Organoid

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File:Intestinal organoid.PNG
Intestinal organoid grown from Lgr5+ stem cells.

An organoid is a three-dimensional organ-bud grown in vitro that shows realistic micro-anatomy. The technique for growing organoids has rapidly improved since the early 2010s, and it was named by The Scientist as one of the biggest scientific advancements of 2013.[1]

History

Attempts to create organs ‘’in vitro’’ started with one of the first dissociation- reaggregation experiment[2] where Henry Van Peters Wilson demonstrated that mechanically dissociated sponge cells can reaggregate and self-organize to generate a whole organism[3]. In the subsequent decades, multiple labs were able to generate different types of organs [4] ‘’in vitro’’ through the dissociation and reaggregation of organ tissues obtained from amphibians[5] and embryonic chicks[6]. The phenomena of mechanically dissosciated cells aggregating and reorganizing to reform the tissue they were obtained from subsequently led to the development of the differential adhesion theory by Malcolm Steinberg [7].

With the advent of the field of stem cell biology, the potential of stem cells in to form organs in ‘’vitro’’ was realized early on with the observation that when stem cells form teratomas or embryoid bodies, the differentiated cells can organize into different structures resembling those found in multiple tissue types[8] .The advent of the field of organoids, started with a shift from culturing and differentiating stem cells in 2D media, to 3D media to allow for the development of the complex 3-dimensional structures of organs[9] .Since 1987, researchers have devised different methods for 3-D culturing, and were able to utilize different types of stem cells to generate organoids resembling a multitude of organs[10].

In 2008, Yoshiki Sasai and his team at RIKEN institute demonstrated that stem cells can be coaxed into balls of neural cells that self-organize into distinctive layers.[11] In 2009 the Laboratory of Hans Clevers at Hubrecht Institute and University Medical Center Utrecht, The Netherlands showed that single LGR5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.[12] In 2010, Mathieu Unbekandt & Jamie A. Davies demonstrated the production of renal organoids from murine fetus-derived renogenic stem cells:[13] subsequent reports showed significant physiological function of these organoids in vitro[14] and in vivo.[15]

In 2013, Madeline Lancaster at the Austrian Academy of Sciences established a protocol for culturing cerebral organoids derived from stem cells that mimic the developing human brain's cellular organization.[16] In 2014, Artem Shkumatov et al. at the University of Illinois at Urbana-Champaign demonstrated that cardiovascular organoids can be formed from ES cells through modulation of the substrate stiffness, to which they adhere. Physiological stiffness promoted three-dimensionality of EBs and cardiomyogenic differentiation.[17]

Takebe et al. demonstrate a generalized method for organ bud formation from diverse tissues by combining pluripotent stem cell-derived tissue-specific progenitors or relevant tissue samples with endothelial cells and mesenchymal stem cells (MSCs). They suggested that the less mature tissues, or organ buds, generated through the self-organized condensation principle might be the most efficient approach toward the reconstitution of mature organ functions after transplantation, rather than condensates generated from cells of a more advanced stage[18]

Properties

Lancaster et al[19]. defines an organoid as a collection of organ-specific cell types that develops from stem cells or organ progenitors, self-organizes through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo, and exhibits the following properties:

  • Have more than one organ-specific cell types
  • Capable of recapitulating some specific function of the organ (eg. excretion, filtration, neural activity, contraction)
  • Its cells are grouped together and spatially organized similar to an organ

Process

Organoid formation generally requires culturing the stem cells or progenitor cells in a 3D media [20].The 3D media can be made using an extracellular matrix hydrogel Matrigel, which is a laminin- rich extracellular matrix that is secreted by the Engelbreth- Holm-Swarm tumor line[21] . Organoid bodies can then be made through embedding stem cells in the 3D media [22].When pluripotent stem cells are used for the creation of the organoid, the cells are usually, but not all the time, allowed to form embryoid bodies [23].Those embryoid bodies are then pharmacologically treated with patterning factors to drive the formation of the desired organoid identity[24].Organoids have also been created using adult stem cells extracted from the target organ, and cultured in 3D media[25] .


Basic research with organoids

Organoids are an excellent tool to study a multitude of biological processes ranging from developmental biology to organ structure, function, and organization. For example, while research on stem cells and regulation of stemness was initially the field of application of intestinal orgaoinds,[12] they are now also used to study e.g. uptake of nutrients, drug transport and secretion of incretin hormones.[26] This is of great relevance in the context of malabsorption diseases as well as metabolic diseases such as obesity, insulin resistance, and diabetes.

