Mini Lab-grown Organs: the good the bad and the mucousy
By Heather Main
For many patients organ transplant is the only hope of survival or of living a close-to-normal life, but there aren’t enough organs for those who need a transplant. Donor incompatibility is another issue. Stem cell research is changing the way we view laboratory research, disease diagnosis and therapeutic possibilities. And when combined with ‘organoid’ and ‘decellularisation’ technologies, stem cell research is pioneering new ways to regenerate, repair or replace damaged organs and tissues that could revolutionize the field of organ transplantation.
Take a look at your hand. Poke it, clench your first, have a chew on your fingernails and marvel at its complexity. While your hand is really more of an appendage than an organ it is a pretty good example of just how complex it is to recreate a part of the body. The first thing you see is skin. Your hand is covered in skin, but underneath you have muscle, tendons, ligaments, bone, cartilage, blood vessels bringing blood cells, lymphatic vessels draining fluids, neurons sensing heat and touch or making the muscles contract, fingernails and all of this is held together with elastic fibre networks…imagine making a hand in the lab and getting all of those parts to function together (Picture 1)! You could apply this breakdown to any other part of the body or any organ to come to the same conclusion - it would be really complicated to make it in the lab!
Let’s go right back to the start, embryonic development. We all began our lives as 3 types of cells, or ‘germ layers’ which form the major components of the following organs:
- endoderm – digestive system, liver, lungs, thyroid etc.
- mesoderm –skeleton, cardiovascular system, fat, vascular, muscle etc.
- ectoderm – skin, eyes, nervous system etc.
To consider the ‘hand’ example again, all of the cells in your hand come from either mesoderm or ectoderm, but to produce distinct cell types may require very different signals and thus not be possible in a single in-vitro culture. This is often illustrated as a tree with a trunk that forks in 3 directions and continues to branch to final leaves, each distinct leaf represents one functional cell type of the body. If you measure the distance to travel between 2 leaves you get an idea of how early in embryonic development they separated from each other. For example the distance between cells of your heart and brain will be more than between your sweat gland and hair follicle. When we make cells from a particular organ in the lab we must decide which ‘branch’ to follow early on, and this restricts what we are capable of producing (Picture 2).
New techniques attempt to create more mixed cultures where one specific type of cell is not aimed for but rather whole branches of cell types are allowed to self-organise themselves into organ-like structures. While it may be more difficult to know exactly how your favourite cell type is behaving when it is mixed with other cells, some aspects of organ function may be easier to assess. The self-organising organ-like structures are less dependent on signals added by a scientist (thus less dependent on a scientist knowing what signals to add) and instead more dependent on the timing and signals produced by their neighbouring cells. A large part of creating organ-like structures in the lab is to have 3D scaffolds for the cells to live in, the elastic fibre networks that hold the cells of our organs together, enabling the system to function. Here we outline two pioneering methods – each with a different approach to recreating functional organs in the lab. The first utilises existing organ structures from donated organs to which cultured cells are added (re-cellularised organs) and the second, cells allowed to self-organise as mixed cell populations lay down their own scaffolds, somewhat disorganised compared to organs in the body (organoids).
In 2008 scientist Harald Ott showed that cells could be removed from an organ, leaving only the elastic fibre network that holds the organ together, and that this network could then be repopulated with new cells1. Potentially a donor organ that is not immune matched with the recipient could have its cells removed, and these cells would then be replaced with cells matched to, or produced from, the recipient. The process of removing the cells or ‘decellularisation’ is relatively simple requiring breaking open the cells with water and cleaning out the cellular debris with enzymes and detergents. The result is around the same size and shape of the original organ, but with holes where the cells once were, a bit like a sponge that can then serve as a framework for remaking a new organ.
This technique has been used with some success to produce a beating heart1-3, a blood filtering kidney4, a gas exchanging lung5 and even a recellularised limb6. The biggest hurdles to having recellularised organs functioning sufficiently for transplantation will be identifying the exact mixture of cells to use to repopulate the organ scaffold and then producing them from donor matched sources. While some functions of specific organs have been demonstrated in the lab, these repopulated beauties are far from functioning well enough to keep a human alive (Picture 3)
The concept of organoids is that organ specific stem cells grown floating in the lab form little balls that somewhat resemble part of the organ they are from. One of the first examples of this was from the lab of the late Yoshiki Sasai in 2008 who purified retinal progenitor cells, grown in the lab to make cells of the retina7. This was followed with the exciting work of Toshiro Sato in 2009 who showed that purified gut stem cells could be grown in the lab to form ‘mini guts’, called gut organoids, which could be grown over several months8. These 3D structures, around a millimetre long, look like part of the internal structure of the gut and showed some of the function of this organ.
Since then organoids have been produced for many organs including liver9, brain10,1, intestine8,12, pituitary3, eye7,14,15, stomach16, thymus17 and lung18. In each case the organisation and identity of the cells in the organoid match that of part of the organ of interest in its normal state in the body (Picture 4 shows an example for the brain).
