26 min read.Updated: 03 Sep 2017, 12:02 AM ISTLivemint
In the first of an infrequent series of Mint on Sunday audio stories, the team at IndiSciComm explores the world of man-made organs. Is this the future of human longevity?
Stem cells have the unique property of developing into any cell of the body under the right conditions. For this reason, there is a growing interest in using them to treat disorders such as hemophilia, diabetes and even neurodegenerative disorders such as Parkinsons.
Apart from their therapeutic potential, researchers have shown that stem cells can be coaxed to spontaneously develop into miniature organ like structures called "organoids". Organoids recapitulate the intricate physical and biological features of organs and hence are important new tools in understanding human tissue development as well as for finding new drugs to treat disorders.
In this podcast, we discuss the biology of organoids, the hope and hype in medical research as well as potential ethical issues surrounding their use.
This podcast is written and produced by IndSciComm, a collective of Indian scientists working on increasing public science awareness.
Shruti Muralidhar is a postdoc at the Picower Institute for Learning and Memory at MIT studying how memory is encoded in the brain.
Navneet Vasistha is a postdoctoral researcher at the University of Copenhagen trying to understand the cellular basis of mental health disorders.
Abhishek Chari is a science writer at the Picower Institute for Learning and Memory at MIT with an interest in microbiology and evolution.
(Scroll down past the references to read a transcript of the podcast.)
Navneet: A long time ago, in a galaxy far, far... Wait, is that how we’re starting this podcast? No, actually in 1975, at the Massachusetts Institute of Technology, scientists James Rheinwald and Howard Green developed a method by which they could indefinitely grow human skin in the lab!
This is the first report of scientists being able to grow an organ in the lab. Their litmus test came five years later, when they were asked to treat two patients admitted to the Brigham Hospital with significant burns. Not only were Green and his colleagues able to graft skin sheets grown from the patients’ own cells, but in six months time, these grafts could no longer be distinguished from the surrounding unburnt skin.
Attempts at growing other organs have not met with a similar degree of success, for a variety of reasons.
However, with recent advances in stem-cell biology, researchers have found that by growing stem cells in just the right way, they can produce tiny blobs of tissue that look and function like organs.
Depending on what “molecular cues" are added, scientists have been able to grow what can lazily be called mini-brains, mini-pancreas, mini-retinal tissues, etc. The collective term given to these lab-grown tissues is “organoids". My name is Navneet.
Abhishek: I’m Abhishek.
Shruti: This is Shruti.
All: And we are IndSciComm. In this podcast, we’re going to be talking about what these organoids are and what they are not, how close are they are to actual organs, what their future potential is and a whole host of other interesting things.
Navneet: So, let’s begin with the basics. What are organoids and why are they interesting?
a. An organoid is a three-dimensional mass of cells that superficially resembles an organ or a gland. Researchers have generated several kinds of organoids using what they know about the development of different organs. Some examples are cerebral or brain organoids, intestinal organoids, pituitary organoids and so on.
b. Essentially, what makes them interesting is that cells grown in a dish with the right nutrient and cell growth factors can form something like mini-organs.
c. Some organoids have been transplanted into mice to restore functions or structures that they are lacking. For example, transplanted pituitary organoids have helped to restore the function of dysfunctional pituitary glands in mice. In fact, scientists have even transplanted a “proto" tooth organoid into the mouth of an adult mouse and watched it develop into a fully grown tooth!
Abhishek: So, cells can form structures of higher complexity like organoids. In essence, simple things (cells) come together to form more complex things (organs). This phenomenon is called self-organization. But how is this possible? Doesn’t the second law of thermodynamics say that entropy has to increase over time?
How can order be created out of chaos, if entropy can only be increased? Entropy, by the way, is just the technical term for randomness. The solution is to rearrange the system using energy. Any decrease in entropy in one part of the system can be compensated by a proportionally larger increase in entropy in another part of the system.
