ORGANOIDS: A NEW WINDOW TO DISEASE
DEVELOPMENT AND DISCOVERY
What are Organoids
Organoids are very small and self-organized 3D cultures of the tissue, which are derived from the cells. Organoids are considered as the mini version of the organs that are produced and developed by in vitro process. These organoids are produced in such a way as to get the three-dimensional structure of the organ, which is produced, in the lab and such miniaturized versions of the organ are used in various biological experiments. The 3D nature of the organoids mimics many of the anatomical and physiological features including cellular movement, and cell-cell interaction. The Organoids are derived from adult stem cells or embryonic or induced pluripotent stem cells(iPSCs)(Driehuis & Clevers, 2017a). Organoids can also be defined as the cells that are developed and derived from a cluster of organ-specific cells and they are generally derived from stem cells, which are considered to be the progenitor of the organs in the body. Adult organoids generated from an adult stem cell can help to study the multicellular composition of the tissue in the culture dish. The organoids include various types that are considered specific to a certain cell, it can function, and behave as an organ.
Figure1 a: Human organoids(Human intestinal organoids for high throughput screening – Nutrition and Health (csiro.au))
Organoids can be grown by obtaining tissues from either a healthy or a diseased patient. Organoids can be grouped as individual cells as well as they can be used in groups to get the desired function and behavior of the organ from where it has been derived. Organoids can also initiate nerve activity as well as can continue to get endocrine secretion to facilitate the release of hormones An organoid derived from the intestine can show gut epithelial responses and explain the process by which the gut microbes can stimulate or prevent gastrointestinal diseases.3D Organoids developed from tumor cells can recapitulate the architecture of tumor of origin. They can allow drug hypothesis testing. Primarily derived organoids (PDO) can be useful models for studying disease pathogenesis and tissue repair. (Drost and Clevers, 2018.). There are various types of organoids, which include cerebral organoids, intestinal organoids, gastric organoids, kidney organoids, liver organoids, and lungs.
By combining organoids and CRISPR CAS9 various diseases can be cured and provide new opportunities for researchers and scientists to produce cures for human diseases such as glomerulosclerosis and polycystic disease associated with the kidney. Tumour organoids derived from cancer patients can be modified with CRISPR and help to track cancer stem cells. These approaches are very helpful in determining the issues and diseases that are associated with chronic human disease.
The organoid culture serves as a link between the in-vivo studies and 2D monocultures. The advantage of organoid culture is that it does not require immortalization before culturing and can allow the researcher to study for a longer duration of time. Adult Lgr5+ cells obtained from the mouse when given proper growth factors and stimuli developed into 3D structures in-vitro that resembled the mini-gut.
Figure1b.Three-dimensional (3D) reconstruction of the midsection of adult stem cell-derived colon(A) and liver(B) organoids, stained for the actin cytoskeleton (red) and nuclei (blue) and imaged by confocal microscopy. Source-(Driehuis & Clevers, 2017)
History of Organoids
In the present time, organoids refer to the growth of cells that are defined as the 3D structure for reassigning in vitro and making an artificial environment for conducting research. In 1907, Henry Peters Wilson first described and attempted the technique of in vitro regeneration organisms. He demonstrated the dissociation of sponge cells that can be auto-generated by the whole organism. A few decades later, the dissociation and re-aggregation were performed by several groups on amphibian pronephros and choke embryos (Takebe and Wells, 2019). In 1964, Malcolm Steinberg introduced the differential adhesion hypothesis. That proposes the sorting of cells, and rearrangement that can be explained by the process of thermodynamics which mediated differential surface adhesion. In stem cell research design that mediated by pluripotent stem cells that were first isolated and injected into the mouse embryo in 1981. Until 1988 scientists were capable of isolating the culture of embryonic stem cells that derived from human blastocysts, this was conducted for the first time. Stem cell research gained importance when PSCs were isolated and cultured using mouse embryos. Later, iPSC was able to establish the reprogramming of mice and human fibroblasts which had a significant impact on the research of stem cells and organoids.
