ASHBi Symposium 2021

Human
Development,
Genetics andEvolution

ASHBi International Symposium 2021
Symposium Time Slots
08 November 2021
Monday, 8 November (Day 1)

Tuesday, 9 November (Day 2)

Wednesday, 10 November (Day 3)JST

PST *1 EST *2 GMT *3

22:00

Claire Rougelle(U Paris)

18:55–19:10

10:05–10:35

Break (30-min)

10:35–10:50

Yasuhiro Takashima

11:15–11:40(ASHBi)
19:25–19:40

Takuya Yamamoto(ASHBi)

19:40–19:55

Tomoyuki Tsukiyama
Break (30-min)Cantas Alev

17:40–18:05

J Gray Camp*(IMO Basel)

Ryuichi Nishinakamura(Kumamoto U)

18:05–18:30

Anne Grapin-Botton(ASHBi)
20:40–21:05

Aida Andres(U C London)

21:05–21:30

Janet Kelso(Max Planck)

Chair: Cantas Alev

17:15–17:40

2:00

17:00-17:25

Takashi Hiiragi

17:25-17:50

James Briscoe

17:50–18:15

Barbara Treutlein*

20:30–20:55

Break (30-min)

Prisca Liberali(F Miescher Inst)

20:55–21:20

0:00

3:00

8:00

1:00

4:00

9:00

2:00

5:00

10:00

3:00

6:00

11:00

4:00

7:00

12:00

5:00

8:00

13:00

(The Francis Crick Instirtue)(ETH Zürich)

18:15–18:45

Break (30-min)

18:45–19:10

Katsuhiko Hayashi(Kyushu U)

19:10–19:35

Azim Surani(Wellcome Trust)

19:35–20:00

Anne Goriely(U Oxford)

20:00–20:25

Mitinori Saitou(ASHBi)

20:25–20:35

Closing Remarks*updated

Toshiro Sato
(Keio U)

21:20–21:45(EMBL/ASHBi)

Break (30-min)

19:30–20:00 Poster Session 2

20:00–20:30

Chair: Ryo Yamamoto

Yasuhiro Murakawa

18:00(-1) 21:00(-1)(Max-Planck Society)(ASHBi)

20:25–20:40

Chair: Fumitaka Inoue

Misao Fujita

1:00

Davor Solter

19:00–19:30 Poster Session 1

Poster Session

19:10–19:25

17:00(-1) 20:00(-1)

Diana Laird(UCSF)Break (30-min)(ASHBi)

0:00(Kyoto Univeristy)
10:50–11:15(U Copenhagen)Yasuaki Hiraoka

16:00(-1) 19:00(-1)(Harvard Medical)(ASHBi)

Chair: Motoko Yanagita

18:00–18:25

Organoid biology/Cancer genome

21:30

Evolutionary mechanisms

21:00(The Francis Crick Institute)

19:55–20:25

20:00

20:30

James Turner

Bhagat Shruti

18:30-19:00

Introduction of ASHBi
Chair: Mitinori Saitou

19:30

17:35–18:00

18:25-18:55

18:30

19:00

(Heidelberg U)

9:50–10:05

17:00–17:15

Organoid biology/Cancer genome

Evolutionary mechanisms

18:00

Chair: Yasuhiro Murakawa

17:30

Henrik Kaessman

Christopher Walsh

Development/Neural development

Opening Remarks
17:10–17:35

9:25–9:50

Chair: James Turner

JST
17:00

Chair: Mitinori Saitou

Development/Neural development

11:00(RIKEN CBS/Kyoto U)(ASHBi)

10:00

10:30

Ryoichiro Kageyama

Germ Cells: Genetic and epigeneitic regulation

9:30

9:00–9:25

Chair: Tadashi Isa

Development/Neural development

9:00

Seishi Ogawa(ASHBi)*1 Pacific Standard Time
*2 Eastern Standard Time
*3 Greenwich Meant TimeMonday, November 8 (Day 1)
Opening Remarks
17:00-17:10
Mitinori Saitou
Evolutionary mechanisms (Chair: Yasuhiro Murakawa)
17:10-17:35

S-1
The evolution of spermatogenesis across mammals
Henrik Kaessmann (Heidelberg University)

17:35-18:00

S-2
The evolution of X-dosage compensation in mammals
James Turner (The Francis Crick Institute)

18:00-18:25

S-3
X chromosome inactivation in humans
Claire Rougeulle (University of Paris)

18:25-18:55

Break

Introduction of ASHBi (Chair: Mitinori Saitou)
18:55-19:10

A-1
Fusion researches between biology and mathematics in ASHBi
Yasuaki Hiraoka (ASHBi, Kyoto University)

