Created By: Jasmit Dhaliwal, Cynthia Yuanxin Gu, Pavlo Navrota, Jasmine Soomal

Organoids are three-dimensional stem cell derived miniature organs produced in vitro. They are an important tool for modeling and finding potential treatments for diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic. Due to the physiological similarities organoids have with human tissue, they are better able to demonstrate how SARS-CoV-2 targets specific cell types.

Figure 1: Magnified image of SARS-CoV-2 (Akirov, 2020).

Coronavirus is a type of virus covered with pointed structures that create a corona or crown-like shape, hence, its name (Sauer, 2021). They are zoonotic, meaning that they can be transmitted between animals and humans. The recent COVID-19 pandemic has been a result of SARS-CoV-2, a type of β-coronavirus. The complete genome sequence recognition rates of SARS-CoV and bat SARS coronavirus (SARSr-CoV-RaTG13) were 79.5% and 96%, indicating that SARS-CoV-2 might originate from bats (Wang et al., 2020). The disease is known to initially cause symptoms, such as fever, cough, and fatigue, but can progress into life-threatening cases. It is also highly contagious and can be transmitted through close contact and droplets, so developing a vaccine is urgent in reducing its spread (Wang et al., 2020).

SARS-CoV-2 infects the host cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptors, which are proteins found on the membranes of tissues. This involves the epithelial cells of the airway, lungs, intestines, kidneys, blood vessels, and more (Andersen, Rambaut, Lipkin, Holmes, & Garry, 2020). Since SARS-CoV-2 is a spike protein, the spikes bind to the receptor to enter the cells (Andersen et al., 2020). The normal function of the ACE2 receptor is to break down angiotensin II (ANG II), whose function is to increase blood pressure and inflammation, to other molecules that counteract the activity of ANG II (Andersen et al., 2020). When SARS-CoV-2 virus binds to ACE2, it prevents the normal functioning of the receptor, allowing ANG II to continue its normal function (Andersen et al., 2020). The number of ACE2 receptors varies person to person and depends on the tissues and cells (Andersen et al., 2020).

Figure 2: SARS-CoV-2 binding to the ACE2 receptor and blocking its ability to bind to ANG II (Sriram et al, 2020).

In order to understand organoids, one has to start with the cells that they are derived from, which are stem cells. Stem cells are undifferentiated cells that do not have a distinct role and can become many specialized cell types (Alison, Poulsom, Forbes, & Wright, 2002). They have both self-renewal and differentiation properties (Nava, Raimondi, & Pietrabissa, 2012). This means these cells have the ability to divide to regenerate the same tissue and the ability to turn into specific mature cell types, respectfully (Nava et al., 2012).

There are three types of stem cells. The first type are totipotent cells. These cells can differentiate into all cell types, including the extraembryonic tissue, which involves the placenta (Alison et al., 2002). Thus, totipotent cells can give rise to an entire embryo (Alison et al., 2002).

The second type of stem cells are pluripotent cells. They have the ability to mature into the cell types from the three germ layers, which involve the endoderm, ectoderm, and mesoderm (Alison et al., 2002). However, they cannot differentiate into the extraembryonic tissue (Alison et al., 2002).

Multipotent cells are the last type of stem cells, which develop into the cell types within the tissue they reside. Therefore, they are particular to their lineage (Alison et al., 2002).

Figure 3: Three different sources stem cells can be obtained from (BMC, 2021).

Stem cells can come from multiple sources. The first source of stem cells are embryonic stem cells (ESCs). These are totipotent in the early embryonic stage and later become pluripotent. ESCs are not totipotent because they cannot become part of the extraembryonic membranes (Alison et al., 2002). They are obtained from the inner cell mass of the blastocyst that’s formed 4-5 days after fertilization. The blastocyst has two parts: the outer cell mass which becomes the placenta and the inner cell mass which develops into the human body (Alison et al., 2002). ESCs are usually obtained during in vitro fertilization (IVF), where multiple eggs are extracted from a woman. These are then fertilized in a test tube and a limited number of eggs that were successfully fertilized will be implanted back into the woman to start pregnancy (Alison et al., 2002).

