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SUPPORTED BY:
DISCOVERING ORGANOIDSSYMPOSIUMABC Center, Pusan National University, Busan, South Korea
Friday 9th August 2019
#ORGANOIDaug19 @CamBioScience
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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PARTNERS
SILVER
P A G E O N E
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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9:45 Plenary Talk: Nicolas Rivron
Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna, Austria
Blastoids: blastocyst-like structures generated solely from stem cells implant in utero
Early mammalian embryos form a blastocyst structure comprising embryonic cells surrounded by a
thin-walled trophoblast cyst mediating the implantation into the mother's uterus and then forming the
placenta. From mouse blastocysts, both trophoblast and embryonic stem cell lines can be derived as in
vitro analogues of the trophectoderm and embryonic compartments, respectively. Our lab showed that
trophoblast and embryonic stem cells self-organize in vitro into structures that morphologically and
transcriptionally resemble E3.5 blastocysts (‘blastoids’), and implant in utero.Blastoids form primitive
endoderm cells thus contain analogues of the three lineages generating the whole organism. Like
blastocysts, blastoids form through inductive signals originating from the inner embryonic cells and
driving outer trophectoderm development. The nature and function of these signals are largely
unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments
along with single cell transcriptomics revealed an extensive list of inductive signals. Among other
functions, the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning
trophoblast epithelial morphogenesis. Altogether, these inductions are paramount to form a
trophectoderm state that robustly implants and triggers a decidualization in utero (formation of a
cocoon). Thus, at this stage, the nascent embryo fuels the development and implantation of the future
placenta. Blastoids open new possibilities to investigate the processes of multicellular self-organization
underlying patterning and cell fate decision.
P A G E T W O
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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10:30 Stefano Giandomenico
MRC-LMB, University of Cambridge, UK
Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output
Cerebral organoids have the potential to improve our understanding of human brain development and
neurological disorders. However, it remains to be seen whether these tissues can model circuit formation
with functional neuronal output. We present an air–liquid interface culture paradigm adapted to cerebral
organoids, which leads to improved neuronal survival and axon outgrowth. The resulting thick axon tracts
display various morphologies, including long-range projection within and away from the organoid,
growth-cone turning, and decussation. Single-cell RNA sequencing reveals a diverse set of cortical neuron
identities, and retrograde tracing demonstrates tract morphologies that match proper molecular
identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate
mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact
organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of
corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant
aspects of human CNS development and disease.
P A G E T H R E E
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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10:55 Sam Wu
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
Using genetic mouse models to study adult tissue behaviors
Imaging-based lineage tracing is a widely used method to trace potency of particular cell types and it is
done by expressing CreER from a gene locus of interest and which can recombine a lox-stop-lox cassette
on a ubiquitous locus (such as the mouse Rosa26 locus) upon tamoxifen administration1. Using the
Rosa26-Confetti allele, which is a conditional multicolor reporter (containing GFP, YFP, RFP and CFP), it
allows simultaneously labeling of different clones and probing the clone-clone competition over time.
Using such approach of multicolor lineage in murine small intestinal epithelium coupled with
mathematical modeling, Snippert and colleagues (2010)2 revealed the neutral drift phenomenon where
each intestinal stem cells have equal chances of taking over the crypt compartment in homeostasis.
Although the confetti allele allows simultaneous tracing of multiple cells, it remains at the observatory
level. To advance the lineage tracing method, we have generated confetti variants called Red2cDNA series
that harbors ectopic gene expression in the red clone-specific manner. We have now generated red
clone-specific expression of various oncogenes (such as Wnt, Notch, Kras, PI3K and Yap) and red
clone-specific loss of functions via FLPe recombinase and a dox-inducible Cas9 endonuclease system.
Upon Cre activation in the tissue-specific manner in adult mice, only the red clones carry oncogenic
signals in an otherwise genetically normal environment. We envision that Red2cDNA series as a genetic
mosaic system will allow precise modeling of clone-clone competition in various dynamic processes such
as development, regeneration or cancer development.
