Organoids.

…finding the middle ground between using a whole genetically modified organism and a flat sheet of cells in a Petri dish…

According to Austin Smith, director of the Stem Cell Institute at the University of Cambridge and also known to his students as the stem cell guy, organoids are “probably the most significant development in the stem-cell field in the last five or six years”.

So what exactly are these futuristic sounding things? In essence, organoids are miniature versions of whole organs that can be grown in vitro, that is, in the lab/outside an organism. Growing these organoids works relatively well and easily for some organ types such as the gut/colon, the liver and the stomach. The main distinguishing feature between an organoid and a real organ is size – organoids rarely grow beyond half a millimetre in diameter. Partly this is because a vascular system, supplying oxygen and nutrients, would need to be integrated to allow larger organoids to grow. However, their three-dimensional organisation and functionality can be surprisingly similar to that of their real counterparts.

OK, great. But where do organoids come from? Although Aristotle proposed that living things could arise from inanimate matter (the theory of spontaneous generation) this way of thinking is generally accepted to have been disproven by Louis Pasteur and others in the nineteenth century. Since organoids do not just pop into existence in scientists’ Petri dishes, one has to grow them from something. Several different starting materials can be used: most easily or intuitively, organoids can be derived from tissue biopsies of the corresponding organ. Alternatively, one can extract adult stem cells from a (mouse) organ and let those cells grow in vitro using special cell/tissue culture conditions. Lastly, it is possible to “persuade” embryonic stem cells or induced pluripotent stem cells to become organoids, provided that one can supply the right signals in the growth medium.

Organoids – so what are they good for? One of the earlier studies (Sato et al., 2009) showed that it is possible to generate intestinal organoids from single intestinal stem cells, in effect backing up the researchers’ claim that there is a well-defined (so-called Lgr5+) stem cell in the gut. Firstly, therefore, organoids help answer questions in the fields of developmental and stem cell biology. A sample “mini-gut” of Sato et al. is shown below: it exhibits crypt-like structures (where stem cells reside and replenish the tissue), villus-like structures and finally, the spheres partly fold in upon themselves to mimic the lumen or interior of the intestine where food would be digested.

A mini-gut - copied directly from Sato et al., 2009

A mini-gut – copied directly from Sato et al., 2009

For the intestine, but also other organs such as the brain, stomach and pancreas, organoids can shed light on questions of basic anatomy: organs are highly complex and often difficult to study in vivo/inside an organism and for some organs we still lack surprising amounts of knowledge. For example, McCracken et al. (2014) used human pluripotent stem cells to grow an organoid that resembles the first half (antrum) of the stomach. Next it will be interesting to find out how to create the second half (fundus), and this, in turn, will help explain how a whole stomach and, eventually, gastrointestinal tract develops.

However, organoids are also being used to study diseases such as cancer, diabetes and neurological disorders. For example, Boj et al. (2015) took biopsies from both mouse and human pancreases (or pancreata, if you are feeling particularly confident) and could obtain corresponding pancreatic organoids. In particular, these organoids recapitulated several different stages of pancreatic cancer and could be genetically modified as well as used to discover new ways in which cancer cells are abnormal.

How to make a pancreatic organoid - copied from Boj et al. (2015)

How to make a pancreatic organoid – copied directly from Boj et al., 2015

In particular, the hope is that, if making organoids becomes fast, cheap and easy, it can be integrated into medical procedures. Van de Wetering et al. (2015) have attempted this for colorectal cancer. In their paper they describe the creation of a “living biobank” of cancerous intestinal organoids from twenty cancer patients. Since the organoids come from a specific patient’s body it can be used for in-depth analysis of mutations, but also for drug testing to see which of the available drugs might work best for the patient in question. This approach is known as personalised medicine and despite being talked about a lot by scientists and the media alike, has yet to really take hold in hospitals.