In 1987, scientists started to improve the culture of cell conditioning by stimulating the microenvironment. The researcher demonstrated that 3D ducts can be formed by epithelial cells that grow in the lumen on EHS ECM extract. From this scientists can collect and synthesize the secret milk protein and make the 2D structure. Equally, in alveolar-type cells, scientists can maintain the differentiation in the presence of the ECM matrix.
It was in the year 2009 that Clever demonstrated the generation of 3D intestinal organoids from adult intestinal stem cells when cultured in Matrigel forming a self-renewing culture that epitomized the intestinal crypt-villus architecture and cell composition. It was seen that in R-spondin culture media LGR5 ligand upregulates Wnt signaling that helps to maintain stem cell population. These functions of the organoids can be observed both in the in vitro and in vivo process, which provides the function and behavior of the kidney organoids. While in the year 2013, at the Institute of Molecular Biotechnology, which is present in Austria it was observed that cerebral organoids were developed from stem cells that were derived from the mimicry of the human brain. This process has provided researchers and scientists to provide treatment and cures for diseases related to the human brain. (Schutgens, and Clevers, 2020).
Figure 2: History of organoid
(Source: (Corrò et al., 2020)
The CRISPR and Organoids
CRISPR is a genome-editing tool that can be used to manipulate genes in the germline. The CRISP-cas9 gives provision to the researcher to introduce double-strand breaks (DSB) at a specific desired location in the genome. The advantage of CRISPR is that DSB introduced can be used to disrupt or knock-in genes efficiently. Double-stranded breaks can be repaired by (i)Non-Homologous End joining or(ii) Homology-directed repair. Initially, CRISP-CASP 9 was used to study genome editing in zebrafish. In a research carried out mouse model was used to study the role of Apc alleles in intestinal cancer. Apc is a tumor suppressor for intestinal cancer and it negatively regulates the Wnt signal. CRISPR targeting introduced frameshifts in two Apc alleles. The result of that the organoids were able to grow in a Wnt-independent fashion.
Organoid culture generated from patients having cystic fibrosis can be useful for in-vitro research purposes. The role of the CFTR gene (Cystic fibrosis transmembrane conductor receptor) in cystic fibrosis can be studied by using CRISPR. Mutation in the CFTER gene results in inactive chloride channel protein. Colon organoids derived from cystic fibrosis patient shows reduced swelling when exposed to forskolin. The crisp technique restored the mutation present in the CFTR gene. The organoid exhibited restored swelling when exposed to forskolin.
Figure 3-Image showing CRISPR as a genome-editing tool to model monogenic diseases and tumorigenesis. Source-(Driehuis & Clevers, 2017)
CRISPR in understanding Tumorigenesis
The formation of tumors is due to genetic mutations which cause the inactivation of the tumor suppressor gene. CRISPR can efficiently modify genes that are mutated in cancer. Diversity is observed between premalignant tumor lesions. CRISPR-modified organoids were developed to study the tumor precursor lesions in-vitroThe effect of TGF-β was studied in APCmin and BrafV600E. (Driehuis & Clevers, 2017b). BrafV600E organoids underwent epithelial to mesenchymal transition when exposed to TGF-β but this was not seen in the case for APCmin organoids.
