Organoids — Preclinical Models of Human Disease
Item Type Article
Authors Li, Mo; Izpisua Belmonte, Juan C.
Citation Li M, Izpisua Belmonte JC (2019) Organoids — Preclinical Models
of Human Disease. New England Journal of Medicine 380: 569–
579. Available: http://dx.doi.org/10.1056/nejmra1806175.
Eprint version Publisher's Version/PDF
DOI 10.1056/nejmra1806175
Publisher Massachusetts Medical Society
Journal New England Journal of Medicine
Rights Archived with thanks to New England Journal of Medicine
Download date 09/08/2022 18:22:57
Link to Item http://hdl.handle.net/10754/631062
1
Organoid Technology Development for Preclinical Models of
Human Disease
Mo Li
1
, PhD and Juan Carlos Izpisua Belmonte
2,
*, PhD
1
King Abdullah University of Science and Technology
2
The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA
92037
*Corresponding Author: belmonte@salk.edu
Word count: 3837
2
Introduction
Currently, an organoid is commonly understood to be a three-dimensional (3D) construct
composed of multiple cell types, originating from stem cells through self-organization, and
capable of simulating the architecture and functionality of native organs. Organoids have
recently emerged as a versatile model that spans the crossroads of in vivo and in vitro
investigation. The technique represents an innovation in the long quest (see historical
notes in Supplementary Appendix) for an in vitro model that faithfully recapitulates
physiological processes of whole organisms. Organoids have many advantages over
traditional 2D cultures. They display near-physiological cellular composition and
behaviors. Many organoid cultures can undergo extensive expansion in culture and
maintain genome stability,
1-4
which makes them suitable for biobanking and high-
throughput screens.
5
Compared to animal models, organoids can reduce experimental
complexity, facilitate precision genetic and imaging techniques, and, more importantly,
enable the study of human development and diseases that is not feasible in animals (Fig.
1).
6-9
To date, organoid technologies have been successfully implemented to address a
wide range of questions–from brain gyrification to personalized cancer therapeutics.
6,7
Here we review the common platforms of organoid technology, highlight recent progress
in their application, and discuss ongoing challenges and future possibilities.
Organoids from Various Stem and Other Cell Sources
Organoids can be generated using somatic cells, adult tissue-resident stem cells
(including progenitor cells) or pluripotent stem cells. Because of limited tissue availability,
expandability, and throughput, somatic cell organoids (recently reviewed
8
) are less widely
used than stem cell organoids and so will not be further discussed.
Organoids from Adult Stem Cells
An important breakthrough in intestinal organoid technology occurred in 2009 when the
group led by Hans Clevers showed that intestinal adult tissue-resident stem cells have an
uncanny ability to proliferate and self-organize in vitro.
9
Intestinal stem cells are
characterized by the expression of the leucine-rich repeat-containing G-protein coupled
receptor 5 (LGR5) gene–a receptor for the Wnt agonist R-spondin.
10,11
Intestinal stem
cells niche factors include Wnt,
epidermal growth factor (EGF), and Noggin, a bone morphogenetic protein (BMP)
inhibitor
.
9
Extracellular matrix is another important constituent of the niche, as dissociated
intestinal cells undergo anoikis.
12
Based on such knowledge, Sato et al.
9
developed a 3D-
embedded culture (often called the R-spondin method, see Fig. 2) to reconstitute an in
vitro niche-like milieu for intestinal stem cells. The cultures that developed with this
method allow single Lgr5+ stem cells to generate a crypt-villus architecture with all
differentiated cell types in a self-renewing fashion.
9
These organoids can expand more
than three months and remain genomically stable, which facilitates the purification of large
quantities of organ-specific cell types.
The R-spondin method has since been adapted to generate organoids from epithelial
tissues from all three germ layers (Supplementary Appendix Table 1). Details about
3
different types of organoids can be found in a recent review
13
and will not be discussed
here. It is worth noting that the presence of Lgr5+ stem cells is not a prerequisite for
organoid generation.
14-16
The liver and pancreas do not have appreciable Lgr5 expression
under homeostatic conditions. Interestingly, Lgr5+ ductal cells are induced during
regenerative responses following liver or pancreatic injury. Such Lgr5+ cells can form
clonal organoids composed of bipotential progenitors (hepatocyte and bile duct potential
for the liver, and ductal and endocrine lineages for the pancreas).
17,18
Bipotent human
liver and pancreas organoids have also been generated.
1,17,19
Thus, the R-spondin
method appears to be applicable to long-term maintenance of adult stem cells of many
epithelial tissues in organoids.
Several organoid cultures of the components of the genitourinary system have been
recently reported—female reproductive and male reproductive tracts, as well as kidney.