This crucial last step – from “bench-to-bedside” – really seems to be the major obstacle stopping cool discoveries in the lab from becoming useful in hospitals/treatment settings. Training more medical doctor/PhDs (MD/PhDs), which is far more common in the US than in the UK, would certainly help. These people are able to appreciate the problems (or challenges, if you like) as well as the needs on both sides. But even then, growing organoids for each and every cancer patient would be a huge logistical undertaking, so there would have to be a lot of investment as well. Maybe initiatives such as these “organs-on-chips” – testing drugs on organoids in an organised, high-throughput manner – will bridge the gap?

References:

Boj Sylvia F, Hwang C-I, Baker Lindsey A, Chio Iok In C, Engle Dannielle D, Corbo V, Jager M, Ponz-Sarvise M, Tiriac H, Spector Mona S, Gracanin A, Oni T, Yu Kenneth H, van Boxtel R, Huch M, Rivera Keith D, Wilson John P, Feigin Michael E, Öhlund D, Handly-Santana A, Ardito-Abraham Christine M, Ludwig M, et al. (2015) Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell 160: 324-338

McCracken KW, Cata EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai Y-H, Mayhew CN, Spence JR, Zavros Y, Wells JM (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516: 400-404

Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459: 262-265

van de Wetering M, Francies Hayley E, Francis Joshua M, Bounova G, Iorio F, Pronk A, van Houdt W, van Gorp J, Taylor-Weiner A, Kester L, McLaren-Douglas A, Blokker J, Jaksani S, Bartfeld S, Volckman R, van Sluis P, Li Vivian SW, Seepo S, Sekhar Pedamallu C, Cibulskis K et al (2015) Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients. Cell 161: 933-945

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CRISPR Digest #7

As mentioned numerous times already, CRISPR is a genome-editing technology that can precisely and efficiently target specific DNA sequences and either delete or make changes to genes. The technique has also been modified so that it can be used to switch genes on or off. However, all these functions rely on the matching of a guide RNA molecule to the DNA sequence. Although this step, finding a guide RNA sequence for the desired DNA sequence, is relatively easy, these guide RNAs will also hit other regions of the genome. These so-called off-target effects can interfere with an experiment and ultimately, also hamper attempts to use CRISPR in therapeutic gene-editing settings. Therefore it is crucial to have a better understanding of how the guide RNAs find their targets so that better guides can be designed. Chari et al. (2015) used 1400 guide RNAs in various human cell lines and analysed the off-target effects, which were affected not only by the sequence of bases but also the three-dimensional structure of the targeted DNA sequences (chromatin status). Using this experimental information they designed a new, freely available software that is better at designing guide RNAs. Other programmes rely on algorithms that only take the raw DNA sequence into account.

HIV_H9_T-cell_II

Human T cell (green) during an HIV (yellow) infection – image from Wikemedia Commons

Although this recent paper (Schumann et al., 2015) did not yet utilise this new software, the researchers around Jennifer Doudna, one of the CRISPR pioneers, did succeed in editing human T cells. These are white blood cells that form part of the body’s immune system and are important for clearing infected or cancerous cells, but are themselves the target of the human immunodeficiency virus (HIV; see image). In particular, HIV particles force entry into some T cells by attaching to a protein receptor called CXCR4. Schumann et al. managed to use CRISPR to drastically reduce the expression of CXCR4 on human T cells – eventually such T cells might be injected back into patients to make them more resistant to HIV infection.

Sometimes T cells can become inactivated or blocked, rendering them unable to perform their functions. Often this blockage is carried out by a protein called PD-1, which is located on the surface of T cells. Some cancer immuno-therapies – notably melanoma treatment – target PD-1, allowing the cells to become active once again and attack the malignant cells. Schumann et al. also used CRISPR to make small changes to the PD-1 gene in T cells, showing that it might be possibly to replace or complement some forms of cancer therapy with gene-editing in the future.

References:

Chari R, Mali P, Moosburner M, Church GM (2015) Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nature Methods advance online publication

Schumann K, Lin S, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GE, Ye CJ, Bluestone JA, Doudna JA, Marson A (2015) Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences