Growing entire human organs was always a dream of the tissue culturers. While Alexis Carrel laboured with his protégé Charles Lindbergh to keep human organs alive outside the body by perfusing them with blood plasma in artificial containers, the ultimate prize was “the culture of whole organs” – the title of an article that the two men published in Science in 1935. That paper didn’t report anything of the kind, but when Carrel saw new tissue growing on a perfused cat ovary, he concluded that organs should be able to make themselves from their constituent cells.
“Isolated cells,” he wrote in his book Man, the Unknown, also published in 1935, “have the singular power of reproducing, without direction or purpose, the edifices characterizing each organ.” These organs, he added, are “engendered by cells which, to all appearances, have a knowledge of the future edifice, and synthesize from substances contained in blood plasma the building material and even the workers.” It seemed almost magical, and even the arch-rationalist Carrel allowed himself a little flourish, saying that “an organ develops by means such as those attributed to fairies in the tales told to children in bygone times.” This kind of magic, he hoped, would lead to another: to immortality, enabled by a constant renewal of organs made in the laboratory.
Thomas Strangeways and Honor Fell at the Cambridge Research Laboratory found that tissues cut from embryos after the cells had differentiated and then cultured in vitro would continue to grow as the same cell type: cells of eyes or bones, say. This was an early form of what today is known as tissue engineering – a striking lexical amalgam of the biological and the industrial, which seems to imply that growing a human (or parts thereof) is at root an engineering problem. In 1926, Strangeways proclaimed that “somatic cells do not require the control of the organism as a whole in order to build up the specific tissues for which they were set apart in life.” There is no clearer statement of the philosophy that motivates, and receives support from, work today on mini-brains and other organoids.
In the late 1910s, the Scottish biologist David Thomson, working in London, travelled to learn from Carrel at the Rockefeller before returning to England to grow nascent organs of chick embryos excised and cultured in vitro. He found that they retained their anatomical structure and argued that this was because a membrane around the growing organ prevented uncontrolled proliferation of cells at the edges. But as Thomson rightly discerned, there was a limit to how big such “artificial organs” could get, because the nutrient would at some stage fail to reach the innermost cells and they would die. He recognized that to overcome this limitation demanded “some means [of] artificial circulation”: the cells needed a blood supply.
Culturing and sustaining organs in vitro was the enabling technology that led biologist J. B. S. Haldane to imagine the production of humans wholly outside the body. As he explained in a lecture of 1923 in Cambridge that he developed the following year into a book called Daedalus, or Science & the Future:
We can take an ovary from a woman, and keep it growing in a suitable fluid for as long as twenty years, producing a fresh ovum each month, of which 90 per cent can be fertilized, and the embryos grown successfully for nine months, and then brought out into the air.
Haldane’s speculations were the prelude to IVF and assisted reproduction technologies, as we will see in the next chapter. But such visions of making humans have never been distinct from work on organ and tissue culture. Haldane himself drew confidence from the results coming out of Strangeways’s lab, and Nature’s review of Daedalus commented that the book didn’t seem far-fetched “if what has already been done with tissue culture is remembered”.
The growth of parts of organs like eyes and bones in vitro at Strangeways led some observers to believe the lab was on the point of making entire organisms piece by piece. “Already other parts of the human body have been grown in test-tubes,” wrote Norah Burke in Tit-Bits in 1938. “Remember that chicken heart that went on growing and growing … It may be that the human species will produce test-tube babies or full-grown human beings, entirely chemically.”
It wasn’t just wild fantasy, given what some scientists were already claiming. By 1959, the French biologist Jean Rostand would assert (with little justification, it must be said) that “it is now possible to construct, as it were, artificial organs, complete with heart and lungs … it has been done by Carrel and Lindbergh … and many others – whose characteristics approach nearer and nearer to the natural.” With such ideas in the air, you could hardly blame the likes of Burke for getting overexcited.
* * *
But could cells really do it all, growing into an entire organ outside the body? Some researchers looked askance at cells cultured in vitro, considering them to be an undisciplined mass: as the Russian histologist Alexander Maximow put it in 1925, “a crowd of various cells without any regular arrangement”.1 There was a suspicion that the whole organism, or at least some substantial portion of it, might be needed to instil the order evident in real tissues and organs.
In any event, despite the hyperbolic and sometimes ominous press reports that accompanied early work on tissue culture at Strangeways and Carrel’s lab, human tissue engineering never took off at that time. There were successes with other mammals and higher organisms, but researchers found that normal human cells were peculiarly difficult to sustain. By the 1960s, efforts to grow human organs and tissues from the cells up had all but disappeared.
A few researchers persisted. In the 1970s, there were efforts to make artificial skin for covering the wounds of burns victims to aid healing and prevent infection. John Burke of the Massachusetts General Hospital developed thin sheets, made mostly from the natural protein collagen, that would support the migration and proliferation of skin cells when placed over the wound. Thus Burke’s material acted not just as an emergency covering but as a kind of biodegradable scaffold to support skin growth in vivo. This technique has now become rather routine: several commercial polymer products exist, typically made from cow collagen, on which skin cells (keratinocytes and fibroblasts) can grow in either in vitro culture or directly on a wound. Materials like this have been used, for example, to treat otherwise non-healing diabetic foot ulcers that would previously have required amputation. Artificial skin has also been grown from umbilical-cord stem cells and stored as ready-made sheets for surgical use. Although there remains the problem of skin grown from one individual’s cells being rejected when grafted onto another person, it can be reduced by immune matching of donor and recipient (see here).
“Synthetic skin” grown from stem cells exemplifies another motivation for growing tissues and primitive organ-like structures outside the body: not for surgical use but for testing drugs and other pharmaceutical products. A good enough mimic could reveal potential side-effects – for example, the propensity for a medication to provoke inflammation and skin irritation or to prove toxic. Such testing has traditionally relied on animal experiments, which are not only ethically controversial but also sometimes of questionable relevance to humans. In vitro cultured skin has already been put to use in this way, and tissue cultures that supply approximations to the human kidney, liver or brain may likewise help to sift through drug candidates for those that work safely and effectively – perhaps even in a way that is personalized to the patient, since drugs don’t always produce the same effects, for better or worse, in every individual. That approach remains to be validated, however – cell biologist Marta Shahbazi warns that “we may find ourselves curing organoids but not curing people.”
The skin is our largest organ, but also in many ways the simplest. Making other artificial organs is more challenging. In the mid-1980s, paediatric surgeon Joseph Vacanti, also working at the Massachusetts General Hospital, became frustrated that his efforts to save children’s lives through transplants were hindered by a shortage of organs. “I watched in agony and completely helpless as several children faded into coma or haemorrhaged to death,” Vacanti recalls. One of the few options available for obtaining livers for transplanting to children was to trim adult livers to size – but that crude approach was fraught with difficulty. “It occurred to me that if we could build liver tissue, we could transplant on demand,” Vacanti recalls. But how?
Vacanti had witnessed Burke’s efforts to grow skin on polymer supports during his stints on the burns ward early in his training. But a liver is a very different sort of tissue to skin – for one thing, like most organs it needs a system of blood vessels to keep the cells alive.2 Vacanti turned to his colleague Robert Langer, a chemical engineer at the Massachusetts Institute of Technology, who had studied ways to control the development of blood vasculature (the process called angiogenesis) as a possible means of shutting down the growth of cancer tumours.
Together, Vacanti and Langer attempted to culture liver cells on an artificial scaffold, using polymer materials similar to the earlier work that were already approved for human use. One way of providing the cultured tissue with a blood supply was to simply build it in: to lace the polymer support with a network of tiny channels, which would be seeded with the cells (called endothelial cells) that make the walls of blood vessels. Langer and his co-workers have made a kind of artificial liver by stacking alternating layers of polymer sheets patterned in this way, one layer seeded with the synthetic blood vessels and the next with liver cells. In this sandwich arrangement, no liver cell is ever too far from a blood supply. Structures like this aren’t intended as long-term artificial livers, but the researchers hope that they might be implanted to sustain patients with liver failure who are awaiting transplants. One big challenge is to connect the body’s own blood-vessel network to that of such an artificial organ. A promising approach is to seed the scaffolding material with proteins called growth factors that stimulate the body to grow vascular networks, so that natural blood vessels might infiltrate it.
Laryngologist Martin Birchall of University College London was, like Vacanti, frustrated by the limitations of conventional transplant surgery. While doing head and neck cancer operations, he says “it became clear that, even with the very best of modern techniques, we were still a long way from being able to restore function and quality of life to patients who had had major treatment to mouth, tongue, larynx, pharynx and oesophagus.” Birchall felt that this impairment in quality of life for his patients could be dehumanizing. “I felt there must be a better way to go,” he says.
