iTR (Induced Tissue Regeneration)

Storm Clouds & Partial Reprogramming In this short video clip (pasted below), Shinya Yamanaka discusses the problem of current partial reprogramming protocols and the risk of over-reprogramming leading to teratoma formation. It’s probably worse than that because there are a host of currently uncharacterized off-target effects. Now for the good news… by studying transitions downstream of pluripotency one can find clockwork genes that reverse the developmental aging of cells and induce regeneration. I believe many people like David Sinclair are in agreement about the need to further advance the tech so long-turn reprogramming can be administered to induce large-scale reprogramming such as to regenerate brain after stroke and myocardium after MI. These will likely require weeks to months continuously in the reprogrammed state, something difficult to achieve with current partial reprogramming formulations. I will discuss that new third-generation technology in coming days, weeks, and months. Comments and debate are welcomed… youtu.be/P_viLHGGFNc?is=bPOk… @jpsenescence @davidasinclair @aubreydegrey @PeterDiamandis

A fun conversation with Tim Ventura about telomeres, age reprogramming, and the prospect of life extension on YouTube. Here is the link: youtu.be/8-GLwqJ1R-I?is=BrHQ…

Salamander Man! That is what one biotech analyst whispered when I described our experiments in reversing developmental aging to turn on the regeneration switches. As I mentioned in my previous post, early in human development we have that “magical” capacity to simply re-grow essentially any tissue is it is damaged. The Mexican salamander is one example in the animal kingdom of what is called “heterochrony,” but there are others. These animals have not switched off that embryonic regenerative trait even as adults. So, the question is, how can we turn it on again to regenerate human tissues? Such a therapy would address upwards of 80% of all health care expenditures in the U.S.   To find the switches (genes), we need a laboratory model. In other words, we need to be able to observe the switches being turned on and off in the lab so we can track down the clockwork mechanisms. The approach we used in the past was to isolated hundreds of diverse cell types from pluripotent stem cells that were still in the regenerative state and compare them with hundreds of diverse cell types derived from non-regenerative adult tissues. Then the interrogation of the cells to discover the important genes is pretty straightforward. We started this work about 10 years ago in collaboration with my friend Alex Zhavoronkov who in those days had a new company called Insilico Medicine (the very early days of AI). We published some of our discoveries way back then (paper linked below).   In the years that followed, we dug deeper and deeper into the biology of regeneration to put the puzzle pieces together. I believe we can safely say most of the important pieces of the puzzle are now assembled and a clear picture has emerged. Approaches like OSKM (global reprogramming, while having off-target effects, can occasionally reset the regeneration switches to the “on” position. But a thorough more precise targeting is possible. It seems clear that partial reprogramming does not activate telomerase (at least in human cells), so it doesn’t really reverse the aging of cells. However, when used in combination with telomerase, a true age-reversal seems feasible. Animals that have telomerase and epimorphic regeneration often do not age. It will be exciting to see what effects these 3rd-generation technologies have on the human aging process. pubmed.ncbi.nlm.nih.gov/2948…

In the previous post I briefly described the 2010 paper that showed it was possible to reverse developmental aging in human cells, reset the telomere clock, and make the all-powerful stem cells from which any young human cell type could be made identical to the patient. This would make a parts supply store available capable of delivering any of our own young cells useful in repairing the body during aging - the same way one can keep a vintage car on the road by periodically replacing the tires or brake linings (i.e. Regenerative Medicine). But the real goal was more profound in scope; could we apply the technology to whole human beings to reverse aging?   Of course, the goal wouldn’t be to reprogram the cells in a human all the way back. We would, after all, turn into a heap of formless stem cells. How about part-way back? This came to be called, “partial reprogramming.” One would have to be careful, of course, because if any of the cells in a human were overly-reprogrammed, it is possible the patient would break out with what are called “teratomas.” They sound terrible and in a sense they are. While benign tumors, they are masses of disorganized teeth, hair, brain, and other organs. Who wants one of those on their nose!   Now, here I need to digress a bit and explain things… Early in our development at the transition point between the embryo and fetal stage, tissues in our bodies can almost “magically” regenerate. You may have seen pictures of the Mexican salamander that can re-grow an amputated limb as well as brain, heart, etc (see video below). That characteristic is called “epimorphic regeneration” and we have it, it’s just that it gets turned off after the first stages of the development of our body is completed. In my opinion, the enormous promise of partial reprogramming would be to induce that tissue regeneration the Mexican salamander enjoys. Indeed, we believe that there are molecular switches that partial reprogramming flip on the trip back in time and these can re-awaken this profound regeneration. Since 80% of health care costs in the U.S. is attributed to chronic disease like the degenerative diseases of aging, a technology that could effectively induce Tissue Regeneration (iTR) could be insanely profitable.   However, it is challenging to apply the first generation genes like OSKM (what I call “global reprogramming,”) in medicine. Long-term application runs that risk I mentioned of forming teratomas. But long-term application is exactly what we need for many of the degenerative conditions of old age. Take the example of the heart. To regenerate a large amount of myocardium after a heart attack, even a small heart attack affecting approximately 5% of a ventricle would, I estimate, take 1-3 months to fully regenerate. That’s way in the danger zone, in my opinion, for risk of teratomas or other undesired complications.   The good news is that today’s molecular technologies are orders of magnitude more powerful than they were just a couple of decades ago. Using them and modern computing it is relatively easy to pin down precisely how to perform precision (segmental) age-reprogramming. The results could, and almost certainly will change the world as we know it. As Richard Goss wrote in his book “Principles of Regeneration: “If there were no regeneration there would be no life. If everything regenerated there would be no death.” youtu.be/yhN0Qy24lFE?si=4sbc…

