. 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.
In 2018, Nelly Olova’s research not only reinforced Belmont’s findings but also definitively showed that partial reprogramming decreased the epigenetic age of the cells under study. Furthermore, Olova’s team discovered that loss of cell identity and the change in epigenetic age occurred at different rates. This suggested the possibility that these two processes could be separated to minimize the risk of cancer while still making cells younger .
Another 2018 study by Markus Doeser’s group found that partial reprogramming could leveraged to improve wound healing and potentially treat fibrotic disease. Fibrotic disease accounts for up to 45% of all disease deaths [7,8].
Yamanaka factors used to improve cognitive function in old mice
In October 2020, another study took us a step closer to partial cellular reprogramming reaching the clinic, as researchers showed that partial cellular reprogramming improves memory in old mice. As previous studies have shown, partial cellular reprogramming is a balancing act: epigenetically rejuvenating cells and resetting their aging clocks without completely resetting their identities, which would make them forget what kinds of cells they are . This balancing act is possible by exposing cells to the reprogramming factors for very specific amounts of time.
As in previous studies, 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 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 .
To reduce cancer risk, they opted to try partial cellular reprogramming without 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. Therefore, they opted to use just Oct4, Sox2, and Klf4 (OSK).
OSK was able to rejuvenate the damaged eye nerves in mice and restore their 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.
Dr. 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!
By 2020, the promise of partial reprogramming caught the attention of investors, and the first companies dedicated to the development of partial reprogramming were born. Turn Biotechnologies developed a proprietary platform using messenger RNA to deliver Yamanaka factors to the epigenome . Building on their academic achievements, David Sinclair and Jaun Carlos Izpisua Belmonte spearheaded the development of Iduna Therapeutics, a division of Life Biosciences. Iduna’s focus honed in on OSK therapy delivered through intravitreal eye injections to target diseases of the optic nerve, such as glaucoma .
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 .
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. These results suggest that this may approximately be the optimal age for cellular function.
2022 was characterized by was characterized by a wealth of new insights and advances, including the discovery that natural killer cells act as a barrier for in vivo preprogramming . Reprogramming early in life was found to rejuvenate cell physiology, improve body composition and tissue fitness, and increase lifespan when the mice got older . Partial reprogramming of muscle cells in the body was found to promote muscle regeneration by remodeling stem cell depots . Reprogramming of heart cells to a fetal state can drive heart regeneration in mice . Additional partial reprogramming experiments in 2022 also demonstrated liver  and intervertebral disc regeneration .
Global versus piecemeal regeneration efforts
Whole-body regeneration is less practical than beginning with individual organs for multiple reasons. First, accomplishments within individual organs not only uncover potential differences in how each organ responds to partial reprogramming efforts, they provide additional data for more global efforts at reprogramming in humans.
Additionally, since aging is not considered a disease by the FDA, the logical workaround is to test partial reprogramming against other treatments for specific diseases, such as degenerative disc disease or fibrosis of the liver. With this approach, it may be possible to acquire funding that would not otherwise be available. Eventually, FDA approval for a specific use will likely be approved. This approval will expedite other approved uses and likely off-label use as well.
2023 and beyond
In 2023, we have continued to gain new insights for improving partial reprogramming efforts. A study on the metabolic requirements of partial reprogramming found that Vitamin B12 depletion occurs during OSKM-induced reprogramming. Unsurprisingly, supplementation with B12 was shown to enhance the efficiency of partial reprogramming .
Possibly the greatest achievement in the effort to improve partial reprogramming in 2023 is the discovery and development of existing and new compounds that mimic the action of Yamanaka factors. This includes the use of chemical cocktails: combinations of specific chemicals in specific quantities delivered over precise time intervals that have successfully enabled researchers to partially reprogram cells [21,22]. Synthetic, self-replicating RNA is also under development .
The reason why this development is so significant is that small-molecule drugs, on average, cost 22.5 times less than biologics (drugs produced using living beings) . Dramatic cost reductions of this nature would enable more research teams to work on partial reprogramming efforts accelerating translation from the bench to the clinic. Furthermore, if these cost savings are passed along to candidates for partial reprogramming, the benefits of this technology will be accessible to more people at a lower cost.
The 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 the body to respond to a particular compound like doxycycline, which would be extremely challenging although plausible, or utilizing transient gene therapy techniques.
Concerns and perspectives
It may be possible to edit the germ line so that our children are born with such a modification to respond to a chosen compound. However, this idea is currently an ethical nightmare to even consider, and there are significant technical challenges for doing so successfully. Whatever the solution is, it needs to be practical.
The other major hurdle is to find a suitable long-term method 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.
The future of Yamanaka factors to combat aging
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.
An early, first-pass use of this approach might be used in a preventative way: older people at risk of age-related diseases could be given partial reprogramming to halt or, at least, significantly slow down this aspect of aging and thus reduce their risk of developing these 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 to totally prevent age-related diseases and keep them healthy, active, and able to continue enjoying life.
Companies such as Google’s Calico are continuing to investigate alternative ways to achieve partial cellular reprogramming without using the Yamanaka factors. This direction of research may prove more practical and safe.
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.
