The train line from mainland Kobe is a marvel of urban transportation. Opened in 1981, Japan’s first driverless, fully automated train pulls out of Sannomiya station, guided smoothly along elevated tracks that stand precariously over the bustling city streets below, across the bay to the Port Island.
The island, and much of the city, was razed to the ground in the Great Hanshin Earthquake of 1995 – which killed more than 5,000 people and destroyed more than 100,000 of Kobe’s buildings – and built anew in subsequent years. As the train proceeds, the landscape fills with skyscrapers. The Rokkō mountains come into view, looming menacingly over the city, peppered with smoke billowing from the dozens of narrow chimneys of the electronics, steel and shipbuilding factories.
Today, as well as housing the Port of Kobe, the man-made island contains hotels, medical centres, universities, a large convention centre and an Ikea store. There are also three government-funded RIKEN research institutions: the Advanced Institute of Computational Science (which is home to what was, until 2011, the world’s fastest supercomputer), the Center for Life Science Technologies, and the Centre for Developmental Biology (CDB).
At the entrance to one of the labs, a faded poster in a thin plastic frame shows the crew of the Starship Enterprise, a young Captain Kirk sitting proudly at the helm. Underneath is the famous Star Trek slogan: “To boldly go where no man has gone before.”
On the other side of the door, scientists in the Laboratory for Organogenesis and Neurogenesis are working on something that has fired the imagination of science fiction authors for many years. They are at the cutting edge of an emerging field: rebuilding the body by growing tissues and organs from stem cells. They hope to develop the next generation of therapies for a variety of debilitating human diseases, and unravel the mysteries of brain development.
Not long after fertilisation, the embryo consists of a tiny sphere of identical, non-specialised cells, referred to as pluripotent stem cells. These have the ability to stay in this state indefinitely, while dividing to produce daughter cells that are capable of turning into any cell type found in the adult body. These embryonic stem cells offered hope for researchers trying to develop disease treatments, but the fact that they could only be obtained from human embryos raised serious ethical questions about their use.
Then, in 2007, a team led by Shinya Yamanaka of Kyoto University demonstrated that connective tissue cells from adult rats could be made to revert to a pluripotent, stem cell-like state and reprogrammed to form different cell types. Others went on to show that cells taken from just about anywhere in the human body can be similarly reprogrammed, into just about any other type of cell.
By 2008, US researchers had taken skin cells from an 82-year-old woman with amyotrophic lateral sclerosis (ALS, a form of motor neuron disease), placed them into petri dishes and reprogrammed them to form the same motor neurons that are destroyed by the disease. By 2010, researchers at Stanford had shown that mouse connective tissue cells could be reprogrammed directly into neurons, bypassing the pluripotent state.
These advances provided a new – and less controversial – way of obtaining human embryonic stem cells. Researchers could grow them in the lab and reprogramme them however they wished, to study the molecular and cellular mechanisms of diseases and to test the effects of newly developed drugs. They also made possible a milestone in regenerative medicine: the first successful transplant of an organ grown entirely from man-made tissue.
The recipient was Andemariam Teklesenbet Beyene, a 36-year-old Eritrean man who was studying for a Master’s in geophysics at the University of Iceland. During his studies, Beyene was diagnosed with advanced cancer, then developed a golf ball-sized tumour that blocked his windpipe. He initially refused the revolutionary treatment that was offered to him, but he agreed after consulting with his doctor in Iceland and his family.
The treatment required the coordinated activity of three teams, each in a different part of the world. First, computerised tomography scans of Beyene’s windpipe were sent to researchers at UCL. They used the scans to build a Y-shaped glass mould, which was coated with a nanocomposite polymer to form a porous scaffold. This scaffold was sent to the USA and Harvard Bioscience, who ‘seeded’ the scaffold with stem cells taken from Beyene’s bone marrow, then incubated it in a specially designed bioreactor for several days; this allowed the cells to infiltrate the pores in the scaffold and to differentiate to form connective tissue. Finally, the scaffold was sent to the Karolinska Institute in Stockholm, where the 12-hour transplant operation took place.
Paolo Macchiarini, a surgeon at the Karolinska Institute, and his colleagues successfully transplanted the first completely synthetic windpipe in June 2011. Beyene remained weak and bedridden for several weeks after the procedure, but he eventually recovered and graduated from university about eight months later.