As a simple analogy, consider the problem of cleaning your room. One way is to throw everything that’s lying around into a cupboard. The room definitely looks more ordered but that doesn’t detract from the mess inside the cupboard. Therefore, you haven’t reduced the net entropy of the system—you have merely re-distributed it.
This isn’t just some quirky, obscure thermodynamics loophole. A Nobel Prize in Chemistry was given for understanding how order can be generated from disorder, to Ilya Prigogine in 1977.
So, there is a theoretical basis to explain the origin of complexity in our universe. Self-organization as a phenomenon has been studied in physics, chemistry, biology and many other disciplines, including economics and sociology.
Now, getting back to the point. The three of us, we are all biologists by training. And just to remind our listeners, we still want to talk about organoids. So, let’s work our way up to organoids by showing you how self-organization is necessary—right from the level of molecules to the level of the organism.
At the simplest level, we have molecules that can self-organize into more complex configurations. This happens with proteins, that are formed as a long, disorganized chain of amino acids. But, they fold themselves into complex nanomachines. Some of these can juggle atoms between other molecules, acting as catalysts for important biological reactions.
Next, molecules can self-organize into mega-structures that form important components in cells. Polymerization of small molecules helps to form the protein-based skeleton inside cells and the protein coats of some viruses.
Moving up from molecules, even apparently simple organisms like bacteria can self-organize themselves into marvels of biological architecture called biofilms. In this combined state, bacteria in biofilms can resist antibiotics, fight off the immune system and demonstrate feats of resilience that single cells are incapable of. You can blame biofilms for everything from the gunk on your teeth after a good night’s sleep, to entrenched catheter infections and many other things in between.
All organisms are dependant, to varying degrees, on self-organization to make them what they are. Every multicellular organism, all the way from slime molds to plants and animals, starts off life as a single cell that has to replicate itself to make an embryo.
Shruti: The early embryo is a mass of stem cells without the defining features of a multicellular organism—like a head, tail, limbs and so on. Provided they get the right “cues" or signals, these stem cells are capable of forming a complete organism. Researchers study these cues and other steps in embryo development using animal models like mice.
Even though we know a lot about the major steps of this transformation happens, there are a lot of unanswered questions. Especially with humans, we still don’t know all the details of every step in embryonic development that results in a fully-formed organism. This is because there are species specific differences between human embryo development and the animal model embryo development. This is complicated by some ethical barriers to studying human embryonic development, which we will discuss later in this podcast.
Despite all this, comparisons between studies done on cell cultures, animal embryos and other model systems have yielded many common steps and major themes. One major result can be stated like this: the embryonic stem cells of multicellular organisms can self-organize.
This self-organization can be broken down into three interacting processes. Self-assembly, self-patterning and self-driven morphogenesis. Its important to note that as research progresses, such terminology and definitions have a way of getting replaced and updated. So, with that caveat in mind, what do these terms mean?
Imagine stem cells as lego pieces that can be grown, added and joined to build both the scaffolding and the substance that is necessary to assemble the entire organism. Of course, biological cells are much more flexible and intricate than legos. But, for now, lego blocks serve as a good analogy.
1. Self-assembly: Stem cells can divide into many cells that can perform something similar to an “Avengers assemble". Over time, they can come together, arrange and rearrange themselves to specific positions relative to each other. You can imagine this as red lego pieces stacking together to build a red wall.
2. Self-patterning: Even though the early embryo is a mass of cells that seemingly has no pattern, each cell of this embryo is capable of forming an entire organism. However, specific roles have to be assigned to the different groups of cells in this mass, so cells in a particular region of the embryo can form specific structures. In lego parlance, this is like making the kitchen of the house with red walls and the bedroom with blue-coloured walls.
3. Self-driven morphogenesis: This process deals with how tissue shapes are developed. Cells react to mechanical stress and other signals to produce tissues of varying shape, stiffness and viscosity. I’m going to stretch my lego analogy a little here, so bear with me! Imagine the lego blocks are made of Play-Doh. Not only can you stack them to make strong walls, you now have the added option of pushing and moulding your structure a little to make curved walls.
