Studies by a team at Stanford University School of Medicine have shown how aged mice regained youthful strength after their muscle stem cells were subjected to a rejuvenating protein treatment and then transplanted back into their bodies. The research also showed that old human cells could be returned to a more youthful, vigorous state when induced to briefly express a panel of proteins, known as Yamanaka factors, which are commonly used to transform adult cells into induced pluripotent stem cells (iPSCs). The results suggested that inducing cultured old human cells to briefly express the nuclear reprogramming factors effectively rewinds many of the molecular hallmarks of aging and renders the treated cells nearly indistinguishable from younger cells.

“When iPS cells are made from adult cells, they become both youthful and pluripotent,” said Vittorio Sebastiano, PhD, assistant professor of obstetrics and gynecology and the Woods Family faculty scholar in pediatric translational medicine. “We’ve wondered for some time if it might be possible to simply rewind the aging clock without inducing pluripotency. Now we’ve found that, by tightly controlling the duration of the exposure to these protein factors, we can promote rejuvenation in multiple human cell types.”

Sebastiano is the senior author of the team’s report in Nature Communications, which is titled, “Transient non-integrative expression of nuclear programming factors promotes multifaceted amelioration of aging in human cells.” Former graduate student Tapash Sarkar, PhD, is the lead author of the article. Co-researchers on the study included teams at the Veterans Affairs Palo Alto Health Care System, the University of California, Los Angeles, and the Molecular Medicine Research Institute in Sunnyvale, California.

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue, and organismal levels, the authors explained. “At the chromatin level, aging associates with progressive accumulation of genetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis.” Nuclear programming of cells to reinstate pluripotency can turn back cellular time—with respect to both the age and identity of any cell—to that of an embryonic cell. Studies have also suggested that even transient reprogramming can reduce the hallmarks of cellular aging and extend lifespan in mouse models of aging. So if the expression of reprogramming factors is applied transiently, but stopped before the “point of no return,” or PNR, then the cells will return to their initiating somatic cell state.

What isn’t known, is how far this approach to cellular rejuvenation might apply to naturally aging human cells. “These observations suggest that, if applied for a short enough time, the expression of reprogramming factors fails to erase the epigenetic signature defining cell identity,” the authors noted, “however, it remains unknown whether any substantial and measurable reprogramming of cellular age can be achieved before the PNR.”

Researchers in Sebastiano’s laboratory generated iPSCs from adult cells, such as skin fibroblast cells, or endothelial cells linking blood vessels, by repeatedly exposing them over a period of about two weeks to a panel of proteins important to early embryonic development. This is achieved by introducing short-lived RNA messages into the adult cells, daily, which encode instructions for the Yamanaka proteins. Over time, these proteins rewind the cells’ fate, effectively pushing them backward along the developmental timeline until they resemble the young, embryonic-like pluripotent cells from which they originated.

During this process, the cells not only shed memories of their previous identities, but they revert to a younger state. The transformation wipes their DNA clean of the molecular tags that differentiate, for example, a skin cell from a heart muscle cell, and also of other tags that accumulate as a cell ages.

Researchers have speculated whether exposing the adult cells to these Yamanaka proteins for days rather than weeks could trigger youthful reversion, but without inducing full-on pluripotency. Researchers at the Salk Institute for Biological Studies found in 2016 that briefly expressing the four Yamanaka factors in mice with a form of premature aging extended the animals’ life span by about 20%. But it wasn’t clear whether this approach would work in humans.

Sarkar and Sebastiano wondered whether old human cells would respond in a similar fashion, and whether the response would be limited to just a few cell types or could be generalizable for many tissues. They devised a way to use mRNA to temporarily express six reprogramming factors—the four Yamanaka factors plus two additional proteins—in human fibroblast and endothelial cells. Messenger RNA rapidly degrades in cells, allowing the researchers to tightly control the duration of the signal. “We utilized a non-integrative reprogramming protocol that we optimized, based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG (OSKMLN),” they wrote.