Windpipe transplants had been performed before but had all involved real windpipes from human donors, stripped down to the cartilage and repopulated with the recipients’ stem cells. Finding a suitable donor can take months, so the use of an artificial scaffold dramatically shortens the time needed. For Beyene, this was life-saving. (It also overcomes another obstacle: because Beyene’s new windpipe is completely synthetic, his body is far less likely to reject it, so he doesn’t need to take the powerful immune-suppressant drugs that other transplant patients take to stop this from happening.)
Tens of thousands of people worldwide are waiting for organ transplants, but there aren’t enough organs to go around – last year, more than 5,600 people were on the waiting list for a kidney transplant in the UK, but just over 3,000 received one. The global shortage of donors has fuelled a lucrative and growing black market: kidneys harvested from living donors can be sold for more than $30,000 and will soon outnumber those taken from the dead.
“Our ultimate goal is to help address the shortage of donor organs available for transplant and to develop therapies for diseases,” says Anthony Atala, director of the Wake Forest Institute for Regenerative Medicine in North Carolina, USA. “I foresee the field advancing so that treatments are gradually developed for a wider range of conditions.”
Atala and his colleagues have used scaffolds to grow bladders, urethras and, most recently, vaginas from patients’ own cells, and have shown that they remain safe and effective for years after transplantation. “We’re now conducting a clinical trial evaluating the safety of muscle progenitor cells for the treatment of urinary incontinence in women,” says Atala, “and we have a variety of projects that aren’t at the trial stage yet, including printing skin cells onto burn wounds and cell therapies for kidney disease, cystic fibrosis and haemophilia.”
Back in Kobe, the Laboratory for Organogenesis and Neurogenesis is growing tissues and organs using an altogether different approach, which doesn’t use scaffolds. Remarkably, they have found that embryonic stem cells can organise themselves into highly complex three-dimensional structures when guided in the right direction. Using a specially developed technique, the team has already coaxed embryonic stem cells to become partial pituitary glands and even bits of brains. Their greatest achievement to date is growing partial embryonic eyes, complete with retinal tissue containing light-sensitive cells, in the hope of developing a new stem cell-based treatment for various diseases that cause blindness.
“We really don’t know where we are going with this,” Yoshiki Sasai, the then director of the lab and Deputy Director of the CDB, told me. “We really are at the final frontier, facing an unknown world.”
© Kyle Bean
In the early 1920s, experiments provided crucial details about the earliest stages of brain development.
At the beginning of an organism’s development, the embryo undergoes a process called gastrulation. This seismic event dramatically alters the developmental landscape through a series of mass cell migrations, transforming the growing embryo from a hollow sphere of identical cells to a solid structure that contains three layers, each of which goes on to form different parts of the adult body. The nervous system initially forms as a flat strip of tissue on the outer layer, or ectoderm. This sheet thickens and expands and then, in a process called neurulation, folds in on itself to form a hollow tube that pinches off from the surface of the embryo and descends below it. This tube will eventually become the brain and spinal cord, while the rest of the ectoderm goes on to form skin.
Hans Spemann, a German embryologist, began investigating embryonic development at the turn of the 20th century. Spemann delighted in making his own microsurgical instruments and encouraged his students to do the same. Using fine loops made from strands of hair plucked from his own daughter’s head, he meticulously split amphibian embryos in two, and found that only the half containing a small piece of tissue would go on to develop into a tadpole. This small piece is destined to form what came to be known as Spemann’s organiser, the structure from which all the mass migrations during gastrulation – and thus your body’s formation – begin.
Spemann also performed experiments in which tissue from the newt or salamander embryos of one species were transplanted into the embryos of another. This allowed him to trace the ultimate fate of the transplanted tissue, since it looked different from that of the host under the microscope. On one occasion, Spemann transferred the organiser tissue from one embryo to another and found it could induce a secondary body axis, complete with a perfectly formed second nervous system.
When Hilde Proescholdt joined his lab as a PhD student, Spemann urged her to investigate the matter further. She did so, using two newt species with different skin colours, and her experiments confirmed that Spemann’s organiser could induce the formation of a second body axis and nervous system upon transplantation – effectively creating conjoined twin tadpoles. Importantly, the second nervous system came from the host tissue, not that of the donor. Thus, Spemann’s organiser somehow caused nearby cells to form nervous tissue.
After working on her thesis, Proescholdt married Otto Mangold, another member of Spemann’s lab. Not long afterwards, a gas heater exploded in the kitchen of their Berlin home, and she died of severe burns. When her results were published after her death, embryologists assumed that Spemann’s organiser secretes a protein that induces the formation of the nervous system and scrambled to identify it.