Organoids as a model for developmental biology

Organoids offer researchers an exceptional model to study developmental biology [27] . Since the identification of pluripotent stem cells, there have been great advancements in directing pluripotent stem cells fate ‘’in vitro’’ using 2D cultures[28]. These advancements in PSC fate direction, coupled with the advancements in 3D culturing techniques allowed for the creation of organoids that recapitulate the properties of various specific subregions of a multitude of human organs[29]. The use of these organoids has thus greatly contributed to expanding our understanding of the processes of organogenesis, and the the field of developmental biology[30]. In central nervous system development, for example, organoids have contributed to our understanding of the physical forces that underlie retinal cup formation[31][32][33], the species-specific dimensionality of the eye[34][35][36], and the factors underlying the control of progenitor cells in the cortex[37][38][39].

Organoid models of disease

Organoids provide an opportunity to create cellular models of human disease, which can be studied in the laboratory to better understand the causes of disease and identify possible treatments. In one example, the genome editing system called CRISPR was applied to human pluripotent stem cells to introduce targeted mutations in genes relevant to two different kidney diseases, polycystic kidney disease and focal segmental glomerulosclerosis.[40] These CRISPR-modified pluripotent stem cells were subsequently grown into human kidney organoids, which exhibited disease-specific phenotypes. Kidney organoids from stem cells with polycystic kidney disease mutations formed large, translucent cyst structures from kidney tubules. Kidney organoids with mutations in a gene linked to focal segmental glomerulosclerosis developed junctional defects between podocytes, the filtering cells affected in that disease. Importantly, these disease phenotypes were absent in control organoids of identical genetic background, but lacking the CRISPR mutations.[40] These experiments demonstrate how organoids can be utilized to create complex models of human disease in the laboratory, which recapitulate tissue-level phenotypes in a petri dish.

Personalised medicine with organoids

Intestinal organoids grown from rectal biopsies using culture protocols established by the Clevers group have been used to model cystic fibrosis,[41] and led to the first application of organoids for personalised treatment.[42] Cystic fibrosis is an inherited disease that is caused by gene mutations of the cystic fibrosis transmembrane conductance regulator gene that encodes an epithelial ion channel necessary for healthy epithelial surface fluids. Studies by the laboratory of Jeffrey Beekman (Wilhelmina Children's Hospital, University Medical Center Utrecht, The Netherlands) described in 2013 that stimulation of colorectal organoids with cAMP-raising agonists such as forskolin or cholera toxin induced rapid swelling of organoids in a fully CFTR dependent manner.[41] Whereas organoids from non-cystic fibrosis subjects swell in response to forskolin as a consequence of fluid transport into the organoids' lumens, this is severely reduced or absent in organoids derived from people with cystic fibrosis. Swelling could be restored by therapeutics that repair the CFTR protein (CFTR modulators), indicating that individual responses to CFTR modulating therapy could be quantitated in a preclinical laboratory setting. Schwank et al also demonstrated that the intestinal cystic fibrosis organoid phenotype could be repaired by CRISPR-Cas9 gene editing in 2013.[43] Follow up studies by Dekkers et al in 2016 revealed that quantitative differences in forskolin-induced swelling between intestinal organoids derived from people with cystic fibrosis associate with known diagnostic and prognostic markers such as CFTR gene mutations or in vivo biomarkers of CFTR function.[42] In addition, the authors demonstrated that CFTR modulator responses in intestinal organoids with specific CFTR mutations correlated with published clinical trial data of these treatments. This led to preclinical studies where organoids from patients with extremely rare CFTR mutations for who no treatment was registered were found to respond strongly to a clinically available CFTR modulator. The suggested clinical benefit of treatment for these subjects based on the preclinical organoid test was subsequently confirmed upon clinical introduction of treatment by members of the clinical CF center under supervision of Kors van der Ent (Department of Paediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Center Utrecht, The Netherlands). These studies show for the first time that organoids can be used for the individual tailoring of therapy or personalised medicine.

Types of organoids

A multitude of organ structures have been recapitulated using organoids[44]. This section aims to outline the state of the field as of now through providing an abridged list of the organoids that have been successfully created, along with a brief outline based on the most recent literature for each organoid, and examples of how it has been utilized in research.