So all in all, various molecular and physical mechanisms are responsible for producing the effects seen in each of these processes—self assembly, self patterning and self-driven morphogenesis. To continue and finish with the lego story, all the molecular and physical mechanisms form a sort of lego instruction booklet.
Not only does it tell you how to make the walls and windows, but it also tells you how to position and shape them to get the final result. And finally, you can get what is on the front picture of the booklet: a colourful house!
The coolest thing about all of this is that the lego instruction booklet is produced by the stem cells themselves! Whether in artificial cell culture or inside an embryo, cells are literally swimming in a “blueprint". This information is made of varying concentrations of cellular molecules, and mechanical forces that vary in four dimensions—length, breadth, depth and time!
Receptors for this information can be found on the stem cells. These receptors “prefer" certain concentrations of the “blueprint" molecules and forces. So, cells encountering the gradient of blueprint molecules and forces will react in a variety of ways. This includes orienting themselves and moving to a position where the preferred level of molecules and forces is found.
While the cells in an embryo assemble and perform various tricks to assemble the complete organism, some also die to contribute to development. Programmed death, known as apoptosis, can be thought of as a kind of orchestrated, ritual suicide that cells perform under certain conditions. This is also an important event in the development of embryos.
So, alive or dead, stem cells in the embryo plan and execute the development of the entire organism.
Navneet: That’s what happens in a normally developing embryo. The complete development of an embryo is of course an extremely complex process. Relatively speaking, the development of a single organoid is simpler. But that doesn’t mean you can get an organoid by putting some cells in a dish with some nutrients, and then forget about them for a few weeks.
A whole lot of ideas from developmental biology (the branch of biology that deals with how organisms grow and develop) as well as cutting-edge cell-culture techniques are needed to make organoids.
So, let’s shift gears and look into two specific examples of organoids that have been generated and what that requires.
A regular human eyeball is a very complicated structure. It is fluid-filled ball that has a biological “lens" made of proteins. This lens projects images from the outside world onto the retina. The retina is made of sensitive receptors that pick up light and colour information that is projected by the lens. All this information is carried to your brain by a set of optic nerves that come out like a “tail" behind the eyeball. And finally, muscles around the eyeball help it to move and focus the lens on the object of your desire.
In place of the marvel of evolution that is the human eye, scientists at present can engineer a structural facsimile known as an optic cup. The optic cup is the entire surface of the retina—without the fluid, lens and muscle. Essentially, the optic cup is like the sensor of a camera, without the lens or the ability to focus.
To begin growing an optic cup outside the body, we need to start with a colony of stem cells. Much like us, stem cells are social cells. They don’t like to grow and stay all alone. They grow best in small clumps called colonies and each colony can contain up to a few thousand cells.
Starting with stem-cell colonies, scientists first separated them into individual cells and then put them back into plates with tiny wells in an optimal proportion. To these wells, they added certain chemicals that modified the ways in which stem cells grew or responded to their environment. These chemicals include growth factors to influence how the cells divide, antioxidants to make sure the cells remain healthy and patterning factors to nudge the stem cells into becoming cells of the retina and not other organs.
To give the growing structures some sort of basal architecture, the scientists also incorporated a gelatinous mixture called Matrigel. Matrigel helped provide a scaffold for the organoids to grow on and organize. Once all these components were in place, they found that they didn’t need to interfere any more. All they had to do was provide fresh nutrient solution to these organoids every two days and by the end of 18 days, the optic cups developed all by themselves.
The best part is that these optic cups had the exact same structures that a normally and naturally grown eyeball would have!
By studying the intermediate steps in the development of optic cups, scientists could appreciate the complex physical forces that lead to its formation. Such detailed study would simply not be possible by other means.
Another very good example is that of intestinal organoids.
