discovering organoids symposium€¦ · discovering organoids - symposium friday 9th august 2019 -...

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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


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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


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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


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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


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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


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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


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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


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12:55 Bon-Kyoung Koo


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


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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


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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


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15:25 Veronica Krenn


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


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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


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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


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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


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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


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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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SUPPORTED BY:

DISCOVERING ORGANOIDS: INTESTINE AND CEREBRAL

#ORGANOIDjun19 @CamBioScience

discovering organoids symposium€¦ · discovering organoids - symposium friday 9th august 2019 -...

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