Adult stem cells (ASCs) or also known as somatic stem cells, are multipotent. They are isolated from the adult tissues, such as the bone marrow, bone, skin, blood vessels, and muscle (Nava et al., 2012). They remain in the non-specific state until the body needs them to become specialized (Nava et al., 2012). Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are two types of adult stem cells. MSCs help create new body tissues, such as bone, cartilage, and fat cells, and HSCs give rise to all the blood cells and cellular blood components via hematopoiesis.

Induced pluripotent stem cells (iPSCs) are obtained by the genetic reprogramming of somatic differentiated cells, in vitro, into a dedifferentiated state resembling the embryonic-like pluripotent stem cells (Nava et al., 2012). This is done by adding the reprogramming genes Oct4, Sox2, Klf4, c-Myc to the ASCs to induce them to become pluripotent. iPSCs are a promised way of obtaining pluripotent stem cells from adult tissues (Nava et al., 2012).

Stem cells are important for various reasons, such as their ability to regenerate and repair damaged tissue by instructing stem cells to differentiate in a certain way (Davis, 2017). They can be transplanted into a damaged body part and be directed to grow and differentiate into a healthy tissue (Stöppler, 2020). This is useful where there are not enough organs for donation. In addition, stem cells obtained via the bone marrow, peripheral blood, and umbilical cord blood, can be donated to other individuals. New drugs can also be tested on tissue grown from stem cells, such as organoids, which will be discussed later on. This can assist with drug discovery and the formation of new treatments. For the future use of stem cells, scientists want to be able to use them for the treatment of cardiovascular diseases and brain diseases, along with cell deficiency therapy (Alison et al., 2002).

Stem cells have been used for the treatment of blood cancers, such as Leukemia, and other disorders of the blood and bone marrow. The process involves the use of HSCs, which are derived from bone marrow, peripheral blood, or umbilical cord blood. First the patient receives chemotherapy or radiation therapy in order to destroy the whole blood cells that are damaged. Then stem cells from a healthy matched donor are injected into the patient and these cells will populate and begin producing new, healthy blood cells to create a new blood system (Stöppler, 2020).

Figure 4: Process of using stem cells to treat leukemic patients (OHSU, n.d.).

Figure 5: Variety of organoids can be created from the two different sources of stem cells: pluripotent stem cells and adult stem cells (Regnard & Hamers, 2020).

Organoids are tiny, self-organized three-dimensional in vitro tissue cultures derived from stem cells, which mimics its corresponding in vivo organs (Souza, 2018). The stem cells can either be pluripotent (embryonic or induced) or adult stem cells. Organoids can range from the size of the width of a hair to four mm (“Method of the Year 2017: Organoids,” 2018).

Organoids have been used for autistic patients, where they have shown abnormalities in the regulation of genes involved in cell proliferation. Organoids have also been used to study the Zika Virus to observe how the virus is associated with microcephaly during early embryo development, where it interrupts normal brain development by promoting the premature differentiation of neuron-producing cells. Furthermore, these structures have been used to compare how normal and diseased organoids respond to certain stimuli (Barbuzano, 2017).

During early embryonic development, the single-layered blastula folds in upon itself and its cells rearrange to form 3 layers, in a process called gastrulation. These layers are known as germ layers and give rise to the different organs and tissues in the body (laesoph, n.d.).

1. The endoderm differentiates into the gut and its associated internal organs.
2. The mesoderm differentiates into muscle cells and connective tissue.
3. The ectoderm differentiates into the nervous system and tissues, like the epidermis

Human pluripotent stem cells (hPSCs) are capable of differentiating into all embryonic tissues that derive from these 3 germ layers. As they differentiate, they lose self-renewal capacity and lineage capacity, and instead, commit to becoming mature functional cells of a certain lineage (Seita & Weissman, 2010).