Thymic organoids recapitulate at least partly the architecture and stem-cell niche functionality of the thymus[47] , which is a lymphoid organ where T cells mature. Thymic organoids have been generated through the seeding of thymic stromal cells in 3-dimensional culture[48]. Thymic organoids seem to successfully recapitulate the thymus’s function, as co-culturing human hematopoietic or bone marrow stem cells with mouse thymic organoids resulted in the production of T-cells[49].
  • Testicular Organoid
  • Hepatic Organoid[50]
  • Pancreatic Organoid[51]
  • Gut Organoid
Gut organoids refer to organoids that recapitulate structures of the gastrointestinal tract. The gastrointestinal tract arises from the endoderm, which during development forms a tube that can be divided in three distinct regions, which give rise to, along with other organs, the following sections of the gastrointestinal tract[52]:
  1. The Foregut gives rise to the oral cavity and the stomach
  2. The Midgut gives rise to the small intestines and the ascending colon
  3. The Hindgut gives rise to the rectum and the rest of the colon
Organoids have been created for the following structures of the gastrointestinal tract:
  • Intestintial organoid[12]
Intestinal organoids have thus far been among the gut organoids to be generated directly from pluripotent stem cells[53]. One way human pluripotent stem cells can be driven to form intestinal organoids is through first the application of activin A to drive the cells into a mesoendodermal identity, followed by the pharmacological upregulation of Wnt3a and Fgf4 signaling pathways as they have been demonstrated to promote posterior gut fate[54]. Intestinal organoids have also been generated from intestinal stem cells, extracted from adult tissue and cultured in 3D media[55] . Intestinal organoids recapitulate the crypt-Villus structure of the intestine, by recapitulating its function, physiology and organization, and maintaining all the cell types found normally in the structure including intestinal stem cells[56]. Intestinal organoids recapitulate the crypt-Villus structure to high degree of fidelity that they have been successfully transplanted to mouse intestines, and are hence highly regarded as a valuable model for research[57]. One of the fields of research that intestinal organoids have been utilized is that of stem cell niche. Intestinal organoids were used to study the nature of the intestinal stem cell niche, and research done with them demonstrated the positive role IL-22 has in maintaining in intestinal stem cells[58], along with demonstrating the roles of other cell types like neurons and fibroblasts in maintenance of intestinal tem cells[59]. Intestinal organoids have also demonstrated therapeutic potential. A 2014 study published in Nature[60], for example, reported that FOXO1 inhibition in intestinal organoids resulted in the formation of glucose responsive insulin producing cells. The data from this study thus acts as a proof of concept of the use of intestinal organoids as a therapeutic approach for diabetes.
  • Stomach or Gastric Organoid
Gastric organoids recapitulate at least partly the physiology of the stomach. Gastric organoids have been generated directly from pluripotent stem cells through the temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling pathways in three-dimensional culture conditions[61] . Gastric organoids have also been generated using LGR5 expressing stomach adult stem cells[62]. Gastric organoids have been used as model for the study of cancer [63] [64] along with human disease[65] and development[66]. For example one study[67] investigated the underlying genetic alterations behind a patient’s metastatic tumor population, and identified that unlike the patient’s primary tumor, the metastasis had both alleles of the TGFBR2 gene mutated. To further asses the role of TGFBR2 in the metastasis, the investigators created organoids where TGFBR2 expression is knocked down, through which they were able to demonstrate that reduced TGFBR2 activity leads to invasion and metastasis of cancerous tumors both in vitro and in vivo.
  • Lingual organoid
Lingual organoids are organoids that recapitulate, at least partly, aspects of the tongue physiology. Epithelial lingual organoids have been generated using Bmi1 expressing epithelial stem cells in three-dimensional culture conditions through the manipulation of EGF, WNT, and TGF-β[68]. This organoid culture, however, lacks taste cells, as these cells do not arise from Bmi1 expressing epithelial stem cells[69]. Lingual taste bud organoids containing taste cells, however, have been created using the Lgr5+ or CD44+ stem/progenitor cells of circumvallate (CV) papilla tissue[70].These taste bud organoids have been successfully created both directly from isolated Lgr5- or Lgr6-expressing taste stem/progenitor cells[71]. and indirectly, through the isolation, digestion, and subsequent culturing of CV tissue containing Lgr5+ or CD44+ stem/progenitor cells [72].

Further reading

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