In Pursuit of Memory

by Joseph Jebelli

  A member of her team showed me some reprogrammed cells under the microscope. They looked like neurons. They behaved like neurons. They even formed synaptic contacts with each other–like neurons. It was surreal. Here, before my eyes, was a hand-made piece of someone’s brain–the innermost recesses of a person’s thoughts and feelings transposed onto a dish. Even after making them for seven years, Wray herself feels the same. ‘When Yamanaka’s discovery was out I remember thinking that it sounded a bit crazy,’ she admitted. ‘This idea that you could have a patient in clinic and then do all this amazing stuff just by taking a bit of their skin. I still sometimes look at them and think, wow, how is this possible?’

  Each morning, Wray washes and feeds vast stockpiles of patient stem cells. If she’s lucky, most will transform into neurons. But they’re extremely sensitive. Indeed, every detail in the lab is designed to give them sanctuary: the incubator is set to body temperature; the fridge stocks gallons of nutrient broth; the surfaces are soaked with sterilising ethanol. On the floor sits a large yellow bin marked ‘Biohazard’, a final resting place for skin that didn’t make the transformation.

  Wray doesn’t know which of her iPS cells are Victoria’s; each line is strictly anonymised. She only knows some must be hers because Victoria stood up and announced her donation–with characteristic zeal–during a patient support meeting some years earlier.

  Researchers are still debating how to use these neurons, but Wray’s using them as a model to better understand the disease. Being this close and personal allows her to use a toolbox of molecular ‘scalpels’ to unveil their routine, everyday functions. By picking away at the cells’ inner workings, she’s able to learn how they become derailed and ultimately fatal. According to Wray, the flaws of other experimental models also define the power of iPS cells. ‘The biggest disadvantage of animal models,’ she explained, ‘is that they don’t have Alzheimer’s. A mouse does not have Alzheimer’s disease. Its brain just isn’t similar enough to ours to call it that. But with iPS cells we’ve got the correct species, which is human, the correct cell type, which is neurons, and the correct levels of the genes we know are involved in the disease. We’re mimicking the disease in real time.’ But Wray has competition.

  In August 2014 Doo Yeon Kim, a leading Alzheimer’s researcher at Harvard University, made an embryonic stem cell culture that replicates the disease so well his model became known as Alzheimer’s-in-a-dish.9 By growing cells in a gel Kim was able to create a three-dimensional culture that looked like ‘mini-spherical brains’, according to one of his colleagues. Although not patient cells, these cultured neurons produced full-blown plaques and tangles in the dish, something iPS cells have yet to achieve. In fact, it was something all other culture models had yet to realise–beta-amyloid would just disperse in the dish, like dust, and tau never quite formed tangles. It was like trying to reproduce a Mexican gunfight without any weapons. It was described as the Achilles heel of the field. But with Kim’s model, scientists could finally begin to determine what actually linked plaques to tangles on a molecular level. Moreover it offered a platform to screen hundreds of thousands of drugs in a matter of months.

  But Wray isn’t fazed. ‘I think both models actually complement each other,’ she said. ‘It’s just about choosing the model most appropriate to the scientific question you’re asking.’

  So what was Victoria’s PSEN1 mutation telling scientists like Wray? ‘Everything we’re seeing makes a strong case to support the amyloid cascade hypothesis,’ she said. ‘But I think we have to target both amyloid and tau, because once the disease starts I think tau takes over. And why not do both: block disease initiation but still assume some tangles have already formed?’

  And what about transplantation? Did she think it would eventually be possible to make new neurons in the dish and then reinsert those into a dementing brain? ‘I think never say never, because even the development of this technology took everyone by surprise. But I’m inclined to think that that’s not where their strength lies. When you look at an Alzheimer’s brain post-mortem the loss of cells is so vast and widespread. So to take these cells and put them back and then expect them to integrate into the kind of brain circuitry that was there before… it’s too big an ask, I think. At the moment anyway.’

