Animal model systems – the traditional in vivo models

• They are the closest to recapitulating body functions and cellular interactions in human tissues.
• They can predict how a treatment or a disease may develop.
• They are limited by the differences among species biology, the differences in sensitivity, the cost of maintenance and limited throughput.

Two-dimensional (2D) monolayer cell cultures – the ex vivo models

• They provide insight into complex diseases with low cost and time required and high reproducibility 
• However, the cell culture model places cells in a non-natural environment without an extracellular matrix (ECM), which hinders the recapitulation of the complexity of the in vivo microenvironment
• They do not mimic the natural development of cells and tissues

Organoids – the ex vivo models

• They can be defined as a collection of organ-specific cell types grown from stem cells that self-organize through cell sorting and spatially restricted lineage commitment in a 3D structure recapitulating the process of self-organization during development in vivo and with functions like the organ that is being replicated.’
  • • They can be obtained from various stem cells
• Cells must be embedded in a specific ECM with specific medium and niche factors
• Medium components depend on the organoid models, examples like:
  • • R-spondin-1 (Wnt agonist)
    • Wnt3A (Wnt signal activators)
    • Other growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF)
    • Nicotinamide
    • Noggin (bone morphogenetic protein inhibitor/BMP inhibitor)
    • Small molecule inhibitors such as Y27632 (Pho kinase inhibitor)
• They can be expanded long term, genetically modified, and cryopreserved, maintaining their phenotypic characteristics

What are stem cells?

• They are undifferentiated or partially differentiated cells that are capable of long-term self-renewal.
• They can produce cells of different lineages.
• These cells are the source of all the structures in our bodies and are responsible for the formation and homeostasis of adult tissues.
• They can be categorized into two types:
  • • Pluripotent stem cells (PSCs), also known as embryonic stem cells (ESC)
      • • They can differentiate into all cell types present in an embryo or an adult
        • They are only present naturally in embryos
        • They can be artificially obtained through the dedifferentiation of other cells
        • Takahashi and Yamanaka were the first to obtain this kind of cell, called Induced pluripotent stem cell (iPSC), in 2006
    • Adult stem cells (ASCs)
      • • They can only differentiate into cells from the organ in which they originated
        • They present in organs and tissues through the majority of postnatal life

How are organoids created?

• It is necessary to have a stem progenitor, either a PSC or an ASC
• Multiple organoid models have been developed using both types of stem cells

Image 1: Mesenchymal stem cells labelled with fluorescent molecules

1. A brief introduction to adult stem cells

• Adult stem cells (ASCs) are responsible for maintaining the homeostasis of body tissues through self-renewal and differentiation
• This kind of stem cell has been found in several tissues
  • • Neural stem cells (NSCs) in the brain
    • Hematopoietic stem cells in the bone marrow
    • Hepatic progenitor cells in the liver
    • Mesenchymal stem cells in adipose tissue
    • Crypt stem cells in the intestine
    • Epithelial stem cells in the skin
    • Germline stem cells in the seminiferous tubules
    • Muscle stem cells in myofibers
• They are embedded in a specific microenvironment called the stem cell niche, which regulates their cellular fate through secreted molecules, cell-cell interactions and physical contact with other cells and the extracellular matrix

2. Characteristics of organoids developed by using adult stem cells

• Organoids generated from adult stem cells (ASCs) or adult tissue fragments more closely resemble the homeostatic and regenerative capacity of the tissue of origin than PSCs
• They are a good model to study diseases, such as cancer or neurodegenerative disorders
• They have also been demonstrated to be more genetically stable than their PSC counterparts

3. Examples of organoids developed by using adult stem cells

• Stomach organoids
• Liver organoids
• Pancreas organoids
• Prostate organoids
• Mammary gland organoids
• Fallopian tube organoids
• Taste bud organoids
• Lung organoids
• Salivary glands organoids
• Esophagus organoids
• Epididymis organoids
• Lingua organoids
• Lacrimal gland organoids
• Thyroid organoids

Image 2: Embryonic stem cells (ESC) under microscope

1. A brief introduction to pluripotent stem cells

• Pluripotent stem cells (PSCs) are known for their self-renewal capacity and their ability to produce differentiated cells from tissues originating from the 3 germ layers
  • • Ectoderm
    • Mesoderm
    • Endoderm        