Our intestines are a high-turnover organ where dead or damaged cells are swiftly removed and new cells are added every couple of days. To secrete enzymes and absorb broken down nutrients from food, the inside surface of intestines contain several finger-like projections called villi.
Fun fact: villi is the plural of villus which in Latin means “shaggy hair". Think about that the next time you imagine food passing through your guts! Now, at the very base of these villi is a specific stem cell that gives rise to all the cells of a new villi during the turnover.
Scientists started by taking some of these intestinal stem cells. Employing a similar molecular wizardry as with the optic cups, they could form intestinal villi outside the body.
Here again, scientists used Matrigel to provide physical support to the growing organoids and found that not only can they grow villi in petri dish, but they can also make other cells of the intestinal tissue. These organoids contained on average about 40 villi that surrounded a central circular structure making it seem like a puffed up plastic glove.
By improving the method and tweaking the added factors,, they managed to keep these organoids alive for up to three months!
These organoids have been extremely useful to study inflammatory bowel diseases like Crohn’s, many intestinal cancers and ulcers caused due to bacterial infection by Helicobacter pylori.
Shruti: Now, we’ve been talking intensively about sampling, harvesting and growing cells. All of these are fraught with ethical issues that need a sustained dialogue between scientists and society for resolution.
The foremost ethical issue with organoid research is regarding the origin of cells used to generate them. Organoid research uses stem cells taken largely from early embryos which are three to five days old—obtained either from abortions or failed attempts at in-vitro fertilization or IVF trials. These are called embryonic stem cells, indicating their source. Debates on using human embryos for research have fallen largely into two camps.
The first camp holds the belief that human life begins at conception. Since each embryo is capable of developing into a fully born child, it has the same moral status as that of an individual human being. Therefore, their argument is that using early embryos for research is immoral.
The second camp believes that the embryo gradually develops into a human being over the gestation period. Therefore, early embryo research is permissible until a certain developmental time point. Beyond this, the embryo will surely develop into an organism.
The ethics advisory board of the US department of health, education and welfare in 1979 suggested the 14-day rule, which allowed research on human embryos until 14 days at which point the “primitive streak" appears. The primitive streak is a visible landmark on the embryo that indicates which end of the cell mass will become the head and which, the tail. This was later endorsed by the Warnock Committee report in the UK in 1984. At present, 17 countries have accepted this rule, with 12 of them having enacted it into law.
Abhishek: This rule has stood firm for decades as scientists were unable to culture human embryos beyond nine to 10 days. Recently though, scientists have been able to grow human embryo-like structures from stem cells for longer periods until 12-13 days. As scientists are fast approaching the 14 day developmental milestone, discussing the ethics and policies of embryo research is even more important.
Of course, the restrictions on embryo research are only in place for humans. Scientists continue to study the development of other organisms in great detail, including tadpoles, fishes, insects and mice. This is because the early aspects of development are highly conserved among all organisms. Studying them gives us important clues about human development.
Nevertheless, for results that can be directly applicable to humans, developmental biologists still need a system made of human cells that can approximate human embryonic development at much better fidelity.
An alternative to the dilemma of sourcing stem cells is the use of induced Pluripotent stem cells or iPS cells for short. These iPS cells look and behave very similar to embryonic stem cells and are increasingly used by researchers. Except that they are derived from skin cells! Sandwiched among its many layers, human skin contains cells known as fibroblasts that can be coaxed to become stem cells.
Scientists like prof. Madeline Lancaster use these iPS cells in cell culture to make “mini-brains". The neurons in these mini-brains, which can be made from a patient’s own skin cells, offer a distinct advantage.
Normally, to study a patient’s neurons, a biopsy or tissue sample has to be taken from their brain. But, using iPS cells, a patient’s neurons can be studied without a brain biopsy. Think about that for a second. The cells that would normally be inside the brain can be studied without taking a tissue sample from the brain. Instead, we can use brain organoids made from skin cells!