  That ‘ask’ pivots on finding a solution to two problems. The first is a structural problem. Born from the outer skin of the embryo, the brain undergoes a series of complex steps to reach maturity, unfolding in an exquisitely delicate process of timing and precision. Because Alzheimer’s undoes this so ruthlessly, iPS cell technology may have to reach a stage where scientists can grow whole sections of brain–replete with blood vessels, myelin and cerebrospinal fluid–to achieve meaningful results upon transplantation. (It’s why Kim’s mini-spherical brains are so encouraging.) The second problem is one of integration. The brain takes a long time to grow. Many of the genes responsible for setting it up are only activated during key windows of development in the first few years of life; conversely, certain genes lie dormant until adulthood. So unless scientists can engineer iPS cells to better reflect such molecular fine-tuning, transplanting them could be, as a professor once put it, like asking an amateur pianist to play Chopin’s last ballade.

  Nevertheless, there are hints that transplantation might work. In 2014 Penelope Hallett of Harvard University did a post-mortem on patients who, fourteen years earlier, had received stem cell grafts for Parkinson’s disease (another affliction involving catastrophic neuronal loss). Amazingly, Hallett found that not only did the grafts remain healthy and intact, they matured and integrated in the host brain as well.10 A year later, she went further, exploring how implantation might actually work in practice. The procedure is known as autogenic stem cell transplantation (auto-SCT): a technique where an injection of iPS cells physically engrafts the cells into the required bodily location. And the reality isn’t far off. Using primate models of Parkinson’s, Hallett’s team has shown that auto-SCT of iPS cells taken from the skin can partially rebuild the substantia nigra, the brain region destroyed by Parkinson’s disease. This improved the animals’ movement symptoms for up to two years.

  Other data suggests that stem cells needn’t engraft at all. Rather, their mere presence may help by providing restorative, ‘neurotrophic’ support to diseased brain regions. That was illustrated in 2009 at the University of California, Irvine, when Frank LaFerla injected stem cells into the brain of Alzheimer’s transgenic mice.11 Within a month the animals’ memory improved. And yet, the stem cells didn’t turn into neurons. Or have any impact on plaques and tangles. Instead, they were quietly churning out the protein BDNF (brain-derived neurotrophic factor), which alone was enough to boost synapse density in the hippocampus by 67 per cent.

  Still, Wray’s concerns are echoed by many in the field. On 6 November 2014 stem cell biologists from all over the world amassed in Durham, North Carolina, for a conference titled, ‘Accelerating the cure for Alzheimer’s disease through regenerative medicine’. The topic under discussion was whether stem cells–both iPS and embryonic–are ready to enter clinical trials as transplants. The audience included delegates from biotech and Big Pharma, as well as senior academics. And feelings were mixed. Some said the technology was still a long way from being safe, let alone effective. Only two months had passed since Masayo Takahashi, an ophthalmologist in Kobe, Japan, had to abandon her efforts to implant iPS-derived retinal cells into the right eye of a woman with macular degeneration, when Yamanaka spotted two potentially carcinogenic mutations in her batch; giving this woman cancer in an attempt to correct her sight would have been disastrous. There were more fundamental uncertainties regarding Alzheimer’s. It still wasn’t known where to deliver the cells, for instance, or how many were needed, and how often. And would they have any impact on plaques and tangles? No one knew.

  But others stressed that clinical trials inform research just as much as research informs clinical trials; indeed, scientists could spend year
s mulling over uncertainties and still have the same basic concerns. And so phase one clinical trials are now being discussed, marking the first, tentative steps in a new era of brain regeneration.

