In a preprint paper, scientists from Calico, Google’s longevity research behemoth, suggest that contrary to our previous understanding, transient reprogramming of cells using Yamanaka factors involves suppressing cellular identity, which may open the door to carcinogenic mutations. They also propose a milder reprogramming method inspired by limb regeneration in amphibians .
Rejuvenation that can give you cancer
In 2006, a group of scientists led by Shinya Yamanaka developed a technique for reprogramming somatic cells back into pluripotent stem cells by transfusing them with a cocktail of transcription factors . These four pluripotency-associated genes, Oct4, Sox2, Klf4, and c-Myc (OSKM), became known as the Yamanaka factors. This breakthrough made it possible to produce patient-specific stem cells from their own somatic cells.
On the other hand, induced pluripotent stem cells (iPSCs), which are the product of cellular reprogramming, are known to acquire carcinogenic mutations. This hurdle has been limiting their use, with scientists all over the world trying to overcome it in order to fully utilize iPSCs’ immense potential .
When somatic cells revert to the pluripotent state, they also shed many features of cellular aging, effectively becoming young again. iPSCs from young and aged donors are almost indistinguishable, and this similarity remains even after the cells differentiate again into various cell types.
This led scientists to attempt cellular rejuvenation with Yamanaka factors but without reprogramming the cells back to a pluripotent state. Such “transient reprogramming”, in which the factors are introduced for a short period of time, stops before the cells reach the Point of No Return (PNR) on the road back to pluripotency – or so it was thought. Transient reprogramming has been shown to improve multiple physiological functions in aged animals and extend lifespan in progeroid mice .
To pluripotency and back
This new paper was published by scientists from Calico, a secretive and well-funded Alphabet (Google) subsidiary in the field of longevity research. Since its inception several years ago, expectations from Calico have been high, but we have only seen a slow trickle of papers. This study is one of the most important to ever come out of the company.
Utilizing their almost unlimited resources, Calico researchers were able to study the effects of transient reprogramming by performing single-cell RNA sequencing for tens of thousands of individual cells. They found that transient reprogramming restored youthful gene expression in adipogenic cells and mesenchymal stem cells, but, at the same time, temporarily suppressed their cell identity programs. These results stand in contrast with the previous notion that transient reprogramming rejuvenates cells without making them revert to a pluripotent state. By analyzing transcription levels of several pluripotency-associated genes on a single-cell level, the researchers showed that such reversion does occur, even if briefly and/or partially, with the cells subsequently reacquiring their cellular identities. These subtle back-and-forth transitions might not have been picked up by previous studies that used less precise bulk analysis.
If cellular identity is indeed suppressed by transient reprogramming, this brings back the specter of oncogenic mutations. The whole idea of transient reprogramming is to rejuvenate cells in vivo, where such mutations cannot be controlled or weeded out.
Can we do with fewer factors?
Since some Yamanaka factors are known to be more oncogenic than others, the researchers analyzed the effects of various combinations of factors to determine whether any of them could be left out. Surprisingly, they found that none of the factors were indispensable – probably because of the way they interact with each other. Apparently, when a combination of factors is introduced to the cell, it activates endogenic transcription of the missing factors. As a result, leaving out any single factor, or even two, only weakens the reprogramming effect, sometimes moderately. As an example, the SO cocktail (half of OSKM), while still effective in transient reprogramming and rejuvenation, was found to suppress cellular identity considerably less than the full array of Yamanaka factors.
The researchers applied the factors both to young and aged cells. While the aged cells were significantly rejuvenated by the treatment, on the transcriptomic map created by the researchers, these aged cells clustered differently from the young reprogrammed cells. This remaining difference probably means that certain features of aging might not be affected by transient reprogramming, but additional research is needed.
Among the gene sets that showed the biggest amplitude of change following the reprogramming was the set that regulates cellular inflammatory response. The genes in this set were upregulated in aged cells and significantly downregulated by reprogramming. As aging is linked to excessive inflammation, a situation known as inflammaging, the ability of transient reprogramming to downregulate these genes is great news.
The amphibian connection
Finally, the researchers attempted an unorthodox approach to transient reprogramming using factors that are associated with multipotency. As opposed to pluripotent cells which can differentiate to almost any cell type, multipotent cells can only differentiate into a small subset of types.
The researchers treated aged murine myocytes (smooth muscle cells) with the multipotency factor Msx1, which also facilitates limb regeneration in some amphibians. This multipotency cellular reprogramming successfully restored youthful gene expression in aged myogenic cells. People probably will not be growing back limbs any time soon, but induced rejuvenation of muscle cells is an important result.
While holding great promise, transient reprogramming apparently is not risk-free. This important paper showcases what can be done with proper funding – such as tens of thousands of single-cell RNA profiles. It expands our understanding of the intricate processes of acquiring and losing cellular identity and of possible ways of using reprogramming techniques to develop therapies.
 Roux, A., Zhang, C., Paw, J., Zavala-Solorio, J., Vijay, T., Kolumam, G., … & Kimmel, J. C. (2021). Partial reprogramming restores youthful gene expression through transient suppression of cell identity. bioRxiv.
 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.
 Abad, M., Mosteiro, L., Pantoja, C., Cañamero, M., Rayon, T., Ors, I., … & Serrano, M. (2013). Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature, 502(7471), 340-345.
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