CRISPR can be used to validate the results of the drug on the oncogene. In a study carried out, the effect of the EGFR and MEK inhibitory drugs was tested on colorectal organoids. The experiment was carried out to analyze the effect of the sensitivity of Ras-mutation status in response to the drugs given. The oncogene Ras decreased sensitivity to the combination of the drugs given (i.e. EGFR and MEK inhibitory drugs).CRISPR was used to validate the results generated. For this, CRISPR was used to incorporate KRASG12D mutation in wild type KRAS colon tumor line. The results showed a loss of drug sensitivity which pointed towards the fact that RAS status was responsible for the loss of drug sensitivity
We know that in our body the cells reside in a complex microenvironment and these are subjected to many signaling interactions. These interactions play a key role in maintaining and regulating cellular phenotypes and functions. Though monolayer cultures layer has been widely used in past their main drawback is that their lack of tissue architecture and complexity fails to show the true biological processes taking place in vivo mouse/human model. It is been known that cells in 3D culture resemble the architectural and functional properties of in vivo tissue because in a 3D culture system there is cell-to-cell or cell-to-extracellular matrix interaction on all three dimensions but in 2D monolayer cultures the interactions are limited to the horizontal plane. ((Corrò et al., 2020)
Lung organoids are derived from primary respiratory cells and cell lines.
The organoids-tracheospheres or bronchosphers are derived from primary basal cells that express p63 and NGFR. These basal cells proliferate and form a spherical organization which is covered by goblet and ciliated cells. NOTCH pathway plays an important role in controlling basal cell differentiation. There is an increase in basal cell markers when the NOTCH1 receptor is blocked but when NOTCH2receptor is blocked there is an induction of ciliated markers (at the expense of goblet cells).
Alveospheres are generated by isolating primary SFTPC-expressing type II Alveolar epithelial cells from mouse lungs. The type II alveolar epithelial cells form 3D aggregate by proliferation and also differentiate into type I Alveolar epithelial cells. The Alveospheres generated capture properties of the alveolar compartment of the lung.
Microenvironment supporting lung organoid formation
For culturing organoids the 3D environment to be given is Matrigel which contains a gelatinous mixture of ECM components-laminin and collagen. Human bronchial epithelial cell lines are mostly cultured in Air liquid interface culture system because differentiation properties like ciliogenesis are retained. The culture is supplemented with fibroblast endothelial cells as these cells secrete certain factors that are essential for determining cell fate.
Figure 4a&4b:- How to culture lung organoids- Lung organoids mimicking features of full-size of the lung(Columbia university medical center- New lung ‘organoids’ in a dish mimic features of the full-size lung (phys.org)
Epidermal growth factors and retinoic acid are other media components that need to be added to the lung epithelial culturable structures derived depend upon where the organoids forming cells in the
3-D environments are positioned. If organoids forming cells are plated on top of the Matrigel layer then the HBECs self organizes into tubular structures. From the tubular structures, branching and budding are initiated. When the organoid-forming cells are embedded within Matrigel, the HBECs organize into spheroids containing cuboid epithelial cells. ( (Nadkarni et al., 2016)
Brain Organoids can be produced by pluripotent stem cells Developing this kind of Organoids needs the self-combination of stem cells and progenitor cells and requires different cells. Organoids help displays the structure that locates the specific region of the brain and controls the specific changes. It helps define specific neurological disorders and create a path for a possible cure. Thus, Organoids become a specific model for analyzing the development of the brain and various neurological diseases.
Image5 -Brain organoid
The non-human primates or animal models have numerous differentiations compared to the human brain sample. By utilizing stem cell expertise, the appearance of a three-dimensional Organoids model attracted scientists and researchers to create medicine. Brain Organoids are a type of Organoids that recreate brain structure, stimulate different brain regions, and emphasize their function in the human body. The critical brain region includes the midbrain, hippocampus, pituitary glands, hypothalamus, and cerebellum. Brain organoids become a great model for the investigation and mechanism of disease. The advancement of gene restriction, cell sequencing, and other critical perimeter technologies have recently developed and brought unprecedented potentiality for framing neurological disease in vitro.
Methodological progress in the culture of brain Organoids
Creating brain Organoids, from human pluripotent stem cells generating brain Organoids that are usually entrenched into the outer cellular matrix, and then the sample is refined into the rotating bioreactor to create tissue intensification and differentiate into various neurological activities (Chhibber et al., 2020). Some studies also showed that using 3D culture systems created human cortical spheroids and organoids derived from pluripotent stem cells without embedding them into extracellular matrices. It also displays functional maturity and synaptogenesis. During the culture, various growth factors and small molecules are generally provided to grow cells. After the beginning of the cell population, neural progenitors and neuroepithelial stem cells are utilized to create Organoids.