In 1997 our work on isolating human embryonic stem cells was well underway. That year, Keith Campbell published in Nature the cloning of Dolly the sheep. This told us that developmental aging was reversible. Cells from an aged mammal (in this case, breast epithelial cells (she was named after Dolly Parton)) were injected into an egg cell stripped of its own DNA. The result was that the breast cell DNA was reprogrammed as if in a time machine, back to the embryonic state so that when it was implanted back into the womb, it could become an identical copy of the original animal the breast cell was taken from.   The burning question was “What in the egg cell could drive this remarkable reversal of developmental aging?” I had to find out. I resigned from Geron and flew to Scotland to meet with Ian Wilmut who was handling the patents associated with Dolly. He suggested we meet in Australia for a stem cell/cloning meeting. At the meeting in Australia I learned of a U.S. cloning company called Advanced Cell Technology (ACT) being managed, believe it or not, by Abel Ponce de Leon. I loved their patents so much I bought the company. At ACT I and my colleagues Jose Cibelli and Robert Lanza and Vince Cristofalo who had taken Hayflick’s seat at the Wistar Institute demonstrated that mammalian telomere length could also be reset by cloning (Science, 2000). Shortly thereafter we reported the first cloned human embryos in an attempt to reverse the developmental aging of human cells. About that time, I was at a birthday party in Boston chatting with the transcription factor expert Mark Ptashne. After a few glasses of wine I asked him how many transcription factors he thought were present in the egg cell that could reverse aging. He thought a minute, then held up four fingers. Odd, I thought, I had seen four genes highly expressed in ES cells (OCT4, SOX2, LIN28A, and NANOG). I decided to file a patent on those genes and Shinya Yamanaka filed on the well-known cocktail of OCT4, SOX2, KLF4, and MYC. This led to the field of induced Pluripotent Stem (iPS) cells where adult cells can be transported back in time to an ES-like state.   In 2010 in collaboration with Homayoun Vaziri, we published a paper titled “Spontaneous Reversal of Developmental Aging of Normal Human Cells Following Transcriptional Reprogramming.” In the image pasted below, you can see the progressive increase in telomere length reported in that paper as a result of taking the cells in this “time machine” as well as my own skin cells going for the ride back in time. I must admit, it gave me goosebumps to see the result, to see my own cells age reversed and my own beating heart cells identical to the ones I had when I was still in my mother’s womb. Think about the implications for human age reversal… I showed the data to Len Hayflick. I asked him if he ever thought he would see a day when we could do this. His reply was, “Not only did I not think I would ever see that day, I thought mankind would never see that day!” Yet, here we are…

Going back to the immortality of germ line cells as described by August Weismann, what if we could capture those cells in the lab dish? Sperm and egg cells don’t replicate and besides they have halved their DNA content. But there are cells called “embryonic germ cells” that contain the entire genome and they DO replicate. In 1995 I tried to isolate human embryonic germ cells from developing testes and ovaries, but the experiments failed. I needed to get them from earlier stages in their development. Another idea was to try to isolate them from the cluster of cells just after the egg cell is fertilized by sperm. Those cells had been isolated in mice and were called “embryonic stem cells (ES cells). They were “all-powerful” in that they could be turned into entire mice! I conjectured that if we could isolate them from the human species, not only could they revolutionize medicine by providing a parts supply store of every cell type in the human body, but they would also be telomerase+ and the only naturally immortal human cell ever isolated. It would enable the endless industrial-scale manufacture of young cells of any kind genetically modified in any way from just a single cell line. There would be no need to continually source new lines. I set up a collaboration with Roger Pedersen at UCSF, Jamie Thomson at UW Madison, and John Gearhart at John’s Hopkins to isolate the cells. Bill Haseltine on hearing the idea in a meeting remarked “This field needs a name… Regenerative Medicine!” The name stuck and the cells became well-known after being published and became the subject of national debate after President George W. Bush devoted his first national address on federal funding for the research to turn them into life-saving therapies. His broadcast given October 9, 2001 is linked below. Over a thousand patients have since been treated with cell-based therapies derived from pluripotent stem cells and the field is expanding year over year especially for their application in age-related degenerative diseases such as Parkinson’s and age-related macular degeneration. Someday, if mankind does not destroy itself in war, we may live in a world where we can access young cells to repair many tissues through the body in the same manner that we keep old cars on the road using a full catalog of available replacement parts. youtu.be/EAiZp5jT04I?is=tpij…