  This is all too late for Victoria, of course. But she’s accepted that with remarkable resilience and unwavering altruism. ‘I know what’s going to happen… but there’s nothing I can… do… about it…’ Eight months had passed since my first visit and she was unmistakably worse: less animated, more introverted, she strained to vocalise her thoughts. ‘So I just hope… they find something… for somebody else.’ Her memory had taken a nose-dive, said Martin. She now forgot what she was doing from one moment to the next. Times, dates and day-to-day norms like shopping or going for walks were fast becoming incomprehensible. One day, Martin found that she’d unwittingly put cutlery in the bin; on another, he observed her hysterically searching for her phone while holding it in her hand all along. Martin, now a bulwark of patience and fortitude, has adapted; he’s ‘just been getting on with it’, he said.

  Given Victoria’s condition, I was amazed to hear that she’d recently resumed her old job. Every few weeks she’s taken to see an old friend, called Iris, who suffers from Alzheimer’s herself. Victoria met Iris more than a decade ago, when she worked as a carer. She’d nursed Iris’s daughter, who had Down’s syndrome, and was now doing what little she could to see Iris, a ninety-one-year-old, through the final stages of a fate she knows awaits her too. She told me that she was bored at home; that she wanted to keep her mind active. ‘I’ve always been in the caring game,’ she said proudly, ‘but… of course… I’m know I can’t make… any mistakes… and I…’ She trailed off and Martin filled in the blanks. For him, the undertaking was not so much to keep Victoria occupied as it was to give her some company while he’s at work. His intuition is well founded: studies show that staying socially active can relieve anxiety and depression in dementia.

  Wanting to offer something, anything, I relayed some of the dazzling work being done using her cells in the lab–impossible were it not for Victoria’s contribution. I still found it hard to digest the knowledge that while Victoria’s mind slowly deconstructs, it was quietly being reconstructed under the nose of Wray and other scientists, creating a portal to somewhere no brain scan can go. It’s hard to say when expectation will meet reality for iPS cells. There is a huge element of luck in biological research. Take Louis Pasteur, the French pioneer of vaccination. His discovery of the chicken cholera vaccine only occurred when he abandoned the experiment out of frustration and took a vacation, returning to discover that leaving the broth was precisely what was necessary to ‘attenuate’, or weaken, the bacteria enough for it to become a vaccine. This kind of thing happens all the time in modern laboratories. It comes from the sheer lawlessness of biology, the ‘most lawless of the three basic sciences’, wrote cancer biologist Siddhartha Mukherjee. ‘There are few rules to begin with, and even fewer rules that are universal.’12

  Nowhere else is this truer than in cell biology. We can imagine cells as microcosms of modern megacities–dynamic, ever-changing entities, constantly generating new and innovative trends in behaviour. With iPS cells, it’s like finding a city on another planet. And so for the time being, the nitty-gritty of how they actually work is a total enigma. Scientists are basically making it up as they go along.

  Nevertheless, some exciting practical developments are emerging. In the decade since Yamanaka unveiled them, iPS cells have been made from hair follicles and even urine. Researchers have envisioned iPS cell ‘banks’, stockpiling varieties that would be compatible for the wider population. This has piqued the interest of the US Department of Defense, which is now funding research to create self-renewing banks of red blood cells for injured soldiers in combat; according to one estimate, only forty donors of varying blood types would be needed to supply the general population indefinitely.13 Outbreaks of tropical infections such as Zika are also reaping the benefits. Researchers at Johns Hopkins University in Baltimore, Maryland, recently used iPS cells to determine how the Zika infection in pregnant women may cause microcephaly, a birth defect in which a baby’s head is smaller than usual.14 In the future we can imagine even more far-reaching applications. As one expert told Nature’s Megan Scudellari in June 2016: ‘The world is watching…’15

  For evidence that even more incredible feats of regeneration are being pursued, one need look no further than the morbid and fascinating events that unfolded in California in the spring of 2012.

  15

  Young Blood

  And so we remained till the red of the dawn began to fall through the snow gloom. I was desolate and afraid, and full of woe and terror. But when that beautiful sun began to climb the horizon life was to me again.

