Extracellular Vesicles (EVs) have only been studied in the 21st century. Think of them as natural lipid nanoparticles, or endogenous viruses. They transmit information around the body and they’re small enough to be exhaled and carried in the air to communicate with other individuals and even other species. EVs are encapsulated in fats that facilitate entry into cells, and inside they contain proteins, RNA, DNA, lipids — all of which carry information. EVs are a universal biological language, a barely-explored medium of communication.

Last week, an extraordinary paper was published demonstrating the rejuvenation potential of EVs from very young animals injected into old animals. The authors come from Smidt Heart Institute in Los Angeles, and their primary field is cardiology, not gerontology, so they focus on heart-derived EVs and benefits for rejuvenating the heart. But the benefits they observe go well beyond the heart and include lifespan in rodents and rejuvenation of human cells from rat-derived EVs.

This research should be understood in the light of parabiosis experiments by the Conboys, Rando, and others and in particular to the rejuvenation technology of Harold Katcher. I am grateful to Harold’s partner, Akshay Sanghavi, for alerting me to this publication. Katcher’s blood-derived rejuvenation serum is called E5, and Akshay has told me that E5 includes EVs as well as a wide range of proteins. The current article suggests the possibility that it is wholly the EVs that are responsible for the benefits of E5. Indeed, the authors repeated their experiments with (1) whole blood plasma, (2) plasma with the EVs removed, and (3) just EVs. They found rejuvenation effects associated with whole plasma and with EVs alone, but not with plasma minus EVs. If this holds up, it is the study’s most important finding. Akshay told me that EVs are easier and cheaper to isolate than the molecular constituents of E5 (proteins).

For all those involved in parabiosis and plasma exchange research, I suggest that it should be an immediate priority to replicate the Smidt findings that all of the rejuvenating power of young blood is contained in EVs. The Conboys might be interested in asking whether the pro-aging effect of plasma infusions from old to young animals is also an effect of EVs.

The state-of-the-art EV separation technique for large throughput is called an acoustic nanofilter. Ultrasound pressure can be tuned to separate a particular size of particle in a specially-designed medium. If I were advising Yuvan, I would suggest that they develop an expertise in acoustic nanofiltration ASAP as a next generation replacement for their plasma fractioning technology.

Exosomes

Exosomes are the most common type of EV, and probably the most relevant to aging applications. They seem to be a general vehicle for inter-cellular and inter-individual communication. The study of exosomes is in its infancy, but it is already known that exosomes are tagged in a way that recipient cells can distinguish and choose which exosomes to pick up and “read the message”.

One area of exosome activity that has been studied is the communication of antigens to the immune system. A particular type of exosome includes foreign proteins that are potential invaders, and they are shared not just within the body but through the air. When you think of herd immunity, consider not just the contagion of people who carry disease but also the information about what diseases are in the air that is transmitted in social interactions.

When communities and whole countries were locked down during COVID, one consequence was to prevent uninfected individuals from learning and preparing an immune response to the virus through the sharing of exosomes.

“In the nervous system, exosomes have been found to help in myelin formation, neurite growth, and neuronal survival, thus playing a role in tissue repair and regeneration.” [ref, ref, ref, ref, ref, ref] “It has been demonstrated that the mesenchymal stem cell exosomes themselves can act as a therapeutic agent to help [repair] tissue injury.” [ref, ref, ref, ref, ref, ref]

“Microvesicles” are, as far as I can tell, a kind of fat exosome. They are as large as bacteria, whereas exosomes are closer to the size of viruses, but the range of contents and hypothesized functions is the same.