19:10-19:25

A-2
Biothics and Science at ASHBi
Misao Fujita (ASHBi, Kyoto University)

19:25-19:40

A-3
Introduction of Single-Cell Genome Information Analysis Core (SignAC) at ASHBi
Takuya Yamamoto (ASHBi, Kyoto University)

19:40-19:55

A-4
Generation of genetically modified monkeys in PRiME.
Tomoyuki Tsukiyama (PRiME, Shiga University of Medical Science)

19:55-20:25

Break

Evolutionary mechanisms (Chair: Fumitaka Inoue)
20:25-20:40

S-4*
Single-cell enhancer identification reveals genetic and evolutionary mechanisms ofhuman diseases
Yasuhiro Murakawa (ASHBi, Kyoto University)

20:40-21:05

S-5
Human evolution: how humans managed to adapt to life around the globe.
Aida Andres (University College London)

21:05-21:30

S-6
The contribution of ancient humans to understanding modern human genetic variation
Janet Kelso (Max Planck Institute for Evolutionary Anthropology)*Selected talks

Tuesday, November 9 (Day 2 - Morning)
Development/Neural development (Chair: Tadashi Isa)
9:00-9:25

S-7
Dynamic transcriptional control of neural stem cells
Ryoichiro Kageyama (Kyoto University / RIKEN Center for Brain Science)

9:25-9:50

S-8
Molecular genetics of human brain development and evolution.
Christopher Walsh (Harvard Medical School

9:50-10:05

S-9*
Elucidating the genetic and evolutionary differences between promoters and enhancers at
high resolution
Bhagat Shruti (ASHBi, Kyoto University)

10:05-10:35

Break

Development/Neural development (Chair: Mitinori Saitou)
10:35-10:50

S-10*
Analyzing human peri-implantation development using naïve pluripotent stem cells
Yasuhiro Takashima (Kyoto University)

10:50-11:15

S-11
A xenotransplantation model for studying the migration of human primordial germ cells
Diana J. Laird (University of California, San Francisco)

11:15-11:40

S-12
Epigenetic Mechanisms Controlling Early Mammalian Development
Davor Solter (Max-Planck Society)*Selected talks

Tuesday, November 9 (Day 2 -Evening)

Organoid biology/Cancer genome (Chair: Motoko Yanagita)
17:00-17:15

S-13*
Reconstituting human somitogenesis in vitro
Cantas Alev (ASHBi, Kyoto University)

17:15-17:40

S-14
Charting human development using a multi-endodermal organ atlas and organoid
modelsJ Gray Camp (Roche Innovation Center Basel / University of Basel)

17:40-18:05

S-15
Kidney organoids for modeling human development and disease
Ryuichi Nishinakamura (Kumamoto University)

18:05-18:30

S-16
Modelling human pancreas development with organoids
Anne Grapin-Botton (MPI-CBG)

18:30-19:00

Break

Poster session
19:00-19:30
Poster Session 1
Room # Poster #Name
Room 1
P-1
Rio Tsutsumi
Room 2
P-3
Mitsuru Sasaki-Honda
Room 3
P-5
Hanna Lu
Room 4
P-7
Shino Shimada
Room 5P-9
Anjani Kumar Tiwari
Room 6
P-11
Shoichiro Tani
Room 7
P-13
Takafumi Ichikawa
19:30-20:00
Poster Session 2
Room # Poster #Name
Room 1
P-2
Shinya Oki
Room 2P-4

Zhe Wang

Room 3
Room 4
Room 5
Room 6

P-6
P-8
P-10
P-12

Masaya Hagiwara
Kenji K. Kojima
Takahiro Suezawa
Shruti Bhagat

20:00-20:30

Break

Affiliation
ASHBi, Kyoto University
CiRA, Kyoto University
The Chinese University of Hong Kong
National Human Genome Research Institute
Babasaheb Bhimrao Ambedkar University
The University of Tokyo
ASHBi, Kyoto University

Affiliation
Kyoto University Graduate School of Medicine
Institute for Frontier Life and Medical Sciences,
Kyoto University
Cluster of Pioneering Research, RIKEN
Genetic Information Research Institute
Graduate School of Medicine, Kyoto University
ASHBi, Kyoto University

Organoid biology/Cancer genome (Chair: Ryo Yamamoto)
20:30-20:55

S-17
Design principles of multicellular systems
Prisca Liberali (Friedrich Miescher Institute for Biomedical Research)

20:55-21:20

S-18
Modeling human gastrointestinal diseases using organoid technology
Toshiro Sato (Keio University)

