No shit - a new way to study diarrhoeal disease

Diarrhoeal disease is awful. I don't think I have to tell anyone that. 

According to the WHO:

Key facts

• Diarrhoeal disease is the second leading cause of death in children under five years old. It is both preventable and treatable.

• Diarrhoeal disease kills 1.5 million children every year.

• Globally, there are about two billion cases of diarrhoeal disease every year.

• Diarrhoeal disease mainly affects children under two years old.

• Diarrhoea is a leading cause of malnutrition in children under five years old.

Behold the wonder of the intestine

 The major causes of disease are infectious agents, namely the likes of noroviruses or rotaviruses. (very good wikipedia article here) But also bacteria, parasites and non microbial assaults. These pathogens enter our bodies via the oro-faecal route where they are able to infect the lining of our intestinal tract. Here they exert their biological effects and manipulate their resident tissue to aid in their replication and spread in the general population. In areas with poor sanitation this is why these diseases are such massive killers. They are acutely adapted to this way of life.

We are somewhat out of our grasp when dealing with - and studying - these organisms. In many cases (noroviruses especially) we cannot even grow the viruses in the lab. Many groups use mouse noroviruses and immunocompromised mice but of course this really isn't optimal for human viruses - especially when pathogenesis and drug development/vaccine studies come along.

So, if we cannot grow them how can we study them? Plus even if we did have the ability to culture them we lack accurate model systems of the human gut to even discover anything worthwhile about the way they grow.

But imagine if we had the ability to study human viruses in the lab, which had been themselves grown in the lab, with human tissue which had also been grown in the lab. 


Enter the human intestinal organoid.  

An intestinal organoid a la Spence et al 2011

An organoid is a structure that resembles an organ. It is not an 'organ' itself taken from a body - it's constructed in the lab to function as one. But organs are pretty complex and none less so than the gut. Its complexity resides in the physical (three dimensional) and biological (cell type/gene expression) planes. So how can we build this 'awesome' work of evolutionary engineering ourselves?

Easy.

You just copy mother nature.

Briefly, the NIH-approved embryonic stem cell line WA09 (originating from the WiCell Research Institute and obtained from the Baylor College of Medicine Human Embryonic Stem Cell Core) was cultured using feeder-free conditions. Stem cells were split at a high density, and, once they reached 85 to 90% confluence, cells were treated for 3 days with a series of differentiation media containing activin A to begin differentiation into definitive endoderm. Definitive endoderm was then treated for 2 to 5 days with growth factors Wnt3a and FGF4, leading to formation of hindgut spheroids. Once spheroids spontaneously detached from monolayers, they were collected, embedded into matrigel (BD Biosciences), and supplied with media supplemented with intestinal growth factors (Wnt3a, R-Spondin1, Noggin, and epidermal growth factor [EGF]; all supplied from R&D Systems). Spheroids matured into intestinal organoids over the course of ~1 to 2 months before they were used for experiments.

It started off like this

Anded up like this

Grown in this way you can 'easily' generate what effectively looks and feels like a human intestinal epithelium (these things also contain some underlying mesenhcymal tissue). They can even be kept alive for over 3 months and the stem cells from which they derive can be frozen and re-animated any time to set up more and more organoid cultures.  


So do these organoids allow viral replication? And can we use them to understand how these viruses infect and cause disease? Well the short answer is yes.


A U.S group from Baylor College of Medicine in Texas were able to cut open the spherical structures and infect them with rotaviruses, even clinical isolates of the virus (OA paper here). In this case the virus needed access to the inside of the structure, the part the resembles in insides of our gut and the place where the cell it likes to replicate in are found. These viruses bound to cells, got in and began replicating and generating new viral particles which could go on and initiate a whole new round of growth. This a whole lot better than using primary monkey kidney cells to isolate and grow the virus or using lab adapted strains.

Organoid structures could be infected with rotaviruses

This was only a preliminary observational study showing a proof of concept that this technique which had previously only been done using mouse stem cells, could work for humans and that they could be used for infection studies. What they didn't show was that rotavirus infection of the organoids bore any resemblance pathogenically to infection in humans. I'm sure this is the next step. What also would be of great use would be to see whether other non-culturable but important human pathogens could be grown this way - I'm thinking noroviruses. What is neat about this work is that we have the ability through recombinant gene vectors to knockdown or over-express any gene we want. The initial paper documented this in 2011. And this is being done with an NIH-approved stem cell line - imagine what could be done with healthy or 'diseased' stem cells.

One problem with this lies in the way they were infected. In order to access the inside of the spherical structure the organoid was cut open using a tungsten needle - an extremely sharp instrument. Who know what kind of effect this would have on nearby cells? But I guess without it this work could not be done and this was probably the safest way of doing so.


Another issue is - like the majority of other in vitro models - there is not immune system component to the structure. And of course the innate immune cells residing in tissues would have a wide ranging effect on the subsequent spread and infection kinetics of the virus. However, you could imagine that in the future it may not be too difficult to add these in to the system. 

Stacy R. Finkbeinera,, Xi-Lei Zenga,, Budi Utamaa,, Robert L. Atmara,b,, Noah F. Shroyerc,, & and Mary K. Estesa,b (2012). Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses mBio, 3 (4) DOI: 10.1128/mBio.00159-12