In this study, both types of pluripotent stem cells were used to derive the 8 differentiated cell types.

1. iPSCs are adult somatic cells that have undergone cellular reprogramming, and they were used to derive liver organoids.
2. The other 7 cell types were derived from human embryonic stem cell lines (H1 and H9). Spontaneous differentiation of hESC cultures produces a fraction of the desired cells (Vazin & Freed, 2010). Instead, the cultures were sequentially supplemented with inducing factors (such as FGF-7 fibroblast growth factor 7) and growth factors to differentiate the hESCs into progenitors and the desired cells. Subsequently, the mesoderm and ectoderm derivatives were stained positively with antibodies that recognized lineage-specific cell-surface proteins (CD31) and neuronal markers, respectively (Vazin & Freed, 2010).

Figure 6: The cell types originating from the three different germ layers (Yang et al, 2020).

As further evidence of successful differentiation, Yang et al. uses immunostaining; a technique that employs antibodies to bind and detect targeted antigens on cells. As mentioned earlier, ACE2 is expressed on hPSC-derived cells as they are a receptor for SARS-CoV-2 infection. Immunostaining will not detect ACE2 expression on the undifferentiated hPSC cells, but should on the derived cells.

When testing each cell type’s permissiveness to SARS-CoV-2, pseudoviruses are used. Pseudoviruses contain the genome of a known virus but are encapsulated with a protein of another virus that does not contain the genetic material needed to produce progeny, meaning that the virus should not infect the inoculated cells (Li, Liu, Huang, Li, & Wang, 2018). In this study, the SARS-CoV-2 Spike protein was carried on a pseudovirus to infect targeted cells (ie. the 8 derived cell types) and analyze their entry based on luciferase activity. The backbone was provided by a VSV pseudotyped delta-G-luciferase virus. Binding of the Spike protein to the ACE2 receptor on the cell surface of the differentiated cells, should release luciferase, an enzyme that produces bioluminescence. Cells with high ACE2 expression will consequently have high luciferase activity. As well, the luciferase activity indicates that the cells are permissive to SARS-CoV-2 pseudo-entry virus infection.

Figure 7: Spike protein on a pseudovirus binding to the ACE2 receptor of a hPSC-derived cell (Cuffari, 2020)..

The Transcriptome is the complete set of RNA transcripts in a cell and their quantity at a specific time or under a specific condition. To analyze the transcriptome one can use qPCR, Microarrays, or RNA-seq. RNA sequencing is a cutting edge high throughput sequencing technique that utilizes massively parallel next generation sequencing (NGS), which in this case is used to deeply sequence target regions. RNA-seq can find alternate splicing patterns, post transcriptional modifications, gene fusion, mutations or SNP, as well as various RNA isoforms. It can also distinguish Intron/Exon boundaries empirically.

The workflow consists of isolating mRNA transcripts which makes up 2-4% of total RNA in a cell. In order to enrich the RNA sample rRNA depletion or poly-A selection can be used to isolate mRNA in the sample (Zhao, et al., 2018). This is then followed by conversion of mRNA transcripts in a cDNA library through reverse transcription. The cDNA is then fragmented and ligated to adapters, which are used to amplify the fragments into clusters within the sequencer. The sequencer uses fluorescently tagged nucleotides to sequence these clusters, with the final output being a FastQ file (“Introduction to NGS”, n.d.). This file contains the sequence and its quality score, which is then used in the computer workflow to generate a map of the transcriptome.

Fragments must be assembled locally to a region on the reference genome, which is carried out through sorting programs that utilize some form of the Burrows-Wheeler transform. Because the entire genome is not being sequenced a greater depth of sampling is achieved allowing for accurate assessment of transcript abundance (McArthur, 2020).

Simple linear normalization can be carried out, measuring fragments per kilobase per million mapped reads. However, RNA-seq is a non linear data space, and to reduce type 1 error, highly expressed and variable genes are downplayed using negative binomial distribution and local regression (McArthur, 2020).