1. Wu SS, Lee JH, Koo BK. Lineage Tracing: Computational Reconstruction Goes Beyond the Limit of
Imaging. Molecules and Cells. PMID: 30764600
2. Snippert HJ1, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein
AM, van Rheenen J, Simons BD, Clevers H. Intestinal crypt homeostasis results from neutral competition
between symmetrically dividing Lgr5 stem cells. Cell. PMID: 20887898
P A G E F O U R
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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11:10 Shuan Rao
Department of Thoracic Surgery, Nanfang Hospital, Guangzhou, China
Modelling esophageal squamous cancer: the demand for new research system
Unlike many other cancers, the genetic drivers for esophageal squamous cancer are still unknown,
therefore, the in vivo system to mimic esophageal squamous’ development is rarely established and
validated. Here we would like to present some preliminary data showing the cytotoxic drug cisplatin
induced pyroptosis in esophageal cancer cells using in vitro and xenograft model, we would also discuss
the limitation of this study and propose the newly developed esophageal cancer organoids system as a
powerful tool to explore the pathogenesis of esophageal cancer development in the future.
P A G E F I V E
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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12:05 Jens Puschhof
Hubrecht Institute, Utrecht, Netherlands
Slithering stem cells - snake venom gland organoids
Recent advances in organoid technology have proven this system to be a valuable tool in understanding
human organ development and pathologies. These adult stem cell derived cultures closely recapitulate
structural and functional properties of their organ of origin. Here, we expand the organoid technology
toolbox by describing a protocol to culture non-mammalian organoids derived from a snake venom gland.
The complexity of venom production, composition and function remains largely unknown for many
species. Organoids derived from an Aspidelaps lubricus venom gland can be long-term expanded and
histologically resemble the gland. Expression of typical venom-related transcripts (3FTx and Kunitz-type
protease inhibitors) can be detected in proliferating organoids with RNA sequencing. Single cell RNA
sequencing reveals distinct venom expressing cell types, as well as proliferating cells with features of
mammalian stem cells. Using mass spectr! ometry, we identify peptides in the culture medium supernatant
that match the composition of the crude venom of the same species. Venom gland organoids furthermore
consist of specialized secretory cells visible by transmission electron microscopy. The system enables
investigation of venom production and function on a cellular level in controlled conditions and without the
need of experimental animals. This study describes the adaption of organoid technology to a
non-mammalian species, providing a model to understand the complexity of the snake venom gland.
P A G E S I X
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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12:30 Ki-Jun Yoon
Korea Advanced Institute of Science and Technology, Daejeon, South Korea
Investigation of human brain development and neurodevelopmental disorders using human cerebral
organoid model
Using both in vivo mouse model and in vitro induced pluripotent stem cell (iPSC)-derived cerebral
organoid model, we have studied on neural stem cell behaviors in normal development and brain
disorders. First, we established human iPSC lines from healthy controls and schizophrenia patients
harboring 15q11.2 copy number variants and used these lines to elucidate the mechanism of
neurodevelopmental defects in neural progenitor cells. Second, Zika virus infection has been shown to
cause neurodevelopmental defects such as microcephaly, but critical components were largely unknown.
To perform a functional screen of all ZIKV-encoded proteins, we electroporated each one individually into
embryonic mouse cortices and human forebrain organoids. As a result, we found that one critical
component, Non-structural protein 2A, is responsible for deficits in NPC proliferation, polarity and
neuronal migration by disrupting adherens junctions. Last, we investigated a fundamental biological
process regulating neural stem cells, post-transcriptional regulation via mRNA methylation.
N6-methyladenosine (m6A) is the most prevalent internal mRNA modification, but its role in neural stem
cells and neurogenesis was previously unknown. Using gene manipulation of m6A methyltransferase,
Mettl14, and transcriptome-wide identification of m6A sites in both mouse and human cerebral organoids,
we revealed that epitranscriptomic m6A-tagging, via regulation of mRNA decay, provides a key
mechanism for temporal control of dynamic gene expression, which in turn regulates cell cycle
progression of cortical neural stem cells. These works show examples to utilize brain organoid technology
for basic and translational research.