Prolonged culture time
Longer culture time; calcium activity is detected after 50 days of culturing. Prolonged cell culturing emphasized the promotion of cell maturation in neuron cells but also emphasizes the growth and segregation of glial cells (Yoshida, 2020). Scientists have reported that Organoids culture requires approximately 229 days in vitro and must be filled with abundant giant cells that have positive for GFAP and GLT1. Thus, long-term cell development promotes cell generation of brain Organoids and computing better development of the human brain. On the other hand, culturing organotypic slices is broadly supported by oxygen of Organoids tissue, which is reduced and forms hypoxic scores.
The liver is composed of epithelial cells like choanocytes and hepatocytes. These cells work together with the mesenchymal, endothelial, and stromal cells to carry out crucial metabolic functions like metabolic, exocrine, and endocrine functions which are necessary for body homeostasis. With the development of stem cell technologies, it became possible to culture and expand functional hepatocytes and choanocytes in the lab. The protocols regarding the distribution of the organoids in the liver thus depend on the conditions of the culture for the initiation and the expansion of the organoids which are hepatocellular. It comes after the second culture environment, which takes out the proliferative signals. The lineage commitment in the duct is inhibited and induces a variety of hepatocytic cells. The latest protocols of differentiation give in the organoids of which near about 50% fulfill characteristics of hepatitis (Brovold et al., 2020). The organoids that are hepatitis are very similar to the primary hepatocytes but they lack the complete functional repertoire of the cell types that have been targeted.
In vitro model, hepatic organoids are functional for making three-dimensional and allow novel platforms that address various research questions that persist in hepatic development and regeneration, detoxification, and studies of metabolism and remodeling for liver disease and adult stem cell biology. Hepatic organoids consist of epithelial cells of spherical monolayers, other than that it preserves the main features of the physiological liver and are obtained by isolating and propagation of stem cells and making progenitor cells from the niches of hepatic cells. PSC-differentiated hepatocytes assemble into 3D organoids to become further mature and they show striking improvement in the expression of genes like CYP450 genes, TAT genes, and albumin. They also show mature hepatocyte marker ASGR1
The majority of liver cells are composed of epithelial cells that mainly work cooperatively with stromal, endothelial cells that can function for crucial metabolic functions, exocrine, and functions for body hemostasis. The research is trying to direct the differentiation inside human studies for the development and understanding of human disease (Wang et al., 2020). Researchers can know that liver cells are full of pluripotent stem cells and during organogenesis, the liver progenitor cells are also known as hypoblasts which are specified for the posterior foregut endoderm. In the signaling factor, the signaling factors are surrounded such as FGF, BMP, and the growth factor of hepatocytes.
The liver can get affected due to mutation in a single gene as in the case of monogenic liver diseases like Alagille syndrome, A1AT deficiency, or due to parasites like Plasmodium parasite or due viruses like hepatitis B virus. Liver organoids which are generated from cells carrying mutations provide new models and help us to study and analyze the developmental abnormalities caused due to the mutations. Liver organoids differentiated from hepatocyte organoids, choanocyte organoids, and hepatobiliary organoids. It has been seen that there is an accumulation of protein aggregates in the liver organoids of a patient having A1AT deficiency, this is similar to the observation seen in the original biopsy. Liver organoids derived from differentiation of induced pluripotent or from biopsies of the patient having Alagille syndrome mimicked in vivo biliary defects that characterize the disease. In a study carried out it was seen that liver organoids from mouse models carrying JAG1 mutation showed a delay in the ability of organoids to differentiate into mature choanocytes and also a failure in the ability to form and maintain biliary ducts. All the studies help us to understand how liver organoids recapitulate the key features of disease in-vitro. Late diagnosis and tumor heterogeneity are the main reasons for high mortality in patients having liver cancer(Sun & Hui, 2020)
Organoids in drug discovery and development
Discovering a new drug that has the potential to prevent, mitigate, and cure a specific disease is essential. Approved drugs go through multiple screening processes, which constitute tens of thousands of compounds before clinical trials (Salick, et al., 2020). Despite various assessments of the process, approximately 80% of potential drugs failed in clinical trials. In cancer drugs, it has been estimated that around 95% or higher compounds are failed at the phase of clinical trials.