While organoids are often referred to as “mini organs”, they are not mini versions of a complete organ. Just as a kitchen would not be referred to as a “mini-house”, the organoid is not a tiny complete organ that just needs time to grow to be a functional organ. For example, a lung organoid may contain cells that produce mucus but not ordered in an open ended tubular network to inhale and exhale air. There will also be other cells required for the lung to function that are missing, like gas exchanging cells. Some organoids contain a huge complexity of cell types, often representing several different parts of the relevant organ, and demonstrate remarkable self-organisation capacity. But still they are not mini-organs. These ones are a bit like an IKEA flatpack: the parts are all there and surprisingly ordered but some assembly would still be needed to make a functional organ and there could be a part or two missing.
There are several different methods for making organoids. The vast majority so far are produced from pluripotent stem cells (PSCs), cells that under the right laboratory conditions can be turned into any cell type of the body. PSCs have been turned into heart2, liver9, brain10,11, intestine11, pituitary13, retina14,15 and stomach16. In all circumstances there is a step where balls of cells ‘self-organise’ to resemble parts of the relevant organ in both the composition of cells within them and the way that these cells are organised. A second method to produce organoids is by purifying out the adult stem cell from the relevant organ and allowing them to grow as balls of cells that also undergo a self-organisation process. This adult stem cell based method has been used to make organoids of gut8,19,20,21, thymus17, lung18 and liver22. Some studies have even started to address the need for cell types from diverse embryonic origins by mixing the separate populations17,18.
Organoids are being used for research to better understand how the organs of our bodies are built and maintained. Organoids can also be used in drug development, as an alternative to animal testing. Twenty percent of drugs in Phase III clinical trials fail due to human liver toxicity23. Liver organoids could be used to test for this in human cells before taking a drug to clinical trials. Organoids could also be used to predict responses to drug treatments. Researchers in the Netherlands are pioneering work to make cancer organoids (tumoroids) in the lab from patients and to test chemotherapy treatments on them19,20. Imagine your doctor testing several treatments on your very own personal tumoroids and from this choosing the most effective, while avoiding the treatments that don’t work and their often serious side effects. While 50% of the population may respond to certain disease therapies, which are then provided as standard, a non-responder profile could be determined before treating you with drugs that won’t work, and alternatives tested. Rather than taking cells from your body and forcing them to grow and respond to signals you provide them, the use of tumoroids allows the cells to behave closer to how they do in the body.
These diagnostic abilities exist outside cancer also, for example in predicting a drug treatment for ‘non-responder’ cystic fibrosis sufferers20. Treatments of the future may include transplantation of recipient-matched organoids to improve the functioning of organs. Organoids are particularly useful with limited availability to tissue biopsies, or in cases like brain diseases where you cannot take tissue biopsies. The field of induced pluripotent stem cells (iPSC) enables any cell type of the body to be made from the skin or blood of diseased individual for study in the lab. When turned into disease relevant cell types any disease behaviours seen can be used for pharmaceutical testing of potential therapies. All of this can happen, identification of therapy, regardless of potential genetic causes being known or not. These new technologies also decrease the use of animals in therapeutic development and decreases artefacts where something that works in rats or mice does not work in humans. Severe autism has been successfully modelled in human brain organoids and showed that the balance of production of neurons in the brain is skewed and this correlates with the severity of the disease11.
Always characterise your stem cells
To a large extent the success of both organoids and re-cellularised organs in mimicking the biological behaviour of the organ depends on the types of cells the researcher starts with. The greatest successes have been seen when a very specific type of stem cell, special to that organ, is used. This mirrors the success of the best-known clinical stem cell treatment, bone marrow transplants. Previously, whole marrow transplants were used. The success of this treatment improved greatly from research that characterised the ‘true blood stem cell’, which is now purified and transplanted. One of the special things about stem cells is that they have a ‘self organising’ capacity, in that they ‘know’ how to build part of the organ in which they reside, something essential to their ability to produce an organ in an embryo or maintain an organ in an adult. As we have seen, scientists are now harnessing this natural program to get their stem cell of interest to create parts of organs outside of the body.
There is no doubt that many improvements can be made from the preliminary studies summarised in the table below, for example replacing artificial laboratory cell lines with adult or pluripotent stem cell derived organ specific stem cells (that retain a self-organising program closer to that of the original organ). Re-cellularisation of a human organ is yet to be trialled and until the ‘mini’ mouse and rat organs are perfected as well as larger mammal trials, it is unlikely that human trials will be allowed. However, though human organoid transplants are yet to occur, the clinical benefits of organoids are already showing and the next years will no doubt see huge successes and clinical advances in disease modelling and therapeutic drug testing in lab grown organoids.