For the kind of airway surgery that Birchall carried out, replacement tissue structures would ideally be personalized in shape to fit the patient. This could be done by shaping a polymer scaffold which is then colonized by cells in vitro before being surgically implanted. In the mid-2000s, Birchall developed a tissue-engineered windpipe this way that could be transplanted to pigs. And in 2008, he and his Spanish collaborators got permission to use the technology on a young Spanish woman who had suffered life-threatening damage to her windpipe after a tuberculosis infection. The researchers seeded a scaffold with the patient’s own stem cells, taken from her bone marrow, to avoid problems of immune rejection. “Considering how little we knew at the time, it worked amazingly well,” he says. The woman is still alive and well today.
Usually such stem cells need signals to trigger proliferation and to guide them towards the required cell fate. For example, so-called mesenchymal stem cells taken from muscle, bone marrow or fat will adopt a different fate depending on how stiff the material is within which they are growing: they develop into the cell type of the tissue with the closest match in stiffness. It’s as if the stem cells give a little tug on their surrounding environment to figure out what they should become. So the differentiation of these cells can be guided by mechanical cues in their surroundings. Alternatively, biochemical agents such as transcription factors can be added to determine the cells’ fate.
Tissue engineering with adult stem cells is still a relatively new technique, but it holds tremendous promise. The several different cell types that might be present in a single organ, such as vascular cells, bile and liver cells in a liver, typically share a common lineage and so might all be made from the same stem cells if given the right cues. An alternative that avoids the difficult business of harvesting adult stem cells from a patient is to use induced pluripotent stem cells cultured from, say, their skin. Langer and Vacanti think that iPSCs could become “the ideal material for building tissue constructs” – so long as their tendency to develop instead into tumours in the body can be kept in check. Embryonic stem cells can be used too, but then you’re also up against the problem of immune rejection, necessitating doses of immune-suppressing drugs.
Today’s architects of artificial organs are upbeat about the prospects. In 2002, Robert Lanza of the American tissue-engineering company Advanced Cell Technologies made the bold prophesy that
If this research [on stem cells] is allowed to proceed, by the time we grow old, this will be a routine thing. You’ll just go and get a skin cell removed at the doctor’s office, and they’ll give you back a new organ or some new tissue – a new liver; a new kidney – and you’ll be fixed. And it’s not science fiction. This is very, very real.
“With every year I see more hope even for the most difficult problems of tissue regeneration, such as brain repair after strokes or spinal-column damage,” says Vacanti. But big problems remain even for relatively simple tissues. For example, although Vacanti and others engineered artificial cartilage in the mid-1990s – which, like skin, doesn’t need an extensive network of blood vessels – the materials still haven’t been used on humans because the wound-healing process after transplantation is complicated. Synthetic cartilage tends to be re-absorbed by the body over time, causing the tissue to deform. Cells have their own agendas, which we don’t yet fully understand, let alone know how to control. “This is difficult science,” Vacanti admits.
* * *
Another option for culturing cells into artificial organs with a predefined shape is to dispense with synthetic polymer scaffolds and use in their place the “skeleton” of an actual donor organ instead. The cells of organs are bound together by a robust network called the extracellular matrix, a web made from a variety of biomolecules that the cells secrete. In animal tissues, these are typically sugar-based polymers (polysaccharides) and fibre-forming proteins such as collagen and the stretchy elastin. Cells have molecules on their surface that stick securely to these matrix components. (It’s precisely because collagen is a component of the extracellular matrix that it makes such a good synthetic scaffold material.)
The idea is to use detergents and enzymes to wash away all the native cells in the donor organ, leaving just the “decellularized” matrix on which the recipient’s cells can then be grown. Since none of the donor cells remain, animal organs such as pig hearts can also provide decellularized supports for growing human organs.
For simple soft tissues like skin, decellularization is already used to make commercially available products, for example using the skin (dermis) and intestine of pigs and cows, and indeed of humans. Martin Birchall has experimented with decellularized trachea scaffolds taken from both pigs and humans; in fact, his 2008 operation on the Spanish patient used such a segment of trachea taken from a deceased 51-year-old woman. For complex organs, studies haven’t yet advanced beyond animal experiments. Lungs, kidneys and hearts have all been grown on decellularized scaffolds from rats, but the outcomes from subsequent transplants have been mixed. The rat kidneys produced a urine-like liquid, for example, but the lung quickly filled with fluid.
In 2013 a team at the University of Pittsburgh School of Medicine reported the growth of a human “mini-heart” on a mouse scaffold. They seeded the decellularized mouse heart with human cells grown from iPSCs into the progenitor cells of cardiovascular tissue. The cells not only spread throughout the scaffold but differentiated into the specialized types of the heart: cardiomyocytes, other muscle cells, and vasculature-forming endothelial cells. The researchers perfused the artificial organ for 20 days with a culture medium that included growth factors, at which point it started showing spontaneous contractions at a rate of 40 to 50 per minute: it was, after a fashion, beating. The artificial heart also responded to drugs known to affect the beating behaviour in humans.