The search went on for almost 70 years. Then, in the early 1990s, two teams of researchers identified two different proteins, Follistatin and Noggin – the latter named after a British slang term for ‘head’ – that are secreted by Spemann’s organiser. To everyone’s surprise, these proteins didn’t induce activity, but did the opposite: they work indirectly by ‘disinhibition’, blocking another group of proteins that normally prevent ectoderm cells from turning into immature neurons and lead them to become skin cells instead. To become a neuron, it turns out, is the default fate for ectoderm cells, unless Follistatin and Noggin lead them otherwise.
“It was a very exciting time,” recalls Andrew Lumsden, founder and former director of the MRC Centre for Developmental Neurobiology in London. “The idea that development proceeds by blocking certain activities rather than enhancing them was quite novel. It was a big wake-up call, because everyone had been looking for a neural inducer since Spemann and Mangold’s work.”
It was around this time that Yoshiki Sasai took a postdoctoral position in Edward De Robertis’s lab at the University of California, Los Angeles. He had graduated with a medical degree from Kyoto University in 1986, and became interested in brain development during a residency in internal medicine. Within a few months of arriving in LA, he’d isolated the chordin gene – which produces yet another protein with the ability to induce nerve tissue – and showed that it works by blocking a signal that induces the formation of skin.
Since then, research teams around the world have identified many more of the molecules and mechanisms of brain development. We now know many of the genes involved in partitioning the developing nervous system and generating the right kinds of nerve cells in the right places; in addition, we know many of the genes that guide migrating young neurons (and then the branched fibres they extend) to their proper destinations.
This body of knowledge formed the foundations of Sasai’s work. What started as a quest to understand brain development inadvertently grew into a potentially useful way to grow complex tissues in the lab. This, in turn, may help to unravel some of the mysteries of morphogenesis – the process by which a flat sheet of tissue is ultimately transformed into a full-sized brain with a highly convoluted cerebral cortex.
When I arrive at the CDB in late January, Sasai is in huge demand: his colleagues have just published a major stem cell discovery, which Sasai played a small part in. Several Japanese television news teams stand between me and my arranged interview, all vying for my subject’s attention.
Beyond the Star Trek poster is a meeting room containing a large table and a standing whiteboard. Books and scientific journals line two of its walls. Behind one door is Sasai’s office, and I watch as two administrative assistants scurry in and out. Behind another door is the main lab area, a large room divided into smaller sections by shelves stacked with bottles of reagents and lab benches crammed with centrifuges and boxes of disposable pipette tips and latex gloves. I take a seat behind the table and wait.
Across the hallway are several other rooms, all with neatly aligned rows of slippers sitting outside. These rooms house more specialised equipment. In one is an atomic force microscope equipped with microscopic cantilevers, which Sasai and his colleagues use to measure the miniscule mechanical forces involved in morphogenesis. In the other, there is a spinning disc confocal incubation microscope, in which the team can film their lab-grown tissues as they buckle and unfurl.
Growing these complex tissues is much more challenging than growing sheets of skin or connective tissue. Sasai and his colleagues have developed a novel way of growing and maintaining embryonic stem cells in a 3D environment – suspended in the culture medium that nourishes them, instead of laying flat on the surface of a petri dish. They have found that embryonic stem cells grown in this way can spontaneously organise themselves to form the complex tissues that make up eyes, glands and brain tissue.
Sasai conceived the method in 2000, after returning to Kyoto and setting up his own lab there. As his interest in brain development grew, he and his colleagues developed a cell culture method for transforming mouse embryonic stem cells into different types of neurons. Their first attempts involved growing the cells in petri dishes alongside ‘feeder’ cells that secrete the signals needed for them to mature beyond their embryonic state and differentiate into mature neurons.
The method wasn’t particularly efficient, however; only a small proportion of the stem cells became mature neurons. Sasai suspected that this had something to do with the contrived environment in which the cells were maintained. The petri dish has been used around the world to grow bacteria and other cells since 1887 – but it’s flat and shallow, and real-life development takes place in three dimensions. Sasai reasoned that the petri dish was constraining the stem cells and preventing their developmental mechanisms from playing out, and he set about devising a ‘floating’ cell culture system.