This data can be presented in standalone heatmaps, or coupled with bioinformatics databases to further process or display results in a clearer and more coherent manner. This can be coupled with Gene Set Enrichment to present changes in whole biological processes and cellular components.

Organoids allow researchers to study the organ in the tissue culture dish without any interference from other organs. These structures assist with developmental biology, which involves studying the process of how stem cells grow into embryos, different tissues, and more. Organoids are useful for disease pathology of infectious disease in which they are used to see how a disease, such as hepatitis B virus, coronavirus, and more, affects a certain organ. These structures are also important for regenerative medicine in which these structures can be used to replace damaged organs or tissues. Furthermore, organoids are important for drug toxicity and efficacy testing in which organoids are used to see the potential toxic effects of drugs against the organs. Finally, organoids are useful for personalized medicine and drug discovery in which drugs can be tested ex vivo on organoids derived from stem cells of individual patients to see how the drugs will interact and respond (Pal, 2019).

Figure 8: Various applications of organoid models (Pal, 2019).

Organoids are unable to reflect the interconnectedness of human organs, so findings from organoid models must be validated with animal models and clinical studies. The lack of immune system and vasculature components also limits an organoid's ability to reflect true human physiology (Xinaris, 2019). Organoids resemble some aspects of the organ, but are not completely the same, and it can be difficult to figure out the precise conditions required to stimulate and promote the stem cell to self-assemble to form the organoids (“Method of the Year 2017: Organoids,” 2018).

Figure 9: Drawbacks of organoid models (BioRender).

Organoids are three-dimensional stem cell derived miniature organs produced in vitro, an important tool for modeling and finding potential treatments for diseases such as SARS-CoV-2. After differentiating hPSC into different cell lines, these cells are maintained and treated to remain organoids. SARS-CoV-2 permissiveness can be tested on the organoids using pseudo virus and analyzing its rate and effects of infection using qRT-PCR analysis, immunostaining, and transcript profiling.

In conclusion, organoids provide valuable, physiologically relevant human models to study SARS-CoV-2 and aid in the development of therapies. Due to the physiological similarities organoids have with human tissue, they are better able to demonstrate how SARS-CoV-2 targets specific pathways in certain cell types.

Figure 10: Overview of how to make a SARS-CoV-2 organoid model (Biorender).

Link: https://docs.google.com/presentation/d/1R7dr9zhNjofZuDIYlPMLF62964OMSxjHu0ULs2Pt4Nc/edit?usp=sharing

Abo-Rady, M. (2020, February 5). HOW IPS CELLS ARE CHANGING DRUG DEVELOPMENT AND DISEASE MODELLING [Image]. Biopharma Excellence.

Alison, M. R., Poulsom, R., Forbes, S., & Wright, N. A. (2002). An introduction to stem cells. The Journal of Pathology, 197(4), 419–423. https://doi.org/10.1002/path.1187

Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C., & Garry, R. F. (2020, April 1). The proximal origin of SARS-CoV-2. Nature Medicine. Nature Research. https://doi.org/10.1038/s41591-020-0820-9

Barbuzano, J. (2017). Organoids: A new window into disease, development and discovery | Harvard Stem Cell Institute (HSCI). Retrieved February 1, 2021, from https://hsci.harvard.edu/organoids

BMC. (2021). Stem Cell Research & Therapy [Image]. Springer Nature. https://stemcellres.biomedcentral.com/articles/10.1186/scrt81/figures/1

Cuffari, B. (2020, July 3). What is a Pseudovirus [Image]. Medical News. https://www.news-medical.net/health/What-is-a-Pseudovirus.aspx

Conesa, A., Madrigal, P., Tarazona, S., Gomez-Cabrero, D., Cervera, A., McPherson, A., Szcześniak, M. W., Gaffney, D. J., Elo, L. L., Zhang, X., & Mortazavi, A. (2016). A survey of best practices for RNA-seq data analysis. Genome Biology, 17(1), 13. https://doi.org/10.1186/s13059-016-0881-8