A beating, mouse-sized human heart in a dish? Well, sort of. Just because a heart contracts doesn’t mean it works properly. All the same, the result bodes well for making human-sized hearts from the corresponding decellularized organs from, say, pigs.
* * *
Tissue engineering treats living flesh as a material for moulding, shaping, transforming. If ever there was a technology that showed just how far we have come with that philosophy, it is 3D bioprinting. Here, cells themselves comprise the “ink” that is dispensed through a fine nozzle just like the coloured inks of your home printer, to build up complex three-dimensional shapes layer by layer.
3D printing is already transforming the art of manufacturing in general. To make objects ranging from machine components to artistic sculptures, the print heads typically squirt out resins or powders of metal, ceramics or plaster that can be treated to weld or set them into robust structures. Under computer control, these systems can produce intricate forms of almost any shape, from pottery to engine parts. They have even been used to sculpt food, producing ornate creations in pasta or chocolate. Textiles too can now be printed rather than spun and woven. As 3D printers become cheaper, companies and even individuals can produce artefacts on demand without having to order them from suppliers, just by downloading the appropriate printing instructions. Some futurologists foresee a time when “shopping” will mean pressing the Print button – although we should hesitate to equate technological possibility with commercial and socioeconomic viability.
Why not, then, make body parts in this way too? In one of the first clinical applications of 3D bioprinting, in 2014 a patient admitted to Morriston Hospital in Swansea, Wales, after a motorcycle accident underwent surgery that used tailor-made, printed titanium components to hold together his damaged facial bones. Bespoke metal implants or biodegradable polymer scaffolds like this, designed to fit a particular patient’s body, could become widespread; they are already used in craniofacial surgery for shaping bone-substitute materials. 3D printers can construct the most convoluted structures or reproduce exactly the shape of the organ or tissue that needs replacing. You can scan a patient’s anatomy before an operation using the technique of computerized tomography – an advanced form of X-ray scanning – and then use 3D printing to produce a material that is exactly the right size and shape. In this way, researchers at the University of Michigan have tailored a purely synthetic polymer sleeve to prevent a malformed section of an infant’s trachea from collapsing and blocking his airway. Larynx (voicebox) implants too have to be rather precisely shaped to fit the patient, and Birchall forecasts that they will one day be made from 3D printed biodegradable scaffolds seeded with the patient’s iPSCs.
The possibilities become even more dramatic when the inkjets dispense biological tissue itself: when we start printing flesh. Here the inks consist of clumps of living cells, typically protected from the trauma of high-speed passage through a nozzle and impact on a surface by being encased within droplets of a soft, biocompatible polymer or gel material. This technology is in its infancy, but it’s clear already that cells can survive the process and be assembled into complex shapes. 3D inkjet printing has been tested on animals to speed up wound healing by spraying cells that make skin and cartilage directly onto the site of injury. Flat sheets of tissue are relatively easy to make, and tubular structures like blood vessels and the trachea are feasible too. Solid 3D organs like livers and hearts would be much more challenging to “print”, and no one imagines that will happen soon.
Another, cheaper bioprinting method is extrusion – like squeezing toothpaste out of a tube, but with a much finer nozzle. Here the “paste” is usually again a soft, biocompatible polymer infused with cells, which is “written” into the desired pattern a layer at a time. Not all cells tend to survive the squeezing, but usually more than half of them do.
3D bioprinting could supply patterned vascular networks for organs and tissues grown in vitro. A team at Harvard University led by Jennifer Lewis has grown tissues that contain three-dimensional grid-like networks of vessels. First they print the grid using a polymer ink that can be washed away later, thus acting like a removable wax mould in a conventional casting process. Then they print the cells (coated with gel) around this network, fusing the gel droplets together to make a robust material. Extracting the “vascular” ink leaves behind open channels running through the artificial tissue. The researchers infuse these passageways with epithelial cells, which form an impermeable, blood-vessel-like coating on the walls. In this way, the team have made tissues of fibroblasts more than a centimetre thick, in which the cells can be kept alive for more than six weeks by blood flowing through the artificial network. By delivering the right transcription factors into the growth medium, they induced the cells to differentiate into a bone-forming variety and could see the beginnings of bone growth. It’s a first step towards making new bones, laced with blood vessels, that could exactly copy the shape of ones that have been irreparably damaged or deteriorated.