I’ve been discussing, in a series of posts, the history leading up to the modern technology allowing the reprogramming of aging. After my PhD at Baylor College of Medicine I entered Medical School in Dallas. I had turned a family company called West Leasing Company into biotech enterprise I called Geron Corporation. In 1992, my third year as a student, I was approached by the Venture firm Kleiner Perkins who offered to finance the gamble that telomerase (if we could find the gene) would enable the extension (or even immortalization) of human cells and its inhibition could treat cancer. Some $30M later we still hadn’t found the gene. Remember, these were the days before DNA was fully sequenced. We were collaborating with Carol Greider at Cold Spring Harbor and Jim Watson and my board of directors were becoming impatient. Carol’s candidate gene in Tetrahymena went nowhere… Finally, Tom Cech’s lab told us of a candidate gene in Euplotes. We looked in our cDNA library and found a hit! In a hectic race to get the entire sequence and patent it, the scientists at Geron worked round the clock, sleeping in a trailer in the back of the building. To test if we had the real thing, I assembled, as I recall, five mortal cell RNAs and five immortal counterparts (four being cancer lines one being from the testicle (germ line). I can’t describe the electricity in the air. What a gamble we had bet on the telomere hypothesis and the search for the gene. The scientists didn’t know the order of the samples I gave them in order to blind the experiment, but I placed them in a logical order: five mortal cell RNAs, four cancer lines, then testis at the end. As the image slowly assembled on the phosphoimager, the sample on the end became visible, then with a collective gasp, we saw five blanks in the mortal and five positives on the right with the last one being the strongest (exactly what one would expect if we finally had the gene).   We then raced to express the gene in human mortal cells (cells from Len Hayflick’s leg being the first) and later a nice study published in Science in collaboration with Woody Wright that showed that indeed, telomeres were the clock behind the Hayflick limit and telomerase alone is capable of immortalizing cells.   This result overturned the long-held belief that the aging of cells was simply entropy at work, wear-and-tear, something we would never completely understand, and certainly something that we could never effectively stop. But for those of us that favored the programmed theory of aging, it was consistent with a mutation of a single letter in the DNA code leading to the premature aging seen in children with progeria and the common immortalization of cells in cancer.   Telomerase was never tested in humans because Geron became focused on telomerase inhibition for the treatment of cancer and in the stem cell program I will describe next. Hopefully in coming years, it will become part of a cocktail of agents that I will describe in future posts that leads to significant extension of human healthspan and lifespan. Below I post a picture of that team of scientists that worked so very hard to be the first to isolate the immortalizing gene telomerase.

Continuing with part 3/10 of the trail that led to developmental age reversal, I began my doctoral research in 1982 with Samuel Goldstein, MD, a brilliant aging researcher being the first to use the then emerging field of recombinant DNA research to study aging using the Hayflick model of fibroblast senescence. Sam had an uncanny instinct that it was changes in repetitive DNA sequences or DNA methylation that was a root cause. Unfortunately, I ended up disproving his published results on the loss of EcoR1 repeats and Inter-Alu (the latter being a provocative paper published in Nature and subsequently retracted).   Not in alignment with Sam on how to handle this conflict, I decided to start my PhD afresh at Baylor College of Medicine and wrote a dissertation on my discovery that senescent cells secreted destructive proteolytic enzymes that tear down tissues faster than they could be regenerated (what Judy Campisi later called the Senescence-Associated Secretory Phenotype (SASP)).   Then later as a medical student at UTSW in Dallas, I stumbled on a paper by a 1986 paper published by Howard Cooke titled “Variability at the Telomeres of the Human X/Y Pseudoautosomal Region” where he showed the age-dependent shortening of telomeres in somatic cells but maintenance at a long length in germ line cells. This fit the Weismann model exactly. This, together with work I was doing with Woody Wright on cells in extreme senescence (Mortality 2) where chromosomes began joining end-on-end, the data convinced me that a wild-eyed theoretical scientist named Alexey Olovnikov was right. The clock of cell aging was the loss of DNA at the ends of the chromosomes and an unidentified protein then called “telomere terminal transferase” (now called “telomerase” for short), and the basis of cell immortality. So, Sam Goldstein instinct was right, I surmised, it was repetitive DNA loss, just not the sequences he was studying. Below is a video where I asked Alexey in St Petersburg to recount how he dreamed up the hypothesis. This theory turned out to be true as we demonstrated once we had found the telomerase gene as I will relate in the next post. youtu.be/w_uS-kmHvwo?si=3LU3…