 Takahashi, K.; Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 2006, 126, 663–676
 Lapasset, L.; Milhavet, O.; Prieur, A.; Besnard, E.; Babled, A.; Ät-Hamou, N.; Leschik, J.; Pellestor, F.; Ramirez, J.M.; De Vos, J.; et al. Rejuvenating Senescent and Centenarian Human Cells by Reprogramming through the Pluripotent State. Genes Dev 2011, 25, 2248–2253
 López-Otín, C.; Blasco, M.A.; Partridge, L.; Serrano, M.; Kroemer, G. The Hallmarks of Aging. Cell 2013, 153, 1194
 Abad, M.; Mosteiro, L.; Pantoja, C.; Cañamero, M.; Rayon, T.; Ors, I.; Graña, O.; Megías, D.; Domínguez, O.; Martínez, D.; et al. Reprogramming in Vivo Produces Teratomas and IPS Cells with Totipotency Features. Nature 2013 502:7471 2013, 502, 340–345
 Ocampo, A.; Reddy, P.; Martinez-Redondo, P.; Platero-Luengo, A.; Hatanaka, F.; Hishida, T.; Li, M.; Lam, D.; Kurita, M.; Beyret, E.; et al. In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell 2016, 167, 1719-1733.e12
 Olova, N.; Simpson, D.J.; Marioni, R.E.; Chandra, T. Partial Reprogramming Induces a Steady Decline in Epigenetic Age before Loss of Somatic Identity. Aging Cell 2019, 18
 Doeser, M.C.; Schöler, H.R.; Wu, G. Reduction of Fibrosis and Scar Formation by Partial Reprogramming In Vivo. Stem Cells 2018, 36, 1216–1225
 Jun, J. Il; Lau, L.F. Resolution of Organ Fibrosis. J Clin Invest 2018, 128, 97
 Rodríguez-Matellán, A.; Alcazar, N.; Hernández, F.; Serrano, M.; Ávila, J. In Vivo Reprogramming Ameliorates Aging Features in Dentate Gyrus Cells and Improves Memory in Mice. Stem Cell Reports 2020, 15, 1056–1066
 Lu, Y.; Brommer, B.; Tian, X.; Krishnan, A.; Meer, M.; Wang, C.; Vera, D.L.; Zeng, Q.; Yu, D.; Bonkowski, M.S.; et al. Reprogramming to Recover Youthful Epigenetic Information and Restore Vision. Nature 2020, 588, 124–129
 Gill, D.; Parry, A.; Santos, F.; Okkenhaug, H.; Todd, C.D.; Hernando-Herraez, I.; Stubbs, T.M.; Milagre, I.; Reik, W. Multi-Omic Rejuvenation of Human Cells by Maturation Phase Transient Reprogramming. Elife 2022, 11
 Melendez, E.; Chondronasiou, D.; Mosteiro, L.; de Villarreal, J.M.; Fernández-Alfara, M.; Lynch, C.J.; Grimm, D.; Real, F.X.; Alcami, J.; Climent, N.; et al. Natural Killer Cells Act as an Extrinsic Barrier for in Vivo Reprogramming. Development 2022, 149
 Alle, Q.; Le Borgne, E.; Bensadoun, P.; Lemey, C.; Béchir, N.; Gabanou, M.; Estermann, F.; Bertrand-Gaday, C.; Pessemesse, L.; Toupet, K.; et al. A Single Short Reprogramming Early in Life Initiates and Propagates an Epigenetically Related Mechanism Improving Fitness and Promoting an Increased Healthy Lifespan. Aging Cell 2022, 21
 Wang, C.; Rabadan Ros, R.; Martinez-Redondo, P.; Ma, Z.; Shi, L.; Xue, Y.; Guillen-Guillen, I.; Huang, L.; Hishida, T.; Liao, H.K.; et al. In Vivo Partial Reprogramming of Myofibers Promotes Muscle Regeneration by Remodeling the Stem Cell Niche. Nature Communications 2021 12:1 2021, 12, 1–15
 Chen, Y.; Lüttmann, F.F.; Schoger, E.; Schöler, H.R.; Zelarayán, L.C.; Kim, K.P.; Haigh, J.J.; Kim, J.; Braun, T. Reversible Reprogramming of Cardiomyocytes to a Fetal State Drives Heart Regeneration in Mice. Science 2021, 373, 1537–1540.
 Hishida, T.; Yamamoto, M.; Hishida-Nozaki, Y.; Shao, C.; Huang, L.; Wang, C.; Shojima, K.; Xue, Y.; Hang, Y.; Shokhirev, M.; et al. In Vivo Partial Cellular Reprogramming Enhances Liver Plasticity and Regeneration. Cell Rep 2022, 39, 110730
 Cheng, F.; Wang, C.; Ji, Y.; Yang, B.; Shu, J.; Shi, K.; Wang, L.; Wang, S.; Zhang, Y.; Huang, X.; et al. Partial Reprogramming Strategy for Intervertebral Disc Rejuvenation by Activating Energy Switch. Aging Cell 2022, 21, e13577
 Vílchez-Acosta, A.; Desdín-Micó, G.; Ocampo, A. Vitamin B12 Emerges as Key Player during Cellular Reprogramming. Nat Metab 2023, 5, 1844–1845
 Guan, J.; Wang, G.; Wang, J.; Zhang, Z.; Fu, Y.; Cheng, L.; Meng, G.; Lyu, Y.; Zhu, J.; Li, Y.; et al. Chemical Reprogramming of Human Somatic Cells to Pluripotent Stem Cells. Nature 2022, 605, 325–331
 Yang, J.H.; Petty, C.A.; Dixon-McDougall, T.; Lopez, M.V.; Tyshkovskiy, A.; Maybury-Lewis, S.; Tian, X.; Ibrahim, N.; Chen, Z.; Griffin, P.T.; et al. Chemically Induced Reprogramming to Reverse Cellular Aging. Aging (Albany NY) 2023, 15, 5966
 Wagner, A.; Mutschler, H. Design Principles and Applications of Synthetic Self-Replicating RNAs. Wiley Interdiscip Rev RNA 2023, 14, e1803
 Heinrichs, M.J.; Owens, G.M. Where Generics and Biologics Meet. Am Health Drug Benefits 2008, 1, 21.