He started growing mouse embryonic stem cells in 96-well plates, which are typically used for storing small amounts of liquids or tissue samples. Initially, Sasai’s team had trouble getting the cells to clump together. “We worked together with a company to optimise the plates for that purpose,” he told me, modestly – belying the challenging nature of the method, which others had struggled to master. They also stopped using the standard culture medium, which contains a cocktail of various growth factors and signalling molecules (including some unknown ones), in favour of their own concoctions.
The team spent five years developing the technique. By 2005, it had been perfected. Groups of around 3,000 embryonic stem cells would clump together when grown under these conditions, forming spherical structures called embryoid body-like aggregates. Because the cells come into close contact with each other, they can communicate in almost exactly the same way as they would in the growing embryo. What’s more, because nervous tissue is the default state of ectoderm, the embryoid bodies quickly become enriched with immature neurons. Embryonic stem cells grown with Sasai’s technique can therefore produce mature neurons far more efficiently than those grown in petri dishes. The team has shown that the technique can also be used to coax embryonic stem cells to differentiate into different types of neurons, including the midbrain neurons that produce the neurotransmitter dopamine (which degenerate in Parkinson’s disease) and cerebellar Purkinje cells (which die off in various movement disorders).
“In the womb, the embryo develops in three dimensions,” Sasai explained, “so embryonic stem cells grown using our method mimic development more smoothly.” Freed from the constraints of the petri dish, and fed the right combination of signalling molecules, the embryoid body-like aggregates go through the motions of development.
Building on their initial findings, Sasai’s team also found that embryonic stem cells grown in this way can organise themselves into layered structures resembling the cerebral cortex of a 15-day-old mouse. The cortex is composed of six distinct layers, each containing certain types of cells arranged in a particular way. These form one after the other from the inside out, as successive waves of young neurons migrate through the embryonic brain at the early stages of development. Embryonic stem cells grown in 3D culture can rearrange themselves to mimic these processes, giving rise to layered tissues with the right types of neurons in the right places. They had succeeded at something at which others had hitherto failed: growing a brain – or at least parts of one – in the lab.
When Sasai arrived for our interview, he was calm, reserved and apparently unflustered by the media chaos in the corridor outside. As he sat down at the table beside me, his assistant brought us green tea. I asked him about the Star Trek poster. He was not a big fan, he said, but he thought it appropriate because he had no idea where his work on lab-grown organs might lead.
Brain tissue is the least complex of the structures that the team created. When grown under slightly different conditions, they mimic another complex organ.
After several days of growth in the suspended culture, Sasai’s ectoderm cells spontaneously change shape. They first protrude outwards, then collapse slightly inwards to form a cup-shaped structure that resembles the embryonic eye and contains immature retinal cells. When this tissue is cut out and cultured separately for two more weeks, it develops further, forming a retina with six layers that resembles the eye of an 8-day-old mouse.
More recently, in 2011, Sasai’s team reported that they had used their cell culture system to grow partial pituitary glands. The pituitary gland is often referred to as ‘the master gland’ because it controls the production of hormones, which then control other glands. Sasai’s lab-grown pituitaries form from interactions between tissues from two distinct regions of the embryo; their meeting causes part of the ectoderm to fold in on itself and detach, making a small pouch. Cells within this pouch continue to change, generating six of the different types of hormone-secreting neurons found in the mature pituitary. It takes about three weeks to grow, and even then it’s still incomplete, but Sasai’s lab-grown partial glands can already fully restore hormone production when transplanted into mice whose pituitaries had been surgically removed.
What Sasai’s team do to grow tissues appears deceptively simple. Embryonic stem cells are harvested from mice and placed directly into the optimised 96-well plates. Each well contains approximately 0.3ml of a specific growth medium – one for brain tissue, another for pituitary tissue, and a third for embryonic eyes. Once these plates are transferred to an incubator, the process begins.
Ultimately, the team aim to grow these tissues on an industrial scale for therapeutic purposes. “One straightforward application is cell transplantation therapy for patients with growth hormone deficiency,” said Sasai. Similarly, the retinal tissues they are growing could eventually lead to therapies for conditions such as macular degeneration and retinitis pigmentosa, which lead to blindness. “We are now testing the functionality of the tissues by grafting them into blind animals.”
Ophthalmologist Robin Ali of UCL and colleagues have already transplanted immature retinal cells from young mice into partially blind adult animals, and recently reported that they can restore some visual function. They are now working with Masayo Takahashi, another colleague of Sasai’s at the RIKEN CDB, to do the same with retinal tissue grown using Sasai’s 3D culture method. Takahashi is also planning to transplant the retinas into monkeys. Last year, she announced a pilot study to test the efficacy of transplanting human induced pluripotent stem cell-derived retinal cells into people with macular degeneration, and she recently started recruiting participants.