Organ functions shown
Primary rat neonatal cardiac cells through intramural injection
Macroscopic contractions and pump function
Human PSC - cardiovascular progenitor cells
Spontaneous contraction and drug response
Primary rat hepatocyte spheroids
Albumin synthesis, glycogen rosettes, improved survival in hepatectomized rats
1) Human umbilical venous endothelial cell (HUVEC) through renal artery
2) rat neonatal kidney cells (NKC) through ureter
Filters blood, produces urine
Human umbilical venous endothelial cells (HUVEC)
Ventilation and perfusion in vivo
Mouse, monkey, human retina
Human PSC derived Rx+ retinal progenitors
Express genes associated with mature phototransduction
Mouse Lgr5+ adult gut stem cell
Self organising epithelial structure
Human PSC - bile duct differentiation
Cystic fibrosis, polycystic liver disease, Alagille Syndrome
Human PSC - forebrain differentiation
Human PSC - brain differentiation
Human PSC – intestinal differentiation
Permeability and peptide uptake in mouse kidney capsule transplantation
Mouse PSC - anterior pituitary tissue
secreted ACTH in response to corticotrophin releasing hormone
Human PSC – retina differentiation
Mouse PSC – retina differentiation
Human PSC – stomach differentiation
Stomach ulcers and gastric disease
1) Mouse induced thymic epithelial cells derived from fibroblasts
2) Mouse immature thymocytes
3) Mouse fetal thymic mesenchyme
Populated recipient immune system with T cells
1) Primary mouse lung endothelial cells (LuMECs)
2) Mouse bronchioalveolar stem cells (BASCs)
Human Lgr5+ adult gut stem cell mutated to form tumors
Aneuploidy, features of invasive carcinoma upon transplantation to mouse
Human Lgr5+ adult gut tumour stem cell
Human Lgr5+ adult gut stem cell
Stem cell transplant
Adult mouse hepatic progenitor cells
Regenerate hepatocytes and biliary epithelia
The development of mini-organs hold promise for novel future therapeutics, however the short term benefits are going to come from improved abilities to mimic normal and diseased processes outside the human body. It’s not time to throw away single cell type pure populations, they can be used to answer different questions compared to organoids, each has its place. In the same thread, re-cellularised organs and organoids will no doubt fulfil distinct but complementary roles in disease research. Development of these three arms of stem cell biology (single cell type, organoid, re-cellularisation) is set to wildly expand our options for moving away from laboratory animal experimentation, meaning more efficient clinical translation of novel therapeutics to humans and more personalised medicine applications. Whether replacing a dead or dying cell type, an organ region or an entire organ, it is the combination of discoveries from doctors and scientists in developmental biology, physiology/anatomy and disease that will bring the next medical revolutions of cell based disease therapies.
This factsheet was authored by Heather Main and reviewed by Paul Knoepfler. Images used by kind permission, credits below.
Picture 1: Anatomy of Hand
Credit: Hikmet Gümüş © Creative Commons
Illustration shows what a hand looks like under need the skin, demonstrating the sight of muscle, tendons, ligaments, bone, cartilage, blood vessels bringing blood cells, lymphatic vessels draining fluids, neurons sensing heat and touch.
Picture 2: Developmental tree
Credit: Frazer Gibson
The developmental tree presents an overview of the development of the organs/tissues, and the "early development" stages through the three germ layers (ectoderm, mesoderm and endoderm). Cells made from a particular organ in the lab must decide which ‘branch’ to follow early on, and this restricts what scientists are capable of producing. Techniques are needed to produce the right types of cells from different branches and then put them together in a way that replicates the elastic fibre networks that hold the organ together allowing the system to function.
Picture 3: Decellularised vs recellularised organs
Credit: Image adapted from figures 1c and 4b from the paper, ‘Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Harald C Ott, Thomas S Matthiesen, Saik-Kia Goh, Lauren D Black, Stefan M Kren, Theoden I Netoff & Doris A Taylor. Nature Medicine 14, 213 - 221 (2008)’.
The left panel of the image shows a photograph of a decellularised whole rat heart. The heart becomes more translucent as the cellular material is washed out. In contrast, the right panel of the image shows a photograph of a recellularised whole rat heart. The heart returns to a pink colour as it becomes repopulated with new cells.
Picture 4: Mouse brain vs. Human Cerebral Organoid
Credit: Madeline A. Lancaster and Marko Repic/IMBA
Since 2009, organoids have been produced for many organs such as the brain: Comparison of neurons (green) hugging neural stem cells (red) in a mouse brain (left) and in a human cerebral organoid (right).
Picture 5: Stomach
Credit: Image adapted from figure 2f from the paper, ‘Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. McCracken KW, Cata EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai YH, Mayhew CN, Spence JR, Zavros Y, Wells JM. Nature 516:400-404 (2014)’.
Since 2009, organoids have been produced for many organs such as the stomach. Human gastric organoid showing similar organisation and folding to a normal stomach organ.
Movie: Beating recellularised heart
Credit: Supplementary movie 3, ‘Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Harald C Ott, Thomas S Matthiesen, Saik-Kia Goh, Lauren D Black, Stefan M Kren, Theoden I Netoff & Doris A Taylor. Nature Medicine 14, 213 - 221 (2008)’.
Movie shows a recellularised heart with contractions (wall movements).
Movie: Unidirectional mucin flow in bronchiolar organoids
Mucin can be seen moving in one direction in a bronchiolar/lung organoids
Credit: Dr. Carla Kim, Principal Investigator in the Department of Genetics, Boston Children’s Hospital and Harvard Medical School. Movie shown at the ISSCR 2015.
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