Organoids as an in vitro model of human development and disease


Do you know that organoids can mimic many features of whole tissue and for that reason they are a useful system to investigate the relationship between cell polarity and tissue organization?

Since I work with organoids in my PhD project, I thought to summarize the key events in the history of organoid cultures and outline the potential of organoid technology for future biomedical research.

In the last ten years, there has been a dramatic surge in the number of publications related to organoids, however, this field of research began many decades ago (Fig. 1) (Simian et al. 2016). Organoids revealed their first popularity in the years 1965-1985, mostly in classic development biology experiments that sought to describe organogenesis by cell dissociation and reaggregation experiments. The past 10 years have witnessed a revival of the organoid with a different definition: an organoid is now defined as a 3D structure grown from stem cells and consisting of organ-specific cell types that self-organizes through cell sorting and spatially restricted lineage commitment (Clevers et al. 2016).

Figure 1. Number of publications per year on organoids and 3D cell cultures according to PubMed. The number of publications per year is graphed for the following PubMed searches: “organoids” is shown in red squares, “organoid” is shown in blue circles, and “3D cell culture” is shown in green triangles (Simian et al. 2016).

The second revival of organoids took place in the research group of Prof. Hans Clevers in Utrecht which resulted in two important discoveries. The first finding was Lgr5 as a stem cell marker by Prof. Hans Clevers and his colleague Dr. Nick Barker in 2007 (Barker et al. 2007). They identified Lgr5+ stem cells in the crypts of mouse small intestine and colon, but not in the villi (Fig. 2).

Figure 2. Restricted expression of an Lgr5-lacZ reporter gene (blue) in adult mouse small intestine (Barker et al. 2007).

These stem cells are inserted between Paneth cells and there are around six to eight Lgr5+ cells per crypt (Fig. 2). Using a mouse in which a green fluorescent protein (GFP)/tamoxifen-inducible Cre recombinase cassette was integrated into the Lgr5 locus, they showed by lineage tracing that Lgr5+ cells can generate all intestinal cell types: globet cells, enteroendocrine cells, enterocytes and Paneth cells. They showed that cells are generated in the crypt and after 3-5 days migrating they reach the villus tip where they undergo apoptosis before being lost into the intestinal lumen (Fig. 3) (Barker et al. 2007).

Figure 3. Lineage tracing in the small intestine. Lgr5-EGFP-IRES-creERT2 knock-in mouse crossed with Rosa26-lacZ reporter mice 12 hours after tamoxifen injection. Histological analysis of LacZ activity 1 day after induction (a), 5 days after induction (b) and 60 days after induction (c). (Barker et al. 2007)

Once identified Lgr5 as a stem cell marker, the second important point took place in 2009 when Prof. Hans Clevers and his colleague Dr. Toshiro Sato developed a method of using Lgr5+ cells to generate a long-term culture in vivo that resembles the human intestine and they called organoid to this artificial structure (Sato et al. 2009). They generated for the first time, gut organoids from adult intestinal stem cells cultured in 3D in Matrigel. From this point onward, an organoid was defined as an in vitro 3D cellular cluster grown from primary tissue, embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) and capable of self-renewal and self-organization, and exhibiting similar organ functionality as the tissue of origin (Fatehullah et al. 2016).

Sato grew the first organoids by suspending mouse intestinal crypts in Matrigel, which supported growth within a 3D matrix, and the laminin-rich nature of Matrigel mimics the microenvironment of the crypt base in vivo (Sato et al. 2009). The Matrigel is an important component of the system that provides a scaffold and additional supplementation of signalling cues via basement membrane ligands to support cell attachment and survival as well as organoid formation (Yin et al. 2016). Crypt growth required the presence of the growth factors: EGF, R-spondin 1 and Noggin and this growth factors were added to the medium to design a microenvironment that resemble the in vivo structure (Fig.4)

Figure 4. Organoids are cultured in Matrigel surrounded by culture media supplemented with niche factors specific to the organoid type.

Organoids can be expanded indefinitely, cryopreserved as biobanks and easily manipulate. Due to these features, organoids are an excellent model system for a wide range of research applications, summarized below.

 

  • Tissue development and disease modelling. Organoid system is a valuable tool in disease modelling and one of the advantages of this technology is the ability to expand both tissue-specific stem cells and their differentiated progeny from very limited amounts of starting material such as biopsies. This system allows researchers to study the processes that govern embryonic development, tissue homeostasis and manifestation of diseases. Organoids can be used as a model to study changes in tissue architecture and tissue polarity during cancer progression (Fig. 5).

    Figure 5. Fatehullah et al. 2013 showed that the loss of adenomatous polyposis coli (APC) (common mutation in colorectal cancers) results in altered tissue organization in organoids. (a) The highly branched morphology of a wild-type organoid is turned into a (b) cyst-like structure in intestinal tissue lacking APC. Paneth cells are arranged in an alternating pattern at the base of the branched crypt-like structures in wild-type organoids (c) (d) stained with anti-lysozyme antibodies (green). However, (e) in APC(fl/fl )organoids, Paneth cells are distributed randomly throughout the organoid.

  • Drug testing. Organoids have the potential to be used for testing efficacy and toxicity of drug compounds and this system enables the discard of ineffective drugs before animal trials.  Moreover, organoids can be grown in a 96-well even 384-well format enabling multiple replicates using a wide range of samples and drug concentrations, thus making them appropriate for high throughput screening (Young et al. 2016).
  • Regenerative medicine and patient derived organoids. It has been demonstrated the feasibility of expanding organoids from adult stem cells (aSCs) followed by safe transplantation into animals. Also, the use of genome editing techniques as CRISPR/Cas9 with patient derived organoids, allows the correction of mutations and the generation of healthy epithelia capable of repopulating diseased tissue and undergo transplantation (Fatehullah et al. 2016).