Apoptotic Bodies — another potential target for EV therapies

There is also a specific EV type that apoptotic cells send to induce apoptosis in other cells. Apoptosis is programmed cell death, playing an important role in self-destruction of diseased and cancerous cells. However, aging involves a cascade of apoptosis in healthy cells that leads to loss of muscle tissue (sarcopenia) and brain cells (neurodegeneration). Apoptotic bodies are the type of EV that triggers the apoptosis cascade, and I speculate that removing apoptotic bodies from the blood will have future potential as an anti-aging therapy. Apopototic bodies are even larger than “jumbo” microvesicles, so it should not be difficult to separate them in medical applications. It is a reasonable guess that the effectiveness of senolytic therapies is directly related to a reduction in apoptotic bodies, which are secreted by senescent cells. (These are my own speculations, not mentioned in the paper that I’m reviewing here.)

The new research

Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence by Grigorian-Shamagian et al, in Nature Scientific Reports. Senior author = Eduardo Marbán.

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The authors extracted EVs from neonatal rat hearts, and applied them to cell cultures and injected them into older live rats (22 months). They used whole plasma and plasma minus EVs as controls, establishing that it is the EVs that carry the benefits. They extracted corresponding EVs from heart cells of neonatal humans, and applied them to cultures of human cells.

They found EKG evidence that hearts were functioning better in treated animals. Insulin resistance was blood sugar were reduced.

Exercise endurance increased in treated animals, while declining progressively in controls.

They found that pliability of tissue was restored toward young levels. Hearts, lungs, muscles, and kidneys all improved their function in treated rats.

Tissue samples (muscle and heart) look better in ways that I don’t pretend to understand.

Old rats were treated with four monthly infusions, then kept for 16 weeks more before sacrificing all animals. During those 16 weeks, 6 of 14 control rats died and only 3 of 36 treated rats. My calculation (Fisher’s exact test) indicates life extension with a 99% confidence level.

Tests were conducted on two human cell cultures: fibroblasts from middle-aged donors treated with human EVs, and cardiac cells treated with rat EVs. Fibroblasts increased reproductive capacity, decreased apoptosis, and a more youthful transcriptome. Similarly, cardiac cells exhibited better self-assembly and a more youthful transcriptome.

The bottom line

In case you haven’t yet read between the lines, I’m excited about this work. I would have liked to see results of some Horvath clocks and a hat tip to the Katcher preprint, but in other respects, I find the research quite thorough and convincing. A major question in plasma exchange research has been the identification of the active component, out of thousands of protein species. This is only one study, but it suggests that researchers look at the activity of EVs rather than proteins or RNAs.

 

A crucial question is whether EVs from other mammals can be used to rejuvenate human tissues. This study suggests yes, but the demonstration was limited to cell cultures and no direct comparison was made between treatment with human-derived and rat-derived EVs. The author has not responded to my email requesting any data relevant to this question. If animal EVs work in humans, the therapeutic market may soon open; EVs should be easy to extract from the blood of young animals that are being slaughtered for meat. If not, we face a major ethical dilemma, as aborted and stillborn infants will support only a tiny fraction of the potential demand for rejuvenating EVs. EVs from young human donors can probably be extracted but it’s a lot to ask of our children. The authors suggest that EVs could be manufactured from human cell cultures. “Given that allogeneic CDCs [cardiosphere-derived cells] are already in advanced testing and have proven safe to date, such cells can be used as manufacturing platforms for EVs, enabling rapid progress to clinical testing in a variety of aging-related disorders.”

The Yamanaka factors (four proteins, abbreviated OSKM) are a treatment that can completely reset any ordinary, functional cell into a pluripotent stem cell, able to regenerate tissues of any type. In the process, the cell loses epigenetic markers of age and reverts to an embryonic state. But differentiated cells are differentiated so they can be functional. Taking them all the way back to stem cells destroys their function. Remarkably, differentiated cells can be taken part-way back, becoming younger but not losing its functional identity. This has been done with pulsed doses of OSKD or with just three of the four factors, OSK. Last year, the Harvard laboratory of David Sinclair reported success rejuvenating the aged retinas of mice with this process.

Rejuvenating a whole body in this way is impractical. Precisely controlling the dosage and the timing of OSK to each cell cannot be achieved outside a Petri dish. To make matters worse, some of the treated cells have a tendency to grow into tumors. 