Navneet: While this sounds like a great solution, it’s still a long way to problem-free experiments. As a society, we still have to decide about the moral status of embryos generated from iPS cells. Further, as iPS cells are an exact genetic copy of the donor, the now-very-real possibility of human cloning raises its head too.
One fortuitous side effect of growing organoids outside the body of the organism is better access to the genetic material. Growing organoids in a controlled cell culture environment makes it that much easier to modify the genes and chromosomes of the progenitor cells.
Scientists have made incredible tools for gene and genome manipulation like CRISPR, that allows very precise removal, addition or editing of any stretch of DNA. This technology could be a boon for patients suffering from hereditary forms of cancers. If an organoid can be grown from a small biopsy of their own afflicted organ, the genes responsible for the cancer can be precisely targeted and modified to make them cancer-free. Finally, these very same “cancer-free organoids" can be transplanted back into the donor.
However, there are myriad ethical concerns here.Will the donors of these iPS cells be aware of the number and magnitude of these manipulations? How much “editing" is permitted before the organoid is an independent entity and not owned by the donor?
Some organoids can also be cryogenically preserved in liquid nitrogen and resuscitated when required. If in the future organoid transplantation becomes a reality, what would the ethics of the procedure be? How will eligibility of the recipients be assessed? As large batches of organoids can be grown in the lab which could potentially be transplanted into several individuals, will the organoids be treated on par with organ transplants? Would the donor or their kin be financially compensated even after they pass away?
A final point about the ethics touches on the question of medical insurance. Researchers are now working to verify the degree to which organoids can reproduce the medical status of a patient.
In fact, as we speak, the Hubrecht Organoid Technology, a not-for-profit organization in Netherlands, is partnering with insurance companies to test just that. They are trying to validate whether the organoid technology can be used to determine the response of the donor to different drug therapies.
As a natural follow-through, do we now see a future where organoid research can potentially be used to allow or refuse medical insurance to an individual?
So as you can see, it is necessary to consider the ethics of organoid research, given the advancements being made using this technology. And to be able to fully harness any benefits, we need to pay equal attention to both sides of ethical arguments before deciding what is best.
Abhishek: Now that we’ve dealt with the ethical aspects of organoids, let’s take a close, hard look at the current reality of organoids. Despite all the hype surrounding this new technology, there are still some crucial scientific hurdles that need to be overcome.
a. Organoids are NOT organs. The important point to remember is that organoids are not just small fractions of the real functioning organs. When we make an organoid, we can only replicate some aspects of the anatomy and function of the original organ. More importantly, we are definitely not at a point where we can grow a fully functioning organ that is ready for transplantation in humans. Conservatively, we can call organoids as “approximations" of organs. This is because they often lack major features of the template organ. For example, lab-grown brain organoids only have a few classes of brain cells. This is problematic in many ways because a normal brain has many, many more different kinds of both neurons and support cells that are connected and arranged for optimal function.
b. Organoids resemble foetal organs more than adult organs. When scientists are using tissues or organoids as a model system to study development or disease, it is important to know what stage of human development they accurately represent. On this front, scientists have found that organoids are closer to foetal and early neonatal forms rather than adult organs. Organoids can still be used to study diseases that have an early onset in life like autism. But late-onset medical conditions such as Alzheimer’s might prove difficult to study.
c. Currently, organoids dont develop in the same way as normal organs do. Growing them in the lab means growing them in tiny wells or flat surfaces of petri dishes—bathed in artificial nutrient medium. However, natural organs develop surrounded by many other types of cells and under the effect of the local chemical and physical forces in their environment. The lack of this special environment leads to some strange effects. For example, lab grown organoids cannot grow their own blood vessels. Thus, after a period of time, the core of the mass of cells becomes deprived of oxygen and nutrients, leading to their eventual death.
d. Another major setback is that they lack the basic morphology and anatomy of normal organs. Recently-grown brain organoids are a good example here. A normally developed human brain is full of deep folds. The peaks and valleys created by these folds are called sulci and gyri respectively. These folds are created due to many reasons. Adhesion among the neurons as they grow in number and size, lack of space in the cranium, etc. Even in the most advanced brain organoids, these sulci and gyri have not been successfully replicated.