I was asked to clarify the history of the first persons’ cells to be immortalized by telomerase. Here’s what happened… About the time we had finally found the telomerase gene, Len Hayflick was visiting the lab and gave a film crew an illustration of how to culture skin cells by hoisting up a pant leg, grabbing a scalpel, and slicing off a piece of skin from his leg! Later he walked into my office and asked, “you want a piece of me?” I said, “yeah, let’s measure the true Hayflick Limit (how many doublings his cells would go before becoming senescent. I cultured his cells and was feeding them at the end of a days work and thought, history should have it that the first cells ever immortalized by human intervention should be Len’s own skin cells. So I took the newly-isolated telomerase gene that by then had been placed into a viral vector, and squirted it into the dish with his cells. I remember in my notebook I titled the experiment “The Immortalization of Dr Hayflick.” Weeks later when it became clear that it had worked, I had dinner with Len and his wife Ruth in a restaurant in Sonoma County on the sea coast. “I said, Len, about that experiment…” He said, “Yeah, what happened with that?” I explained how his untreated cells had senesced but his treated cells were growing exponentially and had clearly immortalized.” He stared out the window for the longest time then whispered “I can’t be that simple!” I tell the story because it’s a good example of how at first glance we think aging must be wear and tear and be very complicated (Len clearly thought that way). But the reality is that a single letter in the DNA code can cause premature aging in children with progeria, and a single gene (telomerase) can rescue cells from senescence. In upcoming posts I will explain how we figured out the other pieces of the aging puzzle, and like telomerase, it is far simpler that most people would have imagined.

In the previous post I discussed how August Weismann proposed that we are all are derived from a an immortal and regenerative lineage of cells called the “germ line.” He predicted that this amazing potential was lost when are bodies are being formed in the womb (something I call “somatic restriction”). Unfortunately, he was too far ahead of his time. About two years before Weismann’s death, Alexis Carrel (the first American to win the Nobel Prize authored a paper titled "On the Permanent Life of Tissues outside of the Organism.” Carrel’s paper convinced the scientific community that however we age, it was not an intrinsic clock within our cells. Charles Lindbergh, the famed aviator claimed that in his first trans-Atlantic flight, invisible beings in the back of his plane told him his destiny was to engineer human immortality. He sought out Carrel and together their theories on aging held sway until the 1960s when Leonard Hayflick overturned the prevailing dogma by demonstrating that body cells actually do age and that cell aging in the dish could serve as a lab model for deciphering the clockwork mechanisms of aging. That is the era I entered the field in search for the key to cell immortality. This was a first step toward human age reversal. I describe some of the drama of those early years in the YouTube video linked below. youtu.be/s7l6d1BclNQ?si=eQ8Q…

In this, the first of 10 posts on age reversal by genomic reprogramming, I begin where the story really needs to begin; namely, the insights of the German naturalist August Weismann. Weismann proposed the term “immortality” to describe the unceasing lineage of cells called the “germ line” that perpetuates the species (that is sperm and egg that make babies that make sperm and egg that make babies, ad infinitum…). Weismann theorized that cells that make up the body (somatic cells) lost that capacity for immortal regeneration, a phenomenon I call “somatic restriction.” He stated “Death takes place because a worn-out tissue cannot forever renew itself, and because a capacity for increase by means of cell division is not everlasting, but finite [in the soma].” If you think about it, this means that the cells that we are made of (trillions of them) have no dead ancestors. Tracing them back in time, the ancestors of the cells that make us “us” have been proliferating since the first dawn of life on Earth, some billions of years ago. They will abruptly die in a few decades in us. The question we began to ask nearly a century after Weismann was, “can we transfer the capacity of immortal renewal characteristic of the germ line cells into our body, into the soma.” In the next post I will describe the first of such steps that led to the modern technology of immortality transfer.

Over the next week I will share in a series of posts some insights and data on the reversal of human aging by transcriptional reprogramming (aka “partial reprogramming,” “epigenetic reprogramming of aging,” or “induced Tissue Regeneration (iTR).” Some of the critical aspects of the technology that need to be addressed are the fine tuning of the process to insure cells in a patient are adequately but not overly age-reversed, how telomere length (i.e. cellular age) can also be reversed, and how to prevent the current plurality of off-target effects of the first generation technology that carry potential risks of cancer. Stay tuned…