The rate of drug failure is high because sometimes drugs are not safe for humans, and most of the time, these drugs are not effective in the human body. It is clear that most cells and animal-based vectors are not sufficient and not giving the correct result of drug efficacy. In the drug screening system, there are various processes. The developer needs a model in which they can be tested and read out.
Scientists are trying to increase the re-examining process and create re-examining processes. Henry Li, chief scientific officer at crown bioscience, explains that the formation of the two-dimensional structure of cells helps to understand cancer, which is not predictive of the drug’s response. Using spheroids, which is defined as Organoids, potentially sidesteps that reduces animal volunteer for screening drugs.
Figure 7a- Bioprinting of organoids for disease modeling, drug development, and for regenerative medicine(Duelen et al., 2019)
By developing a fluorescent reporter-based quantitative screening platform we can quantify the fluorescence intensity in the multiwell plate. The mitochondrial health in the retinal organoid can be determined by measuring the fluorescence generated using JC-1(a mitochondrial depolarized marker). The effect of drug treatment on retinal organoids can be screened by using various assays like colorimetric assays which measure the number of viable cells, and fluorescence-based dihydroethidium staining assay which measures oxidative stress in the retina. Induced pluripotent stem cells can generate multiple tissue types and the organoids derived from iPSCs mimic the human condition. Therefore, iPSCs can be used to see the efficacy of drugs. Due to recent advancements in microfluidic technology, now microfluidic chips have been developed to measure the efficacy of the drug, side effects, and toxicity. (Aasen & Vergara, 2020)
Three-dimensional cellular models can be used for advancement and figuring out new therapeutic paths for the cure of the disease from cancer or cystic fibrosis and even COVID-19.
Image 5– Drug development and pre-clinical trials using retinal organoids. Source(Aasen & Vergara, 2020)
Mini-organs and the big picture
Organoids are defined as self-organizing clusters of cells that can grow in three-dimensional forms that exhibit architecture and function precisely in the live tissue and organs. Organoids are referred to as nearly native and perform as mini-organs in the dish. It is clear that most cells and animal-based vectors are not sufficient and not giving the correct result of drug efficacy. Organoids are mainly found in embryonic or adult stem cells, which can segregate into different types of cells that have multiple functions that depend on the culture and cell condition.
On the other hand, Organoids are gnomically stable when kept in the culture. Besides, it is more physiologically stable and relevant to the system (Bai, and Wang, 2020). After cryopreservation, the cell’s stability persists, which depends on the specific condition of the cell culture and helps prevent the stem cell from exhaustion and helps promote self-renewal and make differentiation. Organoids are a particular type of three-dimensional structure. On the other hand, spheroids can be generated from other cells such as tumor cells, and create other lines of primary cells with or without scaffolding. Scientists prefer to use primary cells, but scientists can create spheroids from cell lines and PDX, known as patient-derived xenograft. A xenograft is a technique in which scientists take live tissue from the human body for experimentation and grow by using the cell culture technique.
Allowing personally tailored medicine, all the scientists have underlined the significance of generating Organoids and spheroids by taking tissues and biopsies. For toxicological drugs, organoids hold great promise for testing drug efficacy and compound screening, which can reduce non-human trials.
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New lung ‘organoids’ in a dish mimic features of the full-size lung (phys.org)
Human intestinal organoids for high throughput screening – Nutrition and Health (csiro.au)
Research writer Ayesha Tungekar