The researchers compared the gene-expression patterns of treated cells and control cells, obtained from elderly adults, with those of untreated cells from younger people. They found that cells from elderly individuals exhibited signs of aging reversal after just four days of exposure to the reprogramming factors. Whereas untreated elderly cells expressed higher levels of genes associated with known aging pathways, treated elderly cells more closely resembled younger cells in their patterns of gene expression.

The researchers then studied the patterns of aging-associated DNA methylation, which serves as an indicator of a cell’s chronological age. “Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and are predictive of a host of age-related conditions lifespan,” they noted. They found that the treated cells appeared to be about 1.5 to 3.5 years younger on average than untreated cells from elderly people, with peaks of 3.5 years (in fibroblasts) and 7.5 years (in the endothelial cells).

The scientists next compared several hallmarks of aging—including how cells sense nutrients, metabolize compounds to create energy, and dispose of cellular trash—among cells from young people, treated cells from old people, and untreated cells from old people. “We saw a dramatic rejuvenation across all hallmarks but one in all the cell types tested,” Sebastiano said. “But our last and most important experiment was done on muscle stem cells. Although they are naturally endowed with the ability to self-renew, this capacity wanes with age. We wondered, ‘Can we also rejuvenate stem cells and have a long-term effect?'” When the researchers transplanted treated old mouse-derived skeletal muscle stem cells (MuSCs) back into elderly mice, the animals regained the muscle strength of younger mice. “Muscles transplanted with untreated aged MuSCs showed forces comparable with untransplanted muscles from aged control mice,” they commented. “… These results suggest that transient reprogramming in combination with MuSC-based therapy can restore physiological function of aged muscles to that of youthful muscles.” Their combined studies using MuSCs from both mouse and human tissue led them to conclude, “Taken together, these results suggest that transient reprogramming partially restores the potency of aged MuSCs to a degree similar to that of young MuSCs, without compromising their fate, and thus has potential as a cell therapy in regenerative medicine.”

Finally, the researchers isolated cells from the cartilage of people with and without osteoarthritis (OA). They found that the temporary exposure of the osteoarthritic cells to the reprogramming factors reduced the secretion of inflammatory molecules and improved the cells’ ability to divide and function. Again, they suggested, their findings could point to new strategies for therapy. “Together these results show that transient expression of OSKMLN can promote a partial reversal of gene expression and cellular physiology in aged OA chondrocytes toward a healthier, more youthful state, suggesting a potential new therapeutic strategy to ameliorate the OA disease process.”

They noted that the epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place, which they stated represents a novel finding in this field of research. “Here we demonstrate that a non-integrative, mRNAs-based platform of transient cellular reprogramming can very rapidly reverse a broad spectrum of aging hallmarks in the initiation phase, when epigenetic erasure of cell identity has not yet occurred,” they wrote. The investigators acknowledged that further research will be needed both to identify the mechanism that drives the reversal of the aged phenotype during cellular reprogramming, uncoupling it from the dedifferentiation process, and confirm that “a multispectral cellular rejuvenation” could be achieved in a cell-autonomous fashion in human cells taken from naturally aging people. Nevertheless, they wrote, “Our results are novel and represent a significant step toward the goal of reversing cellular aging, and have potential therapeutic implications for aging and aging-related diseases.”

The researchers are now optimizing the panel of reprogramming proteins needed to rejuvenate human cells and are exploring the possibility of treating cells or tissues without removing them from the body. “Although much more work needs to be done, we are hopeful that we may one day have the opportunity to reboot entire tissues,” Sebastiano said. “But first we want to make sure that this is rigorously tested in the lab and found to be safe.”

Commented study co-author Thomas Rando, MD, PhD, professor of neurology and neurological sciences and the director of Stanford’s Glenn Center for the Biology of Aging, “We are very excited about these findings. My colleagues and I have been pursuing the rejuvenation of tissues since our studies in the early 2000s revealed that systemic factors can make old tissues younger. In 2012, Howard Chang, MD, PhD, and I proposed the concept of using reprogramming factors to rejuvenate cells and tissues, and it is gratifying to see evidence of success with this approach.” Chang is a professor of dermatology and of genetics at Stanford.

 

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