In the female reproductive tract, the human endometrium provides the microenvironment
for implantation and nutritional support for the early conceptus. Because in vivo study is
impractical, long-term culture models are needed to study the role of endometrial
secretion and endometrium-placenta interactions during early pregnancy. To culture
isolated endometrial epithelia, Turco et al. started with the R-spondin method, and
supplemented the medium with growth factors to mimic the in vivo niche of glandular
progenitor cells (Supplementary Appendix Table 1). Endometrial organoids were
established from human non-pregnant endometrium and decidua. They are genetically
stable, mount an appropriate transcriptional response to sex hormones, and recapitulate
characteristics of gestational endometrium when stimulated with early pregnancy
signals.
20
Organoids have also been obtained from malignant endometrium, although
clonogenic and xenotransplantation assays need to be performed to substantiate their
value as a model for endometrial cancer. To realize their potential in basic research of
human pregnancy, and in developing therapies for endometriosis and endometrial cancer,
these organoids need to be further characterized for their secretory function and their
ability to model implantation of in vitro cultured blastocysts.
There also has been progress in the development of organoids of the male reproductive
system. A recent study showed self-organization of dissociated human testicular cells
under conditions similar to organotypic culture of neonatal mouse testis.
21,22
The
dissociated cells formed a condensed spheroid that has been termed testicular organoids.
21
Despite the lack of native tissue topography, niche cells and spermatogonia persisted
in testicular organoids
21
(Supplementary Appendix Table 1). However, differentiation of
spermatogonia, meiosis, and sperm formation were not reported. The testicular
constructs do not undergo long-term expansion, which makes them more akin to primary
organ culture. Demonstration of clonogenic potential and functionality is critically needed
to move the field forward.
The functional unit of the kidney composed of a renal corpuscle and a renal tubule
(together termed the nephron)–depends on an intricate tissue architecture for its function.
During development, nephrogenesis requires reciprocal interactions between two kidney
progenitor populations in the intermediate mesoderm–the metanephric mesenchyme and
4
the ureteric epithelium. The spatiotemporal unfolding of mesenchyme-epithelium mutual
induction, cell movement, proliferation, and cell adhesion suggests a genetically encoded
self-organization program.
23
Indeed, dissociated embryonic kidney cells self-organize into
their tissue of origin with high spatial fidelity.
24
The developing kidney contains transient
amplifying nephron progenitor cells (NPCs) that give rise to all nephrons.
25
Such nephron
progenitor cells have not been found in the adult human kidney, which cannot regenerate
nephrons once these are lost.
26
However, several putative adult kidney progenitors
capable of tubulogenesis in organoids have been reported,
27-29
but, there is considerable
disagreement on their identity and potential fate.
27,28
Embryonic nephron progenitor cells, on the other hand, are much better characterized
and have been successfully used to create kidney organoids.
30-33
Despite such studies,
many hurdles still remain—first, the loss of differentiation potential in cultured nephron
progenitor cells; second, limited self-renewal of these cells; third, a lack of evidence for in
vivo nephrogenic potential; and, finally, dependence on transgenic markers. Based on
previous observations,
34-36
we developed a long-term 3D culture of genomically stable,
self-renewing nephron progenitor cells
4
(Supplementary Appendix Table 1). It is worth
noting that these developmental progenitors are distinct from adult stem cells, as they do
not appear to exist in the adult kidney and can only self-renew under artificial culture
conditions. In the classic spinal cord induction assay, clonal nephron progenitor cell lines
formed nephron-like structures with proper spatial orientation, indicating an intact
nephrogenic potential.
4
Cultured nephron progenitor cells can differentiate into organoids
with numerous tubular structures that express major nephron segmental markers. These
kidney organoids can contribute to nephrogenesis in neonatal mice and chick embryos.
They generate ectopic nephron-like structures that connect with the host vasculature and
filtrate urine-like fluid when transplanted into the omentum of immunodeficient mice.
4
Human nephron progenitor cell cultures with similar properties can be derived from fetal
kidneys between 9 to 17 weeks of gestation. Nephron progenitor cell lines are amenable
to CRISPR-based genome editing to study human organogenesis and genetic diseases
(Supplementary Appendix Table 2).
4
Pluripotent Stem Cells
Pluripotent Stem Cells can self-renew indefinitely and differentiate into any cell type in the
body, thus offering an attractive alternative to the use of primary tissues to create
organoids. Pluripotent stem cell-derived organoids are formed through directed
differentiation of a homogeneous population, so tissue-specific cell types and their
microenvironment must be created de novo in a dynamic process reminiscent of
embryogenesis. Accordingly, pluripotent stem cell organoid culture must provide stage-
appropriate niche signals during the differentiation. Because the differentiation cues are
not strictly limited to the desired cell fates, pluripotent stem cell organoids often contain
cell types that differ from those in a given organ and that may complicate the signaling
environment and self-organization of the target tissue.
37-39
Early work in directed differentiation established signaling requirements for germ layer
formation, patterning, and induction of tissue identity in 2D cultures. Pluripotent stem cells