Organoids: Mini-Intestine And Mini-Brain

21. February 2017

They won't replace animal experiments and clinical trials, but organoids derived from stem cells already offer many advantages. Zika virus could be studied in a mini-brain. The bacterium Helicobacter pylori is best able to be studied in a mini-gastric system as well.

Even though patients with cystic fibrosis usually reach adulthood these days due to improved treatment methods, the disease is still one of the major unsolved problems in medicine. Nearly 2,000 different mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene have been previously described. The prospects for an effective therapy are low, especially for patients with rare variants.

Test model for rare diseases, spare parts warehouse in cases of damage

In October last year Science Translational Medicine carried an article by a Dutch research group led by Hans Clevers, who could possibly end the roulette game nature of the treatment of these rare variants. Working with intestinal stem cells derived from two cystic fibrosis subjects, the researchers from Utrecht cultivated a “mini-intestine” on which the effects of different therapeutic approaches could be tested in the laboratory. Looking at this mini test at least, the laboratory data has actually been able to be successfully transferred into clinical practice.

Already in 2007 Clevers and his colleagues discovered stem cells, working with which “organoids” up to two millimetres in size could be cultivated. These intestinal stem cells, according to Clevers, “are probably the most active in our body”, but they are by no means the only pluripotent immortal cells that can be used to produce “laboratory organs”. Suitable cells can be isolated from the prostate and pancreas, as they also can from the stomach, kidney, or even the central nervous system as well.

It’s not always the case that such organ model systems are quite so discernibly associated with clinical use; the cell clusters are often first employed in making more detailed studies of functions and circuits in healthy or diseased state of certain organs. However, with respect to the model system “skin”, production has already reached serial scale. “Laboratory skin” obtained from the machine is now often used as a clinically proven cover material on burns.

Tumorigenesis in glass

Two years ago, Clevers began to assemble a collection of colorectal tumour cells in creating an organoid-biobank, in order to test out tumour treatments using this model. Together with his colleague David Truveson from Cold Spring Harbor, he also developed a similar bank for mini organs constituted from pancreatic cancer cells. Organoids have also become an important tool in developing models for the development of carcinomas. Inserting targeted mutations using CRISPR/ Cas9 technique is today no longer a great feat. Two independent studies have for instance showed how four sequential mutations create an adenocarcinoma from a wild-type strain.

ImageJ=1.44o unit=micron

Mini kidney from one patient © Benjamin Freedman & Joseph Bonventre labs

In 2014 James Wells from Cincinnati Children’s Hospital succeeded in cultivating a mini stomach in a petri dish. The stomach pest Helicobacter pylori felt at home within this structure and was thus able to be well studied. In the fall of 2015, news arrived from the Australian city of Melbourne about the cultivation of a tiny kidneys which resembles those found in the early embryo.

Two precursor cell populations for laying the arrangement of renal tubules and plant nephrons formed a network of connective tissue and provisional vascular structures, with a gene expression pattern that resembles the early human embryo. Given how many different tissue types exist in the adult kidney, the authors did not want to conjure up a picture of a potential renal replacement born in a retort. However, the mini-kidney served as a good model for studying problematic E.coli infection with consequential haemolytic uraemia due to the respective toxins present.

Food for zika viruses

The replication of influenza viruses was the subject of impressive studies conducted by virologists on a lung model derived from a patient with a signalling-defect. Most recently, the development of a neural organ model permitted further progress in research on the dreaded zika-virus. At the Johns Hopkins University in Baltimore, scientists infected an iPSC forebrain-organoid and were thus able to modify the microencephaly induced by the virus. The typical symptoms of virus-associated cell death, slowed proliferation and reduced cell layer volume appeared in the laboratory model as well as in-vivo infected embryos. Thus it also seems to be possible to test various treatment options better than before prior to use in humans. Finally, researchers at Yale University through their work on mini-brains were able to identify differences at the molecular level between normal patients and those on the autism spectrum.


Infection of mini-brains with zika virus © Qian and Nguyen et al./Cell 2016

Great difficulties however have been experienced by experts in regenerative medicine in the cultivation of liver cells. “We cannot even maintain them in culture for a few hours”, says Takanori Takebe from Yokohama City University. At any rate, the cultivation of small lentil-sized “liver buds” through a coculturing of hepatoblasts from induced pluripotent stem cells (iPS), together with mesenchymal and endothelial stem cells, was successful; the product can be compared to the liver of a six-week-old embryo. While one of these individual buds does not regenerate an organ, the restoration of a poisoned organ has at least been successful in animal studies, if dozens of these organoids collaborate.

No alternative to animal experiments

With some of these organ systems clinical application is not too far away, nevertheless with many of them their primary application is in basic research in studying the development of diseases, or in studying the respective organ’s physiology. In the long run they will not completely replace animal experiments. Moreover, interaction with neighbouring and distant organ systems is absent when working with organoids born in the petri dish.

When compared to other 2D organ cell cultures, organoids from stem cells are, however, closer to the physiology of the living body. Their cultivation has several advantages over transformed cell lines in that their genomes show little of the mutation impacts which arise in established cell lines through excessively lengthy culturing periods in the incubator. Finally, many organoid cell cultures often need only a few stem cells, which then differentiate in the presence of appropriate factors into the desired tissue.

Unexplained interaction with neighbours

However, 3D organ cultures are only partially suitable in studying inflammatory disease paths. Interaction with the immune system can only be simulated in the culture to a limited extent. Likewise, biomechanical forces can hardly develop in the culture dish as they do in living organisms. 3D organ culture requirements for specific growth factors are still generally insufficiently studied. This explains, for example, why the cultivation of a mini-ovary has so far still failed. Furthermore, the large phenotypic differences between mini organs developed in one culture and another, and sometimes even within the one culture, mostly remain unexplained. A major role in this context may also be being played by the structural substance used.

Nevertheless, experts predict a great future for the three-dimensional creations derived from circulatory system cultures. This is also made evident by the awarding of the prestigious Körber Prize for European Science. Almost ten years ago Hans Clevers had the fortitude to produce small cell organs from intestinal stem cells which he discovered, and which are playing an indispensable role in the testing of medications and the study of tumours. Worldwide, it is estimated that around 200 laboratories today produce artificially born small organs. This number will certainly become even greater.

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