P A G E S E V E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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12:55 Bon-Kyoung Koo
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
CRISPR/Cas-assisted genetics in intestinal organoids
The identification of LGR5+ intestinal stem cells helped us to understand various aspects of adult stem
cells and led to the establishment of primary 3D intestinal organoid culture system from mouse and
human tissues. This novel culture system faithfully recapitulates various aspects of the intestinal
epithelium in vitro with remarkable long-term expansion capacity and genetic stability. Thus, the model is
recognised as a suitable in vitro model system for genetic studies. To exploit all the potential of this
culture, protocols have been fully optimised for primary establishment, maintenance, cryopreservation,
plasmid transfection and viral transduction. A number of examples will be shown to introduce how to
apply CRISPR technology and organoid models for genetic studies, including simultaneous paralogue
knockout, functional genetic screening and precise gene correction.
P A G E E I G H T
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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14:35 Minsuh Kim
Department of Pathology, Asan Institute for Life Sciences, Seoul, South Korea
The patient-derived lung cancer organoid as an in-vitro model predicting patient therapeutic response
Ideal tumor models for precision cancer medicine require maintenance of genetic and phenotypic
heterogeneity of human tumors and ease of handling. Patient derived xenograft model may maintain
tumor heterogeneity of their original tumors. Cancer cell lines have been widely used as cancer model
with robustness of experiments. Recently, cancer organoids have been reported as in vitro models of
various human cancers having important advantage of PDX and cell lines. We developed methods to
generate a biobank of patient-derived lung cancer organoids from five different histological types of lung
cancer including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, adenosquamous
carcinoma and large cell neuroendocrine carcinoma. Histologically, adenocarcinoma organoids maintained
typical glandulo-papillary structures or mucin-containing tumor cells as their corresponding tumor tissues.
Organoids of squamous cell carcinomas showed keratinization and intercellular bridges. Organoids of
small cell carcinoma displayed typical neuroendocrine morphology with expression of neuroendocrine
markers such as CD56 and syneptophysin. Using NGS cancer panel sequencing, the organoid lines
maintained genetic alterations of major driver genes including EGFR, TP53, and RB in the original human
tumors. In xenograft experiments, the organoid lines revealed stronger tumorigenesity than direct graft of
human tumor tissues. Molecularly targeted drug testing using the lung cancer organoids showed drug
responses according to their genomic profiles. In conclusion, our lung cancer organoid lines are an
alternative cancer model recapitulating genotypic and phenotypic heterogeneity of original human
tumors for predicting patient therapeutic responses.
P A G E N I N E
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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15:00 Eunjee Kim
Pohang University of Science and Technology, South Korea
Three-dimensional reconstitution of miniature bladders that structurally and functionally recapitulate in
vivo tissue regeneration and cancer
Current organoid models are limited by their failure to account for factors, such as mature organ
architecture and tissue microenvironment. Here, we reconstitute tissue stem cell-based, multilayered
miniature bladders that structurally and functionally mimic mature mammalian urinary bladders. These
mini-bladders recapitulate the in vivo tissue dynamics of the regenerative response to bacterial infection;
heightened activity of signalling feedback between the urothelium and stroma and the associated
increase in cell proliferation cause the regenerative portions of urothelium to arise from single cells
through oligoclonal expansion. Further, using three-dimensional bioprinting technology, we developed
multilayered tumor organoids with stroma that recapitulate the in vivo pathophysiology of patient-derived
invasive urothelial carcinoma, including tumor–stroma interaction, slower drug response, immune cell
infiltration and muscle invasion. Thus, our study provides a conceptual framework for the reconstitution of
multilayered, functional organoids derived from tissue stem cells or tumor cells that mimic the biology of
native tissues.
