Organoids in immunological research

Organoids in
Immunological
Research
Hans Clevers et.al
Hubrecht Institute, Netherlands
Nature Reviews, Immunology,
December 2019

Overview
Presentation is Structured into 3
Sections
Tissue
Architecture and
basics of
Organoids
Organoids and
Immunology
Organoids in
Personalized
Medicine and
Future Perspectives
2
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Advanced Immunology (BIO 609) Ayush Jain
Nature Method of the Year 2017:
Organoids

1. Tissue
Architecture
and basics
of
Organoids
Powerful tool to maintain different cell
types in a near native state.
3
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3D cultures of Adult Stem Cells
• Organoids are small and self-organized, three-dimensional
tissue cultures (3D) or cell clusters that are derived mainly
from Adult stem cells.
• Adult stem cells or somatic stem cells are
undifferentiated cells found throughout the body (Tissue
specific) that divide to replenish dying cells and regenerate
damaged tissues.
• 3D cell culture are artificially created environment (Hydrogel
or Matrigel) that allows cells in-vitro to grow and interact
with its surroundings in all directions, similar to how they
would in-vivo.
• Self-organizing capacity of vertebrate cells.
Even after complete dissociation, cells can reaggregate and
reconstruct the original architecture of an organ.11 April 2020
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What are
Organoids?

• Organ specific
• Recapitulates function of the organ
• Spatially Organized similar to the
organ
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Properties of
Organoids
M. A. Lancaster and J. A. Knoblich, Science 345, 1247125 (2014). DOI: 10.1126/science.1247125

1. Cell sorting Out-Based on Steinberg
Differential Adhesion Hypothesis
2. Spatially Restricted Lineage
Commitment
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Principles of
Self-
organization

Scaffold and Scaffold free
Basically two types of 3D culturing Techniques
1. Scaffold techniques. Use Solid scaffolds such as
Hydrogels. Hydrogel matrices mimics natural
Extracellular Matrix (ECM) structures. Ex: animal ECM
extract hydrogels, protein hydrogels, peptide hydrogels,
polymer hydrogels, and nanocellulose hydrogel.
2. Scaffold free techniques. Are independent of Scaffolds
like Hanging drop method, low adhesion plates,
Rotating Bioreactors, Magnetic Levitation.
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Types of 3D
Cell-Cultures

Development of 3D Cell culture
technologies and knowledge of
Developmental Biology lead to
Organoid production
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History of
Organoids

Toshiro Sato Developed Gut Organoids in Clevers lab
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History of
Gut
Organoids
Sato, H. et al. Nature 459, 262–265 (2009).

• EpithelialTissue lines Boundaries of Mammalian Body
like in Gastrointestinal tract (Gut).
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Organization
of Gut tissue

Superior morphology
• This technology allows long term proliferation and
differentiation of stem cells under defined conditions
(serum free cultures).
• Organoids are cellularly heterogenous, genetically stable
and retain their tissue phenotype over time.
• Inherent differences between humans and mice
complicate the translation of basic research for direct
human applications, which can be overcome by use of
organoids.
• Cell lines generally show differences in various omics
profile when compared to normal cells, but organoids are
setup from specific tissue(biopsy) and research on them is
directly applicable.
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Advantages
of
Organoids

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Different
Organoid
systems

2. Organoids
and
Immunology
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Concentration of Immune cells is
highest in the Epithelia (except blood
and lymphoid organs) as they are body’s
1st point of Contact with pathogen

Epithelial cells collaborate closely with
Immune cells to maintain Homeostasis
• Epithelial cells collaborate closely with immune cells to
safeguard homeostasis and provide a quick response to
infection.
• Intricate interactions between epithelia and immune cells
prevents pathogenic infection, excessive immune activation
and help in repair process.
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Epithelial
Cells and
immune system

Epithelial Organoids provide a platform
to study primary epithelial cells that
closely resemble in-vivo situation
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Epithelial
Organoids

Complex tripartite interaction between
epithelium, commensal microbiome and
immune system
• Epithelial cells express various PRR’s that recognize
pathogens.
• But epithelial cells from different segments of gut have
different pattern of TLR expression, so organoids from
different segments retained this expression pattern,
independent of Microbiota and Immune cells.
• Stimulation of Organoids through immune cell secreted
cytokines (Not TLR) lead to specific Antimicrobial gene
expression (ex: TLR5 expressed by Paneth cells does not
induce its Degranulation and extrusion but by IFNƳ).
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Epithelial cells as
immune sentinels
for bacteria

Immune cell derived cytokines and the
microbiome influence differentiation of
intestinal epithelial cells
1. Differentiation of M-cells (Micro-fold)
• IL-22 has indirect effect on M-cell development while RANKL
has direct effect.
• Transcytosis by M-cells has important role in inducing immune
responses.
• Early M-cell Marker SPIB (Tf) and Mature M-cell marker GP2
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Unravelling
Complex
interactions with
Organoids
Stromal cell
Nagashima, K. et al. Nat. Immunol. 18, 675–682 (2017).