Sinclair has been searching for other chemicals (besides the Yamanaka factors) that might have a rejuvenating effect at the cellular level without risking cancer and without problematic sensitivity to dosage and timing. With modern, computerized laboratories, it is practical to test thousands of chemicals shotgun-style in separate cell cultures, automating both the delivery of chemicals and the measurement of outcomes.

Recently, Sinclair reported the results of this process: Of the many combinations assayed in his shotgun experiments, six were identified that successfully rejuvenated cells in culture without the cells losing their identities and without causing cancer. “Thus, rejuvenation by age reversal can be achieved, not only by genetic, but also chemical means.”

This sounds like breakthrough science. Let’s look more closely.

What is the evidence that the cells are rejuvenated?

The new Sinclair article cites two lines of evidence

• Nucleocytoplasmic compartmentalization (NCC)
• An aging clock based on which genes are being transcribed

 

NCC was a new idea for me. There are certain proteins that belong in the nucleus, and they tend to leak into the surrounding cytoplasm as one sign of aging in the cel. 

Aging clocks based on transcription are a promising idea but not as well developed as methylation clocks.

Maybe I’m too suspicious, but I’m inclined to ask: Why didn’t Sinclair choose one of the well-established, validated aging clocks as a target, for example the robust Horvath multi-tissue or multi-species clocks? If he was going to use a transcriptomic clock, why did he have to invent a new one, rather than use Lehalier’s clock? If he was going to introduce a new clock, why wouldn’t he validate it by reference to established aging clocks?

I wonder if there is an untold history of this research, in which the Sinclair lab tried and failed to rejuvenate cells as measured by the more conventional aging clocks. 

Aging is not cell-autonomous

In different laboratories, aging research has been pursued via two pathways: top-down and bottom-up. Top-down researchers are looking for central coordination of the age state of the body, broadcast probably via signal molecules in the blood. (Michael Levin thinks that it may also occur through persistent patterns of electrical voltage.) Bottom-up researchers are looking for ways that individual cells lose focus and function with age.

The deep problem with Sinclair’s approach is that it derives from an overly reductionist perspective. Aging of the body is reduced to aging of individual cells. Aging of cells is understood as a blurring in the precision chemistry that the cell relies on for optimum health. 

Other researchers (including the Conboys, Harold Katcher, and Izpisua Belmonte) are pursuing the top-down approach to rejuvenation, based on a paradigm in which aging is coordinated systemically, and not controlled at the cellular level. Interventions are in the bloodstream, through which biochemical signals transmit information about the age state of the body. 

Sinclair repeats without qualification his prejudice that “aging is a loss of epigenetic information.” He cites his previous experiments as support for “the idea that a loss of epigenetic information, resulting in changes in gene expression, leads to the loss of cellular identity.” But we have learned that methylation changes and other epigenetic changes with age are a redirection as much as a loss of information. Some of the changes are predictable and programmed, and, indeed, the idea of a methylation clock depends on the regularity of methylation changes. 

For the future

I like Sinclair’s idea for high-throughput screening of rejuvenation molecules. I would add that combinations should be tried after the first step of identifying promising single candidates. The example of OSKM demonstrates that combinations are able to achieve what no single chemical can do alone. In the discussion section, Sinclair indicates that his lab is already doing this, and that the work is guided by an investigation of the mechanisms of action of the chemicals that his screening procedure discovered. 

The present work was done with human fibroblasts (cells from connective tissue) and the discussion section mentions that ongoing work to repeat the test with cells from other tissues. The screening procedure requires the extensive laboratory resources that Sinclair is able to command. 

A consistent theme in my work is that aging is evolved, programmed, and centrally coordinated. I’ve cited diverse evidence for this, based on observation, not theory. As a paradigm to guide anti-aging research, there is an additional reason to research central control rather than cell-autonomous interventions. That is that engineering the rejuvenation of every cell in the body is a daunting task. In contrast, if aging is centrally controlled and communicated via biochemical signaling, we can expect that modification of the signaling environment ought to be far more accessible goal.