P A G E T E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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15:25 Veronica Krenn
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
Organoids as models for infectious diseases in the developing central nervous system
Human brain organoids are in vitro 3D culture systems derived from pluripotent stem cells that have the
potential to recapitulate many aspects of human brain development and brain disease. In particular, brain
organoids are emerging models for infectious diseases to study the teratogenicity of pathogenic viruses,
such as ZIKA virus, in a human context. However, little is known about the antiviral defenses and
host-virus interactions in human brain organoids. We have established various organoid infection models
that reproduce the teratogenic effects of neurotropic viruses on brain development. We have used these
models to characterize the antiviral responses and uncovered that brain organoids fail to mount strong
interferon responses against viruses as well as against the immunostimulant polyI:C. Nevertheless, we
have found that organoids retain the ability to respond to exogenous interferons and treatment with low
doses of interferons reduces viral loads and ameliorates disease outcomes. Our work identifies an
“immature” status of intrinsic immunity that contributes to the vulnerability of brain organoid cultures to
viral infections and provides a platform for the identification of novel antiviral factors that can be
exploited for antiviral therapy.
P A G E E L E V E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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15:50 Il Ho Jang
Department of Life Science in Dentistry, School of Dentistry, Pusan National University, Republic of
Korea
Dental Organoid: The First Steps
Dental tissue exhibits fast regeneration without scar formation and harbors extraordinarily high activity of
adult stem cells in terms of variety and frequency. Developmentally dental stem cells originate from neural
crests, thus displaying characteristics of ectomesenchyme distinct from those of mesenchyme. Dental
pulp, soft tissue inside tooth analogous to bone marrow, is an excellent source of adult stem cells, in which
dental pulp stem cells maintain the integrity of tooth through differentiation to odontoblasts. The
locations of dental pulp stem cells were suggested as perivascular or Schwann cell-derivative. In our
attempt to generate dental pulp organoid with isolated human dental pulp stem cells, reticular network
formation was observed toward the periphery of the matrix. In the whole explant culture of human dental
pulp, reticular network grew outward from dental pulp with hard tissue formation in the matrix after serial
transfer. Electron microscopy analysis revealed a well-organized dentinal tubule formation as well as
unstructured osteodentin formation. In 3D imaging of mouse incisor after tissue clearing, Sox2-positive
cell cluster was identified near root apex where high stem cell activity has been detected. Spheroid
formation with human dental pulp stem cells generated SOX2-postivie cluster. Though premature, current
progress in generating dental organoid reflects the ectomesenchymal and perivascular nature of dental
pulp stem cells.
P A G E T W E L V E
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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16:45 Shan Bian
Institute for Regenerative Medicine, Shanghai East Hospital; School of Life Science and Technology,
Tongji University, Shanghai 200092, China
Modeling brain tumor formation using Cerebral organoids
Brain tumours are among the most lethal and devastating cancers. Clinical treatment of brain cancers is
currently limited by genetic heterogeneity and the lack of appropriate laboratory models. To address this,
we developed an in vitro 3D organoid model that recapitulates the formation of brain tumors with defined
genetic aberrations. By introducing tumorigenic mutations into cerebral organoids via transposon
insertion and CRISPR/Cas9-mediated mutagenesis, we are able to recapitulate brain tumor formation in
vitro. Screening through a variety of gain- and loss-of-function mutations that were identified in brain
cancer sequencing projects, we define one oncogene that induces primitive neuroectodermal tumour
(CNS-PNET)-like neoplasms in vitro. In addition, we identify glioblastoma (GBM)-relevant gene
aberrations that result in a glial neoplasm-like over-proliferation, while CNS-PNET organoids showed many
features observed in human CNS-PNET specimens. Furthermore, our results show that the newly
developed in vitro brain tumor models can be used to evaluate drug efficacy on tumours with specific
DNA aberrations. Our results demonstrate that brain neoplastic organoids can be used to test strategies
for brain tumour therapy in a personalized manner.