One of the earliest uses of the term ‘regenerative medicine’ was in a 1992 article by healthcare futurist Leland Kaiser, as the subheading to a short section describing a “new branch of medicine…that attempts to change the course of chronic disease and…regenerate tired and failing organ systems”. Through the work of Sasai and others, that branch now appears to be budding.
Not everyone is convinced, however. “There’s a lot of hype about ‘building brains’,” says Lumsden. “You can’t build a brain. You can grow blocks of tissue which contain neurons, but they can’t get any bigger than a pea.”
“The problem is that these things depend on size,” he says. “You can’t grow a big mass of tissue without a blood supply, so a cell has to be within about five cell diameters of the nearest capillary to stay alive.” As a result, the size of lab-grown tissues is strictly limited to several millimetres. It’s unlikely that Sasai’s 3D culture method could be extended to anything much beyond embryonic eyes and partial pituitaries.
Others question the strategy of transplanting lab-grown organs into patients. “As neat as self-assembling brains and eyes are, I don’t think that’s a therapy in itself,” says Chris Mason, a professor of regenerative medicine bioprocessing at UCL. “Why wait for the patient to go blind before performing something so major? We should intervene early and as minimally as possible. If a fire starts in your house, you put it out immediately – you don’t wait for the whole house to burn down and then rebuild.”
The real potential of regenerative medicine, according to Mason, lies in the ability to grow cells derived from patients. “I think the value is that we can better understand diseases,” he says. “You could make neurons from induced pluripotent stem cells taken from patients with Parkinson’s or motor neuron disease. They’ll give us a real opportunity to understand the disease process better, and to look for new drugs that either reverse the disease or prevent it from occurring.”
Yet when I met Sasai, he believed that his method would ultimately lead to what he less modestly called “next, next generation” therapies. He hoped that specific types of lab-grown neurons could be used to develop new treatments that replace the cells that die in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and motor neuron diseases, or as a result of stroke or any other kind of brain injury.
On 5 August 2014, Yoshiki Sasai was found dead next to his lab. His death was an apparent suicide: the tragic result of a scandal surrounding the January stem cell discovery, which was published in Nature, and its eventual retraction. “The scientific world has lost a talented and dedicated researcher, who earned our deep respect for the advanced research he carried out over many years,” said Ryōji Noyori, President of RIKEN, in a statement on Sasai’s death. At the time of writing, an independent committee had recommended that the CDB be dismantled.
Sasai was cleared of any involvement in the misconduct, but he was criticised for failing to properly supervise the work and the researcher responsible. He had helped establish the centre in 2000, and his team’s work had put it on the map as a world-class research institution. He reportedly felt “deeply ashamed” about the incident, which had engulfed his institution and himself.
Sitting with me in the meeting room on that rainy January morning, Sasai acknowledged the challenges facing his research but was determined and optimistic about its potential. “We are now attempting to generate neurons from human embryonic stem cells,” he said, “but efficient replacement is still technically demanding, and we are trying to monitor the behaviour of transplanted cells using optical imaging.” He predicted that his lab-grown retinal tissue would be ready for testing in humans within five years, and that replacement organs grown outside the body would be commonplace within the next ten years.
Yet, he said, he still could not explain how cells organise themselves into such complex tissues. “It’s surprising to see ordered structures emerging without any external forces or influences,” he told me, likening it to politics – a kind of cellular democracy. Throughout development, the cells act upon and influence each other’s behaviour; pushing and pulling, they jostle for space and compete for the limited supply of the resources that they need to grow.
“Self-organisation means that all these processes are democratically regulated,” he said. “The whole process is entirely self-driven. The cells know how to make the optic cup or layered cortex. I do not tell them what to do – they talk to each other and decide for themselves.”
Sasai believed that self-organisation only emerges from populations of a certain size. “This kind of thing can only be seen in groups of about 1,000 to 100,000 cells,” he said. “At this level, cells can be directly democratic, and don’t need a special governor or president to orchestrate them. In a village of several hundred, the people can probably get together and decide what to do, but a country would be a total mess without a government.”
“Self-organisation is so mysterious,” he continued, his reserved demeanour giving way to a child-like curiosity. “We still can’t explain why the cells come together to make an eye. There must be more principles that we still don’t understand yet. It’s something that makes me completely in awe of life.”