2. Characteristics of organoids developed by using pluripotent stem cells

iPSCs

• If iPSCs are exposed to the right combination of growth factors and signals and provided with a 3D scaffold, these cells can start differentiating into different cell types and self-organizing into organoids
• The development of iPSCs has translated into an important advancement in the generation of organoids
  • • iPSCs are easy to obtain, highly reproducible
    • They can generate multiple tissue types
    • They produce a kind of organoid that more closely resembles the fetal tissue stage
    • They can be exploited as a model for developmental processes and organogenesis and their associated diseases
• One of the main problems with this type of organoid is finding a way to provide all the cells with oxygen and nutrients and remove waste substances
  • • Multiple options have been proposed to overcome this issue like recreating an in vitro vascular circulation
• Other challenges to overcome in this type of organoids are the heterogeneity and the lack of maturation that they present

ESCs

• ESCs exist in embryos during the first days post-fertilization
• They can divide indefinitely and originate from every other cell of the organism
• This kind of cell can also originate from organoids
  • • However, their differentiation into a specific tissue is a complicated process
    • They require a long timespan and different cocktails of growth factors to generate these structure
    • They also raise considerable ethical concerns, especially in the case of human ESCs
• Despite these problems, ESCs constitute a quite satisfying model for the development of genetic, and infectious disease research, especially for tissues with low regenerative capacity
• ESC-derived organoids show high complexity because they can include mesenchymal and endothelial components, in addition to the epithelial component

3. Examples of organoids developed by using pluripotent stem cells

Image 3: Human induced pluripotent stem cell colony. Researchers at NIH’s National Eye Institute (NEI) developed the first patient-derived stem cell model for studying eye conditions related to oculocutaneous albinism (OCA). This image (Credit: National Eye Institute/NIH) is used under Public Domain Mark 1.0: https://creativecommons.org/publicdomain/mark/1.0/

Like what happens in normal tissues, it has been proposed that tumours have some cells responsible for the generation and maintenance of the rest of the cells. In the context of cancer, they are called cancer stem cells.

• Cancer stem cells can shift between a proliferative and a quiescent state.
  • • Their properties can be altered by interactions with their microenvironment, and processes such as inflammation, hypoxia or wound healing might favour carcinogenesis.
    • They may also change their environment in their favour.

• Cancer organoids have been successfully established from both ASCs and PSCS, including:
  • • Prostate cancer organoids
    • Breast cancer organoids
    • Pancreas cancer organoids
    • Liver cancer organoids
    • Ovary cancer organoids
    • Esophagus cancer organoids
    • Colon cancer organoids
    • Stomach cancer organoids
    • Fallopian tubes cancer organoids

• Patient-derived organoids (PDOs) from different regions of the same tumour retain the heterogeneous genetic composition.
  • • However, they need to be characterized in-depth before experimental procedures
    • High efficiency in organoid culture from patient-derived tumours cells and matching healthy cells has enabled the generation of highly characterized PDO biobank that make these resources available to the research community
      • • They are very promising for the discovery and testing of treatments.
        • They can be used as a tool for high-throughput drug screenings, for the identification of epigenetic or genetic causes of drug resistance, and the comparison of different compound toxicities in normal versus tumoral tissues.

• The applications of PDOs pave the path for improving drug discovery and testing and avoiding the use of animal models.

• There are multiple lines of evidence that organoids are an advancement for research and clinical applications, holding great potential to substitute or complement models currently used.

• They are being employed to achieve a better understanding of basic biology and to investigate numerous diseases.

• They are more representative than cell lines and less costly and time-consuming than animal models, they can be used for high-throughput drug screenings.

• PDOs are a remarkable option for facilitating genetic screening and treatment testing.

• Considerable research still needs to be performed to improve organoid systems, like
  • • Standardization
    • Maintenance and tracking

• In the future, the creation of assembloids (combinations or organoids) could serve as an instrument to deepen the understanding of the interaction between different organs and systems in our bodies.

• Emerging genetic manipulation tools, such as the CRISPR/Cas9 system, can employ organoids as a testing platform for modifications before translation to the clinic.

• There is still much to investigate in organoids, but undoubtedly, they will become a considerable help in research in the following years.