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CRITICAL REVEW ON ORGANOIDS: A PROMISING TECHNIQUE IN NEED OF
PROTOCOL OPTIMISATION
Introduction
,Since the discovery of stem cells, regenerative medicine has become increasingly captivating
with all the promises and hopes to regenerate tissues and model pathogenic diseases.
Particularly, there has been great enthusiasm surrounding the recent technical advances in
the three-dimensional, self-organising stem cells culture, namely organoids. This technique
has shown promising results in personalised medicine and improved disease modelling and
it has also created expectations to substitute transplants in the future. Hence, the aim of
this critical review is to present the theoretical basis and protocol behind the creation of
organoids together with its application in the field of neuroscience, highlighting the
advantages and limitations of this breakthrough method.
The theoretical basis of organoids? Stem cells!
Stem cells (SCs) are unspecialised cells with the potential of proliferating indefinitely and,
most importantly, differentiate into any cell type of the body. The potency of SCs refers to
their ability to differentiate into any (pluripotent), many (multipotent) or one cell type
(unipotent) of the three embryonic germ layers. Pluripotent SCs (PSCs) can be only found in
the early embryo, however, the adult body conserves the ability to regenerate. The
regenerative process is carried out by progenitor cells which are descendants of SCs that can
only give rise to restricted subset of cell types, but do not self-renew indefinitely, and adult
multipotent SCs. Indeed, the list of tissues reported to comprise adult SCs is expanding and
involves the brain, spinal cord, bone marrow, umbilical cord, peripheral blood, skin and so
on. These adult SCs are necessary to overcome the ethical issues arising from the use of
embryonic SCs.
Adult SCs, by being autologous, overcome the alloreactive immune response previously
observed in allogeneic transplants. In a similar way, induced pluripotent SCs (iPSCs) which
are obtained by reprogramming fully differentiated cells to a pluripotent state, can avoid the
immune response from happening1.
SCs are exploited for their regenerative potential, however, they can also be used for
disease modelling or test cellular drug responses. Undoubtedly, two-dimensional cultures of
SCs are valuable tools, but they are not an accurate representation of the complexity of cell-
extracellular and cell-cell interactions happening in vivo.
, For this reason, organoids provide a potential solution to the issues that arise not only from
the simplicity of SCs but also by experimental animal use.
Organoids are a new research tool derived from pluripotent (embryonic or induced) or adult
SCs in vitro to form small, self-organising three-dimensional structures that simulate many
of the functions of native organs10. With time, all types of organoids have been generated,
including intestinal, hepatic, retinal, renal, endometrial, cortical and so on 2,3,4. Certainly,
their creation has been supported by the gradual optimisation of protocols which is
fundamental to mimic as closely as possible the parent organ.
When looking at the history of organoids, a landmark study was conducted in 2009 by
Clevers and colleagues which revealed the potential of adult intestinal SCs to self-organise
and proliferate in vitro. Indeed, the characteristic of intestinal SCs cells is the expression of a
gene (LGR5) that encodes the receptor for the Wingless-related integration site (Wnt)
agonist R-spondin; besides Wnt, the fate of these SCs is regulated by other molecules such
as epidermal growth factor and a bone morphogenetic protein inhibitor 5,6. Wnt are secreted
factors responsible for the regulation of cell growth, mobility and differentiation and they
act by activating specific signalling cascades within the target cells. Another key role in the
environment of intestinal SCs is played by the extracellular matrix (ECM) which is required
to prevent anoikis, which is a type of programmed cell death happening in anchorage-
dependent cells when they detach from the ECM7.
Based on the conclusions reached by Clevers, Sato et al. were able to demonstrate that
intestinal crypt-villus organoids can be generated from a single SC without the presence of a
mesenchymal niche8. The characterising features of these crypt-villus organoids are the
capacity to expand for farther than 3 months while maintaining a stable genome.
Subsequently, this method, also termed R-spondin method, has been used and adapted to
create organoids from epithelial tissues of various organs (Fig. 1).