Vladimir Mironov, who heads a Russian startup company called 3D Bioprinting Solutions, is convinced that bioprinted human organs are on the way. His company is currently aiming to make a mouse thyroid gland this way – and in the longer term, a human kidney. Mironov has no qualms about extrapolating the vision all the way to what he calls “the realization of Pygmalion’s dream”: printing an entire functional human body. (The legendary sculptor Pygmalion made a statue of a woman so beautiful that he fell in love with her, whereupon a blessing from the goddess Aphrodite brought the statue to life when he kissed it.) The self-organizing cells will take care of the details themselves, Mironov says; all the printer need do is put them in more or less the right place with the right density.
I’m not sure we need take this notion too literally. It would have been grist for the mill of Amazing Stories – “The Man Who Printed His Wife” would be just the kind of unexamined metaphor in which that barometer of techno-cultural change revelled. But a printing process for humans hardly addresses any burning clinical or social need.
Cynics might therefore see Mironov’s suggestion as sheer hype. But we might more generously regard it as a provocative thought experiment. For the point about 3D printing is that you can make any shape you like, and it’s not obvious that a 3D-printed organ would have to recapitulate the exact shape or cell organization of the natural variety in order to work properly – Jennifer Lewis’s printed vasculature does not, for example. A simplified, idealized structure might be perfectly adequate, perhaps with greater geometrical regularity that makes it easier to manufacture. No one knows how amenable tissues and organs might be to redesign.
You might then ask the same questions of an entire bioprinted body. Perhaps this too needn’t be shaped exactly like a human body, or even have a recognizably humanoid form at all. Again, rest assured that this is purely hypothetical; no bizarre bioprinted organism is going to twitch, groan, and sit up on the manufacturing platform any time soon. But the scenario can be posed, and it shows that the technologies of cell transformation and construction are allowing us now to formulate and even to begin investigating profound and disconcerting questions in biology. What are the limits of what we can, and should, grow? What are the design constraints of a human body? What qualifies as a human?
* * *
The instincts of an engineer are to build. Those of a biologist are to grow.
So far, making artificial organs from cultured cells involves a bit of both. Whether by using shaped or decellularized scaffolds, or by positioning cells using 3D printers, the idea is to guide the culture towards the correct structure for a tissue or organ by artificial means. Neither approach, though, truly reflects what happens in the body. Here, as we saw, tissues are shaped by other tissues through a complicated dialogue between cells involving chemical and mechanical cues, movement, adhesion, self-organization.
In the early days of tissue culture, there was much debate about whether or not the whole organism is needed to impose order on proliferating cells. Research on organoids has now delivered an answer, and it is the one very familiar to biologists: yes and no. Cells of a particular tissue type can do an awful lot of the arranging via their mutual interactions, but they don’t get it quite right without the correct signals from their environment within the whole growing organism. The more you can make an organoid “think” it is part of an embryo, the more it is likely to resemble the real thing. As with children, you can force cells to be a certain way but it might be better to let them find their own way there, with just enough gentle instruction.
And the best instructor of all is a (developing) body.
Where, though, can you get a body that will patiently and expertly tell cells how to grow into organs? Well, it needn’t be a human one. We can grow human organs inside other animals.
Your instincts might rebel here, and rightly so. We learn in basic biology that species are fundamentally incompatible, made distinct by the fact that they can’t interbreed. In truth, it’s a little more complicated than that, for some closely related species can have progeny – that’s how mules are made from the crossbreeding of a horse and a donkey. Indeed, cross-species breeding is more widely possible in principle than you might imagine: tigers and lions, for example, can produce either a tigon (male tiger/female lion) or liger (vice versa). Neither are such hybrids necessarily sterile: the mule is, but tigons and ligers are not. Such crossbreeds are rare in nature more because of instinctive mating habits and separation of habitats than because they are biologically impossible.
All the same, species that can interbreed to make hybrids must be very closely related in evolutionary terms: a tiger can’t mate with a horse. So how on earth could a human organ grow in another animal?
Yet it can. Take, for example, the work reported in 2013 by Takanori Takebe of the Yokohama City University Graduate School of Medicine in Japan and his co-workers. They made human iPSCs and guided them into becoming precursor cells for making liver tissue, called hepatic endoderm cells. Then they cultured these cells in a mixture with endothelial cells taken from umbilical tissue and mesenchymal stem cells. Contrary to the prevailing belief that cells in culture can’t mimic the complex interactions that lead to organ formation, this mixture of cell types organized itself into structures that look like the early form of an embryonic liver, called liver buds. In a normal human embryo, such structures appear around the third or fourth week of gestation.
