Providing the Right Cues for Organoid Development

Jul 2, 2018 | Trends

Organoids are in vitro-derived miniaturized organs that exhibit self-organization and recapitulate the functions of the in vivo organ they represent. For organoids to mimic their real-life counterparts as much as possible, they must receive appropriate physical and biochemical cues. These cues might include:

Physical cues:

These provide support for cell attachment and survival.

Examples include laminin, collagen, and fibronectins. These factors are often derived from non-human species (e.g., mouse, rat, or cow), but they may also be by-products of human tissues, for example, laminin and collagen from human placenta, and laminin from human fibroblasts. Alternatively, they may be human proteins recombinantly expressed in heterologous hosts, for example, human collagen expressed in tobacco plants.

Biochemical cues:

Molecules that modulate the signaling pathways governing proliferation, differentiation, and self-renewal.

Examples include:

  1. Growth factors such as epidermal growth factor, fibroblast growth factor 10 (FGF10), hepatocyte growth factor, and others
  2. Inhibitors of glycogen synthase kinase 3 beta (GSK3β), involved in nerve cell development and body patterning, transforming growth factor beta (TGF-β), histone deacetylases, a family of enzymes that plays widespread roles in cell signaling, signal transduction, cell growth, cell cycle and death, and transcriptional control WNT3A, a key member of the canonical Wnt signaling pathway that plays roles in embryonic and neural development, cell differentiation, proliferation, and tumorigenesis. R-spondin, which belongs to a family of WNT regulators, may also be used as a cue for organoid development.

The list of physical and biochemical cues above is not exhaustive and the optimal combination will depend on the type of tissue/organ in question and the level of differentiation required. In any case, the cues are delivered to organoid progenitor cells (most often embryonic or induced pluripotent stem cells, or adult stem cells) through materials that mimic the extracellular matrix.

Matrigel – The Universal Extracellular Matrix for Organoid Culture

Matrigel is probably the most widely used extracellular matrix material in organoid generation nowadays. This is a soluble extract of basement membrane proteins secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells that resembles the complex extracellular environment of most tissue types.

Standard Matrigel contains laminins and collagen IV, which are the major support proteins required by the basal lamina. Heparin sulfate, another major component of Matrigel, is a proteoglycan found near the cell surface that binds protein ligands to regulate a number of biological processes, including but not limited to, developmental pathways, blood clotting and blood vessel formation, and tumor metastasis. Other components present in Matrigel include growth factors and entactins (also known as nidogens), a family of glycoproteins located in the basal lamina that play an important role during organ formation in late embryogenesis. Matrigel can also be supplemented with other materials depending the level of differentiation desired, the source of the cells to be cultured, and the desired tissue type.

Matrigel polymerizes in response to temperature, and forms a 3D gel at 37 °C. Besides the roles described above, entactins also connect laminins and collagens to reinforce the hydrogel structure of Matrigel. Polymerized Matrigel supports cell morphogenesis and differentiation in epithelial, endothelial, muscle and nerve cells, as well as supporting tumor growth, making it also ideal for cancer organoid generation. Matrigel is produced and marketed by a range of vendors, either as Matrigel or other trade names. Most preparations include an antibiotic such as gentamicin for sterility.

Which Cues to Use and When?

For organoids where minimal differentiation is required in downstream applications, organoid progenitor cells are provided with the minimal information they need to differentiate into the desired tissue type, along with suitable nutrients and growth conditions, and are then left to their own devices, where intrinsic self-organization is allowed to direct the tissue towards its 3D form.

Let’s take brain organoids as an example. They can be generated from human pluripotent stem cell-derived neuroectodermal embryoid bodies embedded in Matrigel without any further cues. The result of this approach is structures that contain many brain regions, including cortex, as well as possible mesodermal and endodermal lineages. For region-specific brain organoids, it is possible to direct hPSCs towards structures that consist primarily of either dorsal or ventral forebrain tissue. Region-specific approaches to organoid generation require solid knowledge about the signals and pathways that govern development of the organ type. This knowledge can be applied to select the cues for delivery for the developing organoids (1).

While we can’t provide a complete overview of the cues necessary for all organoids here, we stress the importance of researching what is right for your tissue type before you invest time and money in organoid generation. Here is a couple of examples that demonstrate the diversity of cues depending on organoid type:

Stomach organoids derived from human pluripotent stem cells

For endoderm induction, these organoids require an inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) and certain members of the human TGF-β family, such as bone morphogenetic protein 5 (BMP5) and Activin A. For spheroid generation, WNT, FGF, Noggin and Retinoic acid are required. Organoid formation requires the presence of Noggin, Retinoic acid, and EGF, while EGF is required for maturation. With this array of cues, and assuming everything works as it should, one can expect to obtain LGR5+ cells, mucous cells and gastric endocrine cells (2).

* LGR5 is a marker of adult intestinal stem cells.

Kidney organoids derived from human pluripotent stem cells

These organoids require WNT and a GSK3α inhibitor for mesoderm induction, and GSK3α inhibition and the presence of FGF9 for organoid formation. If all goes well, this approach should yield organoids that contain nephrons and epithelial cells (3).

We hope that this brief overview highlighted the great diversity and flexibility that exists in organoid generation, and we would be happy to hear about your own experiences. Have you generated any obscure organoid types and how did you do it? Drop us a line in the comments!


  1. de Souza N. Organoids. Nature Methods. 2018;15:23.
  2. McCracken KW, Catá EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature. 2014;516(7531):400-4.
  3. Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 2015;526(7574):564-8.

Article by Karen O’Hanlon Cohrt PhD. Contact Karen at 

Karen O’Hanlon Cohrt is a Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD,  Karen moved to Denmark and held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for writing helps her to keep abreast of exciting research developments as they unfold. Follow Karen on Twitter @KarenOHCohrt. Karen has been a science writer since 2014; you can find her other work on her portfolio.