P A G E T H I R T E E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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17:10 Hyung-Sik Kim
Department of Life Science in Dentistry, School of Dentistry, Pusan National University, Republic of
Korea
Enteroendocrine cell-derived hormone A is involved in the intestinal homeostasis by directing
differentiation of the mouse intestinal organoid
Organoids can be utilized as a modeling system for the investigation of stem cell biology, organ
development and disease progression, as well as for drug discovery. Here, we isolated mouse small
intestinal crypts and cultured intestinal epithelial organoids, one of the most well-established organoid
system. It develops a highly organized structure with both adult stem cell niches and fully differentiated
populations. Interestingly, as the largest endocrine system in the body, enteroendocrine cells(EECs)
produce the highest level of hormones and bioactive molecules despite that they comprise only 1% of the
intestinal epithelium. In this study, we focused on EEC-derived endogenous signals to evaluate their
impact on the intestinal homeostasis. It was noted that one of EEC-secreted hormone A was impeded the
normal generation of intestinal organoids; upon treatment of A, organoid growth was retarded and the
typical budding pattern was almost disappeared, resulting in the round to oval shaped-organoids. The
epithelial lining was intact and budding ability was restored after A withdrawal, suggesting that A did not
induce epithelial cell death. We found that the morphology of A-treated organoid was similar to that of
IWP-2(Wnt inhibitor)-, DAPT (Notch inhibitor)-and U0126(MEK inhibitor)-treated organoid. Since these
signaling are important to maintain ISC population, we performed qPCR to screen the ISC and
differentiated intestinal cell markers. Importantly, both active ISC and proliferation markers are
down-regulated, while secretory lineage markers were increased upon A treatment. These data suggest
the novel endogenous impact of EEC-derived hormones on ISC maintenance, differentiation and intestinal
homeostasis.
P A G E F O U R T E E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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17:35 Yoohee Jin
Department of Biotechnology, Yonsei University, Seoul, South Korea
Bioengineering approaches for organoid cultures and applications
Organoid technologies offer powerful insights into the biological processes of the tissues with high
complexity. Despite their huge potential, there are many challenges that remain. This presentation will
show recent advances in bioengineering approaches to improve organoid maturation, scale-up culture,
and reproducibility. This talk will draw on several examples of organoids to illustrate how bioengineering
can contribute to drug discovery and tissue formation as well as remaining challenges we should
overcome. This study was supported by the Bio & Medical Technology Development Program of Korea
National Research Foundation (NRF) funded by the Koran government, the Ministry of Science and ICT
(MSIT) (2018M3A9H1021382).
P A G E F I F T E E N
DISCOVERING ORGANOIDS - SYMPOSIUMFriday 9th August 2019 - Busan, South Korea
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18:00 Plenary Talk: Cantas Alev
Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Japan
Recapitulating the human segmentation clock in vitro
Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis
and organ formation. Despite recent advances in the in vitro induction of major mesodermal lineages and
mesoderm-derived cell types, experimental model systems that can recapitulate more complex biological
features of human mesoderm development and patterning are largely missing. Here, we utilized induced
pluripotent stem cells (iPSCs) for the stepwise in vitro induction of human presomitic mesoderm (PSM)
and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling
the human segmentation clock, a major biological concept believed to underlie the rhythmic and con-
trolled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton.
We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and
DKK1, and identified novel oscillatory genes in human iPSC-derived PSM. We furthermore determined the
period of the human segmentation clock to be around five hours and showed the presence of dynamic
traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based
genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial
skeletal development and segmentation defects of the vertebrae such as spondylocostal dysostosis have
been reported (e.g. HES7, LFNG, DLL3, MESP2). Subsequent analysis of patient-like iPSC knock-out and
point mutation lines as well as patient-derived iPSCs together with their gene-corrected isogenic controls
revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating
the overall utility of our model system.
P A G E S I X T E E N
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