But the organoids could get no further in vitro because of the restrictions imposed by lack of a vascular network. To get around this problem, the Japanese team transplanted their human liver buds into mice. To suppress immune rejection, these mice had been genetically engineered to have a dysfunctional immune system. In the initial experiments, the researchers transplanted the buds to the cranium of the mice, which sounds perhaps like a weird and even grotesque choice but was made purely because it was then easier to examine the subsequent growth. That choice of transplant location shows, however, that bodies can be remarkably tolerant of what goes where, once the developmental process is well underway. For indeed, the liver buds not only survived but began to grow blood vessels in response to the signals received from the mouse tissues. These were human blood vessels, made from the endothelial cells already in the organoids. The grafts also took and grew when implanted in the intestinal-abdominal region of the mice (the so-called mesentery), round about where a liver really should go.
Of course, a mouse-sized organ can only do a mouse-sized job. But Takebe and his colleagues have since scaled up their process to make large batches of the organoids: what they call “a manufacturing platform for multicellular organoid supply”.
Once implanted with a human organoid, the mice in these experiments are not hybrids (like mules) but chimeras: organisms containing the cells of more than one genotype. In a mule, all the cells have the same genome, composed of a mix of horse and donkey genes. But chimeras are genetic mosaics.
We saw earlier (here) that some humans are chimeric: their bodies are mosaics of cells with different genomes. For example, some of their cells may come from the mother, having found their way into the fetus through the placenta. But just as the mythical beast of that name exemplified, chimeras can contain tissues of unrelated species. The ancient Greek Chimera was described by Homer in The Iliad as “lion-fronted and snake behind, a goat in the middle”.
The viability of chimeras (in the biological, if not the Homeric, sense) seems less puzzling when we acknowledge our cellular nature. Sex is a very particular and rather specialized way of cells proliferating, and it mixes the genomes of the parent cells. Now, there is no prohibition on a gene from one species being incorporated into the genome of another; industrial genetic engineering of bacteria relies on that. But intimately blending the entire genomes of two species is in general too much for biology to handle: the resulting set of “instructions” makes no sense. That’s why egg cells have a mechanism for checking that an arriving sperm is of the right species before the two can merge. You can’t make a chimera like the Minotaur by the mating of a woman and a bull.
If there is no actual merging of genomes, however – if every cell in an organism retains its own genotype – then there’s no problem. If the individual cells are viable, all that then matters is whether they can get along together. And we know cells of very different types can do that: it’s why bacteria thrive in our gut, and indeed all over our bodies. Chimeras are simply another kind of diverse yet harmonious cell community.
* * *
Growing tiny human organoids like Takebe’s proto-livers in mice is a remarkable feat, but it’s not clear how you could ever grow a complete, full-sized human liver, gut or brain that way.
But what if they were grown in human-sized animals – in a pig, cow or sheep? The idea seems feasible in principle. In practice, needless to say, it’s complicated.
For one thing, the whole enterprise sounds ethically challenged. Should we rear animals like this as mere carriers of human spare parts? The yuck factor is considerable: many people will find the notion of a pig with a human liver to be disturbing, even obscene.
Is it possible in the first place, though? The scenario would run something like this: you make induced pluripotent cells from a patient in need of a new liver, kidney, pancreas or whatever, transplant them into a pig embryo, and hope they grow into the corresponding organ in the piglet, to be harvested when – forgive the harshness, but such is the reality of livestock farming – it is time to make bacon. But why would the human stem cells choose the particular fate you want, and not some other?
Several years ago, Japanese biologists Toshihiro Kobayashi and Hiromitsu Nakauchi saw a way to guide that decision. The idea is to create a “niche” for the required organ in the host animal: engineering the embryo so that it lacks the ability to make the organ itself. The idea stemmed from work in the early 1990s in which mouse embryos lacking a gene that was crucial for the development of white blood cells involved in the immune system were injected with embryonic stem cells taken from other mice that had this gene. The embryos developed into mice capable of producing the white blood cells using the injected cells.
This shows a remarkable smartness on the part of the host embryo. The donor stem cells aren’t initially committed to a particular fate: they could develop into any tissue type. But as the embryo develops, it’s as if its cells say, “Time to make our immune cells – but wait, we don’t have the right gene. Ah, but look, here are some foreign cells that do. Let’s give the job to them!”
That is, of course, quite a burden of anthropomorphization for cells to bear. Sometimes, though, it is hard to tell a comprehensible story without a narrative like this, precisely because cells display the kind of responsiveness that looks like foresight, intelligence, collaboration. It is this “looks like” that holds the tension about how we think of identity and autonomy in life.