Davis, C. P. (2017, October 10). Stem Cells Definition, Therapy, Types, Uses & Function. Retrieved February 1, 2021, from https://www.medicinenet.com/stem_cells/article.htm

Gastrulation. (n.d.). BioNinja [Image]. https://ib.bioninja.com.au/options/option-a-neurobiology-and/a1-neural-development/gastrulation.html

Gentry, S.N. & Jackson, T.L. (2013) A Mathematical Model of Cancer Stem Cell Driven Tumor Initiation: Implications of Niche Size and Loss of Homeostatic Regulatory laesoph. (n.d.). 13.2 Development and Organogenesis.

Li, Q., Liu, Q., Huang, W., Li, X., & Wang, Y. (2018, January 1). Current status on the development of pseudoviruses for enveloped viruses. Reviews in Medical Virology. John Wiley and Sons Ltd. https://doi.org/10.1002/rmv.1963

McArthur, A. G. (2020). RNA-seq [PowerPoint slides]. Retrieved from https://web.microsoftstream.com/video/289c511f-9f04-47b6-a8df-9c0897c74276?list=user&userId=bac902c8-b8c1-4008-ac1d-c98e58db4086

Method of the Year 2017: Organoids. (2018, January 3). Nature Methods. Nature Publishing Group. https://doi.org/10.1038/nmeth.4575

Molnar, C. & Gair, J. (2015) Development and organogenesis [Image]. Concepts of Biology.

Nava, M. M., Raimondi, M. T., & Pietrabissa, R. (2012). Controlling self-renewal and differentiation of stem cells via mechanical cues. Journal of Biomedicine and Biotechnology. Hindawi Limited. https://doi.org/10.1155/2012/797410

OHSU. (n.d.). Understanding Bone Marrow/Stem Cell Transplant [Image]. OHSU Knight Cancer Institute.

Pal, R. (2019, August 29). Key Applications of Organoids. Retrieved February 1, 2021, from https://blog.crownbio.com/key-organoid-applications#_

Regnard, G. & Hamers, S. (2020, May 28). Organoids: definition, culturing methods and clinical applications [Image]. CytoSMART. SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids. Cell

Sauer, L. (2021). What Is Coronavirus? Retrieved February 1, 2021, from https://www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus

Seita, J., & Weissman, I. L. (2010, November). Hematopoietic stem cell: Self-renewal versus differentiation. Wiley Interdisciplinary Reviews: Systems Biology and Medicine. NIH Public Access. https://doi.org/10.1002/wsbm.86

Stöppler, M. C. (2020, December 10). Stem Cells Definition, Therapy, Types, Uses & Function. Retrieved February 1, 2021, from https://www.medicinenet.com/stem_cells/article.htm

Vazin, T., & Freed, W. J. (2010). Human embryonic stem cells: Derivation, culture, and differentiation: A review. Restorative Neurology and Neuroscience. NIH Public Access. https://doi.org/10.3233/RNN-2010-0543

VCH. (2021). Zika Virus [Image]. Vancouver Coastal Health. http://travelclinic.vch.ca/handouts/zika/

Wang, L., Wang, Y., Ye, D., & Liu, Q. (2020). Review of the 2019 novel coronavirus (SARS-CoV-2) based on current evidence. International journal of antimicrobial agents, 55(6), 105948. https://doi.org/10.1016/j.ijantimicag.2020.105948

Xinaris, C. (2019). Organoids for replacement therapy. Current Opinion in Organ Transplantation, 24(5), 555–561. https://doi.org/10.1097/MOT.0000000000000680

Yang, L., et al. (2020, July 2). A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids. Cell Stem Cell, 27, 125-136. https://doi.org/10.1016/j.stem.2020.06.015

Zhao, S., Zhang, Y., Gamini, R., Zhang, B., & von Schack, D. (2018). Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion. Scientific Reports, 8(1), 4781. https://doi.org/10.1038/s41598-018-23226-4