2.Diffrentiation of Tuft cells(chemosensory) and Goblet cells
11 April 2020Advanced Immunology (BIO 609) Ayush Jain
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Regulation of immune responses by tuft cells
Christoph Schneider. Nature Reviews September 2019
Alarmin
IL2-Paneth cell
differentiation

Epithelial cell-immune cell interactions
in fetal development.
• Mouse peripheral tissues are colonized by adaptive
immune cells only after birth, so to study interaction of
human fetal intestines with cells of immune system
Organoids are used for example:
1. (CD4+CD69+) Effector Memory cell produce TNF (low
level) and other express other genes promoting epithelial
growth and development.
2. High TNF leads to ISC(Intestinal Stem Cells) apoptosis
• This Organoid system modelled Development of
Necrotizing Enterocolitis (part of intestine dies in new-
borns), babies born High TNF producing cells lead to
disease.
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Epithelial
Organoids in
studying fetal
development

3D environment of thymus crucial for
modelling its function is achieved
through thymic organoids
• Thymus is central location of T-cell selection (+ve and –ve),
Maturation and Differentiation.
• Recreating the 3D environment (cortex and medulla) of
thymus has been shown to be crucial for modelling its
function, which was achieved through thymic organoids.
• These Organoids were functional upon transplantation to
nude mice (athymic mice).
• Thymic organoids containing both cortical TECs and
Medullary TECs have not yet been generated from single
stem cell.
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Thymic
Organoids to
Study T-cell
Development

1. ISC subset can function as Non-
classical APC to CD4+ T-cells via
MHC class II.
2. Microorganisms derived metabolites
(mainly SCFA) greatly affect mouse
Intestinal organoids in terms of gene
transcription
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Organoid-immune
cell co-culture
systems in basic
Research

Established Organoid-immune cell co-culture systems
11 April 2020Advanced Immunology (BIO 609) Ayush Jain
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Organoids Support Replication of viruses
tropic to Cultured organ ex:
HSV and HPV - oral mucosa organoids
Human Norovirus- small intestine
organoids RSV- Airway Organoids
cultured under air-liquid interphase
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Studying infection
through Organoids
1. Modelling Helicobacter pylori Immune evasion
CagA- cytotoxin associated gene A
MET- Hepatocyte growth factor receptor
Leads to Phosphorylation of
MET and epithelial cell
proliferation

2. Modelling Cryptosporidium parvum
virulence
3. Modelling Clostridium difficile virulence
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Damage to
Paneth cells
• Toxin B binds to Frizzled proteins in
intestinal crypts
• Stat5 Signalling reduced Epithelial
Damage by inducing differentiation of
Paneth cells from ISC
Increased Transcription of type-I
Interferon-related genes by epithelial cells

Organoid cultures provide accurate
assessment of behaviour of healthy
epithelium both under steady state
and tissue injury
• ILC3-Derived IL22Protects intestine from Damaged caused
by Radiation, Chemotherapy or Graft-vs-Host Disease.
• Recovery from tissue injury induces fetal like developmental
programme in ISCs depending on Pro-inflammatory factors.
• Low grade Inflammation is necessary for epithelial
regeneration, but is not required under steady state conditions.
• Complement has role in intestinal regeneration upon injury
in-vivo.
• Complement Cascade has direct effect on epithelium
• Use of Organoids explained that disease phenotype is not
only dependent on hematopoietic cells, therefore their lack of
response to Immunosuppressive therapy is explained.
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Organoids in
Tissue Repair and
Regeneration

• Holistic Approach: cancer
Organoids directly from tumours
Preserving endogenous immune cells
• Reductionist Approach: cancer
organoids with (separately isolated
and expanded) immune cells
• Modelling tumour Micro-environment in-vitro is a
problem as they are insufficient to recapitulate the
heterogeneity of tumour epithelial cells.
• Patient derived xenografts models typically depend on
mouse immune system, which does not adequately
replicate human immune system.
• While Creating tumour Organoids from patient derived
tumour tissue are much reliable and does not have the
above mentioned problems.
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Studying Tumour
Microenvironment

• In 2016 Organoids from human epithelial
breast tissue retained IEL’s
• Hematopoietic stem cells could be
maintained up to 8 days in co-culture with
generated human tumour organoids.
• Tumour Organoid culture helped in
identification of Secreted Chemokines that
may be associated with ineffective
antitumor immunity
• Blockade of CDK4 and CDK6 promoted T-
cell activation and killing of tumour cells.
• Cellular Immune components like NK,
NKT, Th, B cells and Tumour associated
Macrophages were readily maintained up
to 30 days in Organoid cultures
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1. Holistic Approach

• Triple Co-cultures of cancer Organoids
with DCs and CTLs in presence of
PDL1 inhibitors Significant death of
Cancer cells.
• Stable Epithelial Organoid Culture for
high mutational burden such as MMR
deficient cancers can be developed
• Cancer Organoids present antigens that
can trigger T-cell Proliferation and
stimulation.
• Recently an assay to test CAR-T cell
medicated cytotoxicity against Cancer
Organoids.
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Reductionist
approach

3.Organoids in
Personalized
Medicine and
Future Perspectives
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• Using Patient derived Organoids to
predict a patient’s response to a therapy
• Using organoid as a platform for
developing and testing personalized
medicine approaches
• Bidirectional interactions between epithelial cells and
immune cells can be studied
• Organoids can also be used to study interactions
between host and microbes
• Multi partite co-cultures has to be developed
• Tumor-derived organoids, which represent the
transcriptional and mutational profile of the original
tumor, provide a new and reliable model system for
the interaction of the immune system with tumour
cells.

Limitations
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• Organoids are often derived from biopsies, which
represent only a small part of the tumor tissue.
Therefore, the complexity of the whole tumor will
always be underestimated by the organoid.
• Tumor-derived organoids are not exposed to the
external pressures that occur in situ, such as
hypoxia or immune selection, which can influence
the outgrowth of tumour clones. leading to a
situation in which a dominant clone in vitro is not
as dominant in situ or vice versa
• Culture conditions can influence cellular behavior in
many ways

Conclusion
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Organoids Currently provide most
accurate in-vitro system for culture of
human epithelial cell of any organ and
show great promise in fundamental and
translational research

Questions??
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Thankyou for your
patience
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34

Organoids in immunological research

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