The point is that growing organisms will indeed display the initiative to fill an empty “tissue niche” with competent cells taken from another organism. In 2010 Kobayashi, working at the Gurdon Institute in Cambridge, showed that the process could be engineered: a niche can be made to order. He and his colleagues knocked out a gene crucial for pancreas development in mouse blastocysts, and then added to these embryos some cells taken from another mouse in which that gene was still active. Sure enough, the embryos developed into mice with a pancreas. Two years later, Kobayashi and Nakauchi showed that the same trick worked for making kidneys.
Making it work across species looks like a challenge of another order. The researchers first “broke the species boundary” by growing pancreases from rat embryonic stem cells in mice that lacked the pancreas-making gene, and vice versa. In other words, the resulting chimeric rodents had pancreases made entirely of cells from the other species.3
Experiments on these mouse–rat chimeras have revealed something that I find extraordinary. When rat pluripotent cells were added to embryonic mice, they were found to form part of the resulting mice’s gall bladders. But rats don’t have gall bladders. How can rat cells make an organ that rats themselves don’t have? Apparently, these cells have an incipient ability to make gall-bladder tissue, and this ability gets unlocked by the “time to make a gall bladder” signals coming from their environment within the embryonic mice. That ability didn’t come from nowhere: the common evolutionary ancestors of rats and mice did have gall bladders, but rats lost them. Their cells, however, “remember”, as if preserving the memory of their evolutionary history. Might our own cells, then, hold a potential to make body parts that are not human – developmental memories of our own evolutionary ancestors?
So organ growth via trans-species chimerism works for rodents. What about humans and pigs? This is tougher for several reasons. One is that the experiments take longer: pigs have a three-month gestation period, as opposed to about three weeks for mice. And humans and pigs have been on separate evolutionary trajectories for much longer than mice and rats.
The first step was to show that organ niches can be engineered in pigs. In 2013, Kobayashi and Nakauchi reported that pig embryos lacking a gene vital for pancreas development could indeed generate a pancreas from the embryonic stem cells of another pig. The male pigs grown this way could even be used as a source of sperm to make more “non-pancreas-making” pigs by IVF, with a ready-made pancreatic niche.
Could this niche be filled by human stem cells?
It took a four-year project to find out. In 2017, biologist Juan Carlos Izpisúa Belmonte of the Salk Institute in California and his co-workers reported that they had made pig fetuses that contain human cells. The human iPSCs were introduced to pig embryos at the blastocyst stage, and the embryos were allowed to develop for up to four weeks.4 (The researchers allowed them to go no further to avoid an ethical outcry about mature pigs with human tissues.) Although the survival rate of the human cells was rather low, some were still present, and were developing into muscle and the progenitors of other organs. The team also found that human iPSCs could survive in cattle embryos, albeit again rather inefficiently.
This remains some way from showing that human organs will grow in pigs. But given the experience so far, I believe it can happen – in principle. Should it?
* * *
Izpisúa Belmonte and colleagues were unable to use US federal funding for their work, because in 2015 the National Institutes of Health placed a moratorium on supporting such research until the ethical questions had been carefully considered. Although the NIH had promised to review this decision in 2016 after consultations, the prohibition remains in place at the time of writing – although, given the current incumbent of the White House, nothing is predictable.
No one was more dismayed at the NIH’s decision than Nakauchi, who had already moved to Stanford University in California from the University of Tokyo in 2014 to escape a Japanese ban on research that involved the insertion of human pluripotent stem cells into non-human embryos. These prohibitions are deeply frustrating to researchers like Nakauchi who are sure that the technique can be made to work. “Animal-grown organs could transform the lives of thousands of people facing organ failure,” he has said. “I just don’t understand why there continues to be resistance.”
But that resistance is surely not so hard to fathom. Chimeras seem to upset the natural order. That was their mythical role, after all: the Chimera of Greek legend manifests as a fire-breathing omen of ill fortune. It is a “monster” in the etymological sense: a signifier of a rift in nature. Biological deformities in the Middle Ages and the Enlightenment, including misshapen human babies, perpetuated this role as harbinger: they were not mere aberrations, but portents. They warn us to beware.
Chimeric organisms in the modern, technical sense aren’t just reminiscent of these mythical origins; they are representations of them. They do actually blend tissues across species. After the NIH imposed its moratorium on this research in 2015, it conducted a public consultation in which a majority among the several thousand people who expressed a view were opposed to research on chimeras. And these misgivings are not wrong; they are not ill-informed, knee-jerk, Luddite reactions. (At least not entirely; many people had the mistaken impression that the creation of chimeras would necessarily involve human embryos.) There is only so much rearrangement of people’s intuitions about the “natural order” that they can be expected to accept all at once.
