Sasai's work is more than tissue engineering: it tackles questions that have puzzled developmental biologists for decades. How do the proliferating stem cells of an embryo organize themselves seamlessly into the complex structures of the body and brain? And is tissue formation driven by a genetic program intrinsic to cells, or shaped by external cues from neighbouring tissues? By combining intuition with patient trial and error, Sasai has found that it takes a delicate balance of both: he concocts controlled environments that feed cells physical and chemical signals, but also gives them free rein to 'do their thing' and organize themselves into tissues. He sometimes refers to himself as a Japanese matchmaker who knows that, having been brought together, two strangers need to be left alone. “They know what to do,” he says. “They interact in a delicate manner, and if the external cues are too strong, it will override the internal ones.”
Sasai's work could find medical applications. Recapitulating embryonic development in three dimensions, it turns out, generates clinically useful cells such as photoreceptors more abundantly and efficiently than two-dimensional culture can, and houses them in an architecture that mirrors that of the human body. Sasai and his collaborators are now racing to implant lab-grown retinas into mice, monkeys and humans. The way Sasai sees it, maturing stem cells in two-dimensional culture may lead to 'next generation' therapy — but his methods will lead to 'next, next generation' therapy.
Sasai and his colleagues discovered that the chordin protein is a key developmental signal released by the Spemann organizer. Rather than pushing nearby cells to become neurons, they found, chordin blocks signals that would turn them into other cell types. The work helped to establish the default model of neural induction: the idea that, without other signals, embryonic cells will follow an internal program to become neural cells.
By the late 1990s, embryonic-stem-cell scientists were also looking at these signals. They wanted to turn stem cells into mature cell types — particularly neurons — that might lead to therapies. The problem, says Sasai, is that scientists generally “push too hard and perturb the system”. Sasai knew that in the embryo, subtracting signals from the system is what counts, not perturbing it. “We tried to minimize external cues,” he says.
Sasai built an experimental system around that philosophy. He threw out the serum usually added to growing embryonic stem cells, which contains a brew of uncharacterized growth factors and other signalling molecules. He also removed physical cues, such as contact with the plastic surfaces of a culture dish, by allowing embryonic stem cells to spontaneously form floating aggregates known as embryoid bodies. “If they're attached, they're like prisoners and can't act out their own desires,” he says. Keeping the cells alive without these support systems was a challenge but, five years of careful experimentation later, Sasai published and later patented his serum-free embryoid body culture — a pared-down life-support system with just the right mixture of ingredients for cells to survive. It would go on to form the heart of his brain-tissue factory.
All this will not create eyes that can be plugged into an eye socket like a light bulb into a lamp. Even if Sasai could get his optic cups to develop into mature retinas, researchers have little idea about how to wire a transplanted retina up to the brain.
What the work does offer is a potentially abundant source of pure, dense, well-organized photoreceptors, the developmental stage of which can be precisely selected — something that has been difficult to achieve in standard two-dimensional culture. Eventually, Sasai hopes, his optic cups will provide sheets of photoreceptors that can be inserted into a retina damaged by conditions such as retinitis pigmentosa or macular degeneration. Sasai demonstrates the procedure by grabbing a stack of papers to stand in for the retinal layers and then slipping one sheet between the others.
Sasai has set his sights on more complex neural tissues. Last November, he reported the formation of a part of the pituitary gland — his “most complicated” tissue yet. In the embryo, the pituitary gland arises when two different tissues integrate to form a pouchlike structure. Sasai managed to recapitulate this in vitro partly by starting out with more than three times more embryonic stem cells than he had used to grow a mouse retina; the adjustment seems to increase the levels of signals that the cells exchange. When transplanted into mice in which the pituitary glands had been knocked out, the rudimentary organs restored the endocrine system and saved the mice. This work, too, might eventually provide a supply of pure, specialized cells, which could be used to treat endocrine disorders.
Sasai hopes to improve on his early efforts by growing a better pituitary gland, equipped with a blood supply; a cerebral cortex with all six layers of tissue; and photoreceptors mature enough to detect light. But his next major task is to culture a cerebellum, which will involve growing and integrating three tissues of different embryonic origins. The matchmaker is already at work, trying to conjure up the right atmosphere. “When a boy meets a girl, they start their own story — but not in a large auditorium full of people,” he says. “You need to put them on a beach or in a disco. Our system is simply going to create this environment.”
What Sasai plans to take on after the cerebellum is a secret, but he hopes eventually to encompass the whole brain. He does not mean building one — that would be enormously difficult and ethically fraught. Instead, he wants to work out how brain parts, with their remarkable capacity for autonomous growth and organization, combine and fold into a structure of such tremendous complexity.
Cerebral Cortex Engineering Paper
Cell Stem Cell journal - Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals
Nature Neuroscience - Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells
Tissue Engineering the Optic Cup of the Eye
Nature - Self-organizing optic-cup morphogenesis in three-dimensional culture
Tissue Engineering the Pituitary Gland
Nature - Self-formation of functional adenohypophysis in three-dimensional culture