Shruti: So, even though we can use organoids to study pathways at a cellular and subcellular level, we cannot use organoids to study issues at a macro scale. At least, not just yet. But, despite the fact that organoid technology is still in its infancy, it has the potential to be extremely useful in many ways. Additionally, a lot of money and effort is currently being invested into improving organoid technology.
a. Organoids are the perfect system for testing new drugs. A newly-developed drug undergoes many different safety checks before it is finally released in the market for humans. These include administering the drug to animal models of the disease, such as mice and rats. Despite being one of the best models, rodent systems are still only an approximation of human systems. Using lab grown human cell derived organoids for these tests gets us one step closer to testing how exactly the drug will act on target cells and tissues. As organoids can be grown from human stem-cells, the only cost involved is to maintain their continuous growth over time. As opposed to growing rats, which requires considerable money, space and manpower allocation.
Also, as organoids are much smaller in comparison to full organs, they can be easily scaled up in production and maintenance. This way, many concentrations of one drug or indeed, many drugs can be screened at the same time, thus minimizing both time and effort. In fact, current studies are working towards making three-dimensional organoids of cancerous growths. The Cold Spring Harbor Laboratory in Long Island, USA has recently received a very generous grant which will help them improve on currently existing technology. This will help immensely in the discovery and testing of new generations of anti-cancer drugs.
b. As much as we scientists pat ourselves on our backs for all the new discoveries and inventions we make, we still have a long way to go when it comes to understanding the biology, biochemistry, genetics and cellular properties of normal organ development. Organoids are a great way to study this. It’s like taking a small snapshot of the organ in question and being able to look at it in much more detail than is usually possible. In the future, molecular and genetic studies of organ development could be done exclusively on organoids.
c. A final point about what organoids have been a boon for: studying mechanisms of new or lesser known diseases. Zika is the perfect example of this. The Zika virus is spread by mosquito bite and pregnant mothers infected with this virus give birth to children with severe brain defects. As foetal brain tissue is extremely difficult to study, scientists infected lab grown brain-organoids with the Zika virus. They found that the virus specifically causes the death of progenitor cells—cells that have the potential to make new and more neurons—something undeniably vital for the growing foetal brain. Efforts are now underway to find drugs that can block Zika virus from infecting these progenitor cells and causing micro-encephaly.
Navneet: We’ve covered many aspects of the organoid issue up to this point. We understand that all the specific examples, complex techniques and finicky ethical issues are too much to remember. So let’s summarize:
While scientists are still far from being able to grow replacement organs for transplantation, stem cell biology and organoid technology are progressing at a rapid pace. Their potential to change humanity has already led to a reassessment of when a developing embryo can be considered a human being.
Any reassignment that happens comes with all the complications that flow from that moral, legal and philosophical status. Irrespective of the ethical flashpoint that this subject has become, medical interventions to treat and prolong human lives cannot be curtailed for long.
Pioneering work has already been initiated in the field of transplantation, with stem cell transplantation for spinal injury, macular degeneration and Parkinson’s disease. While early reports about the safety of such therapy have been positive, only time will tell about their efficacy.
Such experimental therapy has always been the vanguard for new medical technologies. But it is equally crucial to mobilize societal support for research, to allow science and medicine to progress to a point where current novelty becomes status quo.
All: Thank you for listening. We hope you enjoyed this podcast. Check out our blog, sciblabber.wordpress.com, for more of our work. That’s s-c-i-b-l-a-b-b-e-r. You can also follow us on Twitter or Facebook by searching IndSciComm. Thats I-N-D-S-C-I-C-O-M-M.