This is not to say that we must resign ourselves to such judgements. Personally, I do not believe that a ban on chimeras for regenerative medicine is the right decision, but I can’t adduce any philosophical calculus in support of that view. I do feel uneasy about the notion of pigs being grown as vectors for human organs, to be slaughtered and dismembered when their time has come. But I realize that this is an illogical position for someone who eats pork and bacon, where the slaughter serves no purpose beyond the gratification of our gastronomical cravings.
I don’t, however, fear that there is something unnatural, some violation of propriety, in the sheer existence of a pig with a human liver. Such a combination runs deeply against our experience, and it can easily morph into much more disturbing visions. Yet our instinctive reaction is one based on an illusion, namely that the human is an integral and inviolable whole, a well-defined entity with a homogeneous biological identity. Once we come to recognize that we are co-evolved, co-developed communities of cells, a pig–human chimera seems no more unnatural and repugnant than the notion that our gut is colonized by symbiotic bacteria. The question is simply: do the cells get along?
The real issue, it seems to me, is less the what than the how and the why. This is not a matter of means justifying ends. Quite the opposite – to navigate the astonishing and sometimes frightening possibilities that cell biotechnology is creating, we should be wary of absolute pronouncements about what is right and wrong, what is natural and unnatural. We should ask instead how well we will be served, as individuals and societies, by what we choose to do. Ignoring animal welfare in order to save lives will in the end be morally corrosive, but so will a refusal to alleviate human suffering on the tenuous grounds that it “feels wrong” to some groups.
This is the problem with being a community of cells that has evolved a sense of unique identity and moral agency. It’s not easy to fit those two characters together. We might do so most wisely and humanely by denying neither of them.
* * *
Even researchers who are impatient to see this technology forge ahead recognize that there are limits to what might be acceptable, at least without very careful ethical consideration. Kobayashi and Nakauchi have suggested what some of those limits might be, and their list is eye-opening. They say we should think twice before launching into any of the following:
We know these three scenarios already. They are the images, the dreams and nightmares, of legends and fiction: (1) takes us to the Island of Doctor Moreau; (2) shows us King Minos’s wife Pasiphaë, under Poseidon’s enchantment, mating with a bull to spawn the Minotaur; and (3) is the Centaur.
Kobayashi and Nakauchi present this list of possibilities in a paper called “Revisiting the Flight of Icarus”, for they suggest that by designing and fashioning for himself wings “to achieve his ambitious goal of flying”, Icarus was “chimerizing” his own body by adding to it the desired part of another species. I guess they did not really know the myth – for of course the wings were made by Icarus’s father Daedalus, and not simply to be able to fly but in order to escape imprisonment by Minos on Crete. Daedalus was incarcerated by the furious king because he had made the artificial cow-structure within which the enchanted Pasiphaë hid herself to have union with the bull. Researchers in this field are likely to find themselves increasingly grappling with myth and dealing with what myth represents. So it might be wise for them to include among their reading lists not just Cell, Nature and Science, but Homer and Robert Graves.
Now, let me be clear that Kobayashi and Nakauchi immediately followed up their list by saying that they are confident none of these outcomes will ever be realized. There seem to be barriers, they say, that will limit such large contribution of cells, tissues and body parts from the donor in trans-species “xenotransplanation” experiments. Besides, there are various measures one can take to ensure that inadvertent colonization of the host body by the donor tissues doesn’t happen.
Perhaps. But not everyone feels that the limits of the possible (let alone the permissible) are so clear. Take a 2013 study in which the progenitor cells of human brain (glial) cells were grafted into newborn mice. When they had matured, the mice showed better learning and memory, for example in how to navigate mazes. This doesn’t mean that the mice had acquired more human-like cognitive powers – but the researchers suggested that the greater complexity and capabilities of the human glia stimulated neuronal processing in the mouse brain networks. In truth we don’t really know what has gone on in the mouse brains to produce these improvements in performance, but nevertheless it does seem fair to say that the presence of the human brain cells made the mice smarter.
I have seen experts on brain organoids talk with all seriousness about the prospect, although certainly not the desirability, of a brain made from human neurons grown inside a pig: a full-sized brain, blessed with a vascular system like the liver buds that Takanori Takebe has grown in mice. Again, consider it merely a thought experiment. What, then, should we think of it? What would a humanized pig think?
I’m not talking about a research proposal, and if anyone were crazy enough to suggest it, it would be rightly rejected. I’m talking about how biological advances are dismantling old certainties and contriving new possibilities – and prompting challenging questions about where we draw lines, and why, and how.