  Bram Stoker, Dracula

  IN 1956, HALF a century before Yamanaka’s work galvanised the concept of regenerative medicine, a gerontologist at Cornell University called Clive McCay performed a ghoulish experiment. Using small scissors and surgical sutures, he stitched together pairs of rats–one young and one old–making their circulatory systems unite.1 It was called parabiosis (from the Greek para, meaning ‘alongside’, and bios, for ‘life’), and was done to investigate ‘the possibility,’ he wrote, ‘of reversing the pathological changes in an old animal by bathing its tissue in the blood of a young one’.

  McCay’s work extended that of the German alchemist Andreas Libavius, who in 1615 proposed joining the arteries of an old man to those of a young one. While it was never determined if Libavius actually carried out the experiment, he evidently had strong convictions about its efficacy, concluding that ‘the hot and spirituous blood of the young man will pour into the old one as if it were from a fountain of youth, and all of his weakness will be dispelled’.2

  French zoologist Paul Bert was inspired by Libavius, and in 1864, using rats, he published a radical study–‘Expériences et considérations sur la greffe animale’–that became the earliest recorded parabiosis experiment.3 It won him an award from the French Academy of Sciences. Bert’s aim, though, was principally to demonstrate the method’s feasibility; he showed that veins formed between the two rats, and that fluid injected into one animal passed into the vein of the other.

  But McCay had the same inclinations as Libavius, and was obsessed with unlocking the biological mechanisms of ageing and longevity. Since antiquity, ageing had been attributed to the decline of a mysterious factor the Greeks called ‘innate heat’, which, when extinguished, left the body cold and dry. And for millennia there have been tales of a fabled ‘Fountain of Youth’, which gives everlasting youth to those who drink from its waters. Throughout history, there have also been rumours that the mysterious elixir of youth resides in young blood. The Roman emperor Constantine was reputedly advised by pagan priests to bathe in the blood of children to cure his leprosy. When children went missing in Paris in the 1700s, it was thought that King Louis XV was bathing in their blood. It was even rumoured that the late North Korean dictator Kim Jong-Il tried to slow down his own ageing by injecting himself with the blood of healthy young virgins.

  These gruesome, extraordinary tales just might have a rational basis despite the horrifying crimes some of them suggest. In his experiments, McCay found that the constant exchange of blood between old and young rats made the bones of the older animals similar in strength to those of their young partners. Something in the blood was rejuvenating them, but what was it? And what else might it revive? For reasons that aren’t entirely clear, though perhaps due to its dark implications, the work was scarcely followed up. It wasn’t until recently that two US universities started to provide answers.

  The first was a Harvard University group led by Amy Wagers, an ambitious stem cell biologist with dusty blonde hair and grey-blue eyes. Wagers resurrected parabiosis while training under Irving Weissman at Stanford University in the early 2000s. Weissman had spent decades studying examples of parabiosis in nature and was fascinated by a sea-dwelling creature called the Star
Ascidian (which reproduces as a bud, grows off its parent, and remains joined to it until the parent eventually dies and is reabsorbed by the offspring). When Wagers expressed an interest in exploring the movement of circulating blood stem cells, Weissman advised her to use parabiotic mice. She pursued the work at Harvard, where she established her own laboratory in May 2004.

  Back at Stanford, word spread of the spooky method Wagers was employing for her experiments, and it wasn’t long before researchers interested in the biology of ageing, led by a neurologist called Thomas Rando, asked her to work alongside them. Wagers agreed, flew back to Stanford, and in 2005 the team discovered that joining young and old mice rejuvenates muscle and liver tissue in older animals.4

  At Harvard, Wagers then went on to show that young blood could even rejuvenate the heart and spinal cord. It was flabbergasting; all the ancient folklore about the merits of young blood suddenly had scientific endorsement. It sparked a media sensation. One headline read: VAMPIRE THERAPY: YOUNG BLOOD MAY REVERSE AGEING.5