CRITICAL REVEW ON ORGANOIDS: A PROMISING TECHNIQUE IN NEED OF
PROTOCOL OPTIMISATION
Introduction
,Since the discovery of stem cells, regenerative medicine has become increasingly captivating
with all the promises and hopes to regenerate tissues and model pathogenic diseases.
Particularly, there has been great enthusiasm surrounding the recent technical advances in
the three-dimensional, self-organising stem cells culture, namely organoids. This technique
has shown promising results in personalised medicine and improved disease modelling and
it has also created expectations to substitute transplants in the future. Hence, the aim of
this critical review is to present the theoretical basis and protocol behind the creation of
organoids together with its application in the field of neuroscience, highlighting the
advantages and limitations of this breakthrough method.
The theoretical basis of organoids? Stem cells!
Stem cells (SCs) are unspecialised cells with the potential of proliferating indefinitely and,
most importantly, differentiate into any cell type of the body. The potency of SCs refers to
their ability to differentiate into any (pluripotent), many (multipotent) or one cell type
(unipotent) of the three embryonic germ layers. Pluripotent SCs (PSCs) can be only found in
the early embryo, however, the adult body conserves the ability to regenerate. The
regenerative process is carried out by progenitor cells which are descendants of SCs that can
only give rise to restricted subset of cell types, but do not self-renew indefinitely, and adult
multipotent SCs. Indeed, the list of tissues reported to comprise adult SCs is expanding and
involves the brain, spinal cord, bone marrow, umbilical cord, peripheral blood, skin and so
on. These adult SCs are necessary to overcome the ethical issues arising from the use of
embryonic SCs.
Adult SCs, by being autologous, overcome the alloreactive immune response previously
observed in allogeneic transplants. In a similar way, induced pluripotent SCs (iPSCs) which
are obtained by reprogramming fully differentiated cells to a pluripotent state, can avoid the
immune response from happening1.
SCs are exploited for their regenerative potential, however, they can also be used for
disease modelling or test cellular drug responses. Undoubtedly, two-dimensional cultures of
SCs are valuable tools, but they are not an accurate representation of the complexity of cell-
extracellular and cell-cell interactions happening in vivo.
, For this reason, organoids provide a potential solution to the issues that arise not only from
the simplicity of SCs but also by experimental animal use.
Organoids are a new research tool derived from pluripotent (embryonic or induced) or adult
SCs in vitro to form small, self-organising three-dimensional structures that simulate many
of the functions of native organs10. With time, all types of organoids have been generated,
including intestinal, hepatic, retinal, renal, endometrial, cortical and so on 2,3,4. Certainly,
their creation has been supported by the gradual optimisation of protocols which is
fundamental to mimic as closely as possible the parent organ.
When looking at the history of organoids, a landmark study was conducted in 2009 by
Clevers and colleagues which revealed the potential of adult intestinal SCs to self-organise
and proliferate in vitro. Indeed, the characteristic of intestinal SCs cells is the expression of a
gene (LGR5) that encodes the receptor for the Wingless-related integration site (Wnt)
agonist R-spondin; besides Wnt, the fate of these SCs is regulated by other molecules such
as epidermal growth factor and a bone morphogenetic protein inhibitor 5,6. Wnt are secreted
factors responsible for the regulation of cell growth, mobility and differentiation and they
act by activating specific signalling cascades within the target cells. Another key role in the
environment of intestinal SCs is played by the extracellular matrix (ECM) which is required
to prevent anoikis, which is a type of programmed cell death happening in anchorage-
dependent cells when they detach from the ECM7.
Based on the conclusions reached by Clevers, Sato et al. were able to demonstrate that
intestinal crypt-villus organoids can be generated from a single SC without the presence of a
mesenchymal niche8. The characterising features of these crypt-villus organoids are the
capacity to expand for farther than 3 months while maintaining a stable genome.
Subsequently, this method, also termed R-spondin method, has been used and adapted to
create organoids from epithelial tissues of various organs (Fig. 1).