21:25-21:45

S-19
Molecular classification and risk stratification of colorectal cancer
Seishi Ogawa (ASHBi, Kyoto University)Wednesday, November 10 (Day 3)
Development/Neural development (Chair: Cantas Alev)
17:00-17:25

S-20
Self-organisation in mammalian development
Takashi Hiiragi (ASHBi, Kyoto University / Hubrecht Institute)

17:25-17:50

S-21
The Dynamics of Spinal Cord Development
James Briscoe (The Francis Crick Institute)

17:50-18:15

S-22
Tracing and perturbing lineages during human brain organoid development
Barbara Treutlein (ETH Zurich)

18:15-18:45

Break

Session 4 Germ cells: Genetic and epigenetic regulation (Chair: James Turner)
18:45-19:10

S-23
Oogenesis in vitro: what we can and cannot reconstitute.
Katsuhiko Hayashi (Kyushu University)

19:10-19:35

S-24
Genomic imprinting, human development and the ‘immortal’ germline
Azim Surani (Wellcome Trust/CRUK Gurdon Institute, University of Cambridge)

19:35-20:00

S-25
The selfish testes of ageing men and de novo mutations
Anne Goriely (University of Oxford)

20:00-20:25

S-26
Mechanism and In Vitro Reconstitution of Mammalian Germ-Cell Development
Mitinori Saitou (ASHBi, Kyoto University)

Closing Remarks
20:25-20:35
Takashi Hiiragi

Posters
P-1

MODELING LIMB SKELETOGENESIS IN ORGANOID CULTURE DERIVED.
FROM MOUSE EMBRYONIC LIMB BUD AND HUMAN ES CELLS
Rio Tsutsumi (ASHBi, Kyoto University)

P-2

Data-driven and technical approaches to understand spatial gene regulation
Shinya Oki (Department of Drug Discovery Medicine, Kyoto University Graduate School of
Medicine)

P-3

Modeling disease-specific gene regulation of facio-scapulo-humeral muscular
dystrophy (FSHD)
Mitsuru Sasaki-Honda (CiRA, Kyoto University)

P-4

Hydrogel for precise manipulation of human pluripotent stem cells microenvironment
Zhe Wang (Institute for Frontier Life and Medical Sciences, Kyoto University)

P-5

TBA
Hanna Lu (The Chinese University of Hong Kong)

P-6

Organoids Platform: Control and Design the microenvironments to achieve organoidarchitecture
Masaya Hagiwara (Cluster of Pioneering Research, RIKEN)

P-7

Cancelled
Shino Shimada (NHGRI)

P-8

Human Transposable Elements in Repbase: Genomic Traces of Human Evolution
Kenji K. Kojima (Genetic Information Research Institute)

P-9

Brain Energy Dynamics with integration of functions
Anjani Kumar Tiwari (Babasaheb Bhimrao Ambedkar University)

P-10 Disease modelling of pulmonary fibrosis using human pluripotent stem cell-derived
alveolar organoids
Takahiro Suezawa (Department of Drug Discovery for Lung Diseases, Graduate School of
Medicine, Kyoto University / Watarase Research Center, Kyorin Pharmaceutical Co. Ltd.)P-11 Elucidating dynamic gene regulatory networks in human skeletal development using
single cell analyses on a human pluripotent stem cell-based model of endochondral
ossification
Shoichiro Tani (Center for Disease Biology and Integrative Medicine, Graduate School of
Medicine, The University of Tokyo / Sensory & Motor System Medicine, Graduate School of
Medicine, The University of Tokyo)
P-12 Elucidating the genetic and evolutionary differences between promoters and enhancers
at high resolution
Shruti Bhagat (Institute for the Advanced Study of Human Biology (ASHBi), Kyoto
University / RIKEN Center for Integrative Medical Sciences / Department of Biosciences andNutrition, Karolinska Institutet )P-13 An ex vivo system to study cellular dynamics underlying mouse peri-implantation
development
Takafumi Ichikawa (European Molecular Biology Laboratory (EMBL) / Institute for the
Advanced Study of Human Biology, Kyoto University)

ASHBi Symposium 2021

S-1
The evolution of spermatogenesis across mammals
Henrik Kaessmann
Center of Molecular Biology, Heidelberg University

The testis is a key male reproductive organ that produces gametes through the process ofspermatogenesis. Testis morphologies and spermatogenesis phenotypes evolve rapidly in mammals,presumably due to the evolutionary pressure on males to be reproductively successful. The rapid
evolution of the testis was shown to be reflected at the molecular level based on bulk-tissue work, but themolecular evolution of individual spermatogenic cell types across mammalian lineages remains largely
uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes
from ten species that cover the three major mammalian lineages (eutherians, marsupials, egg-laying
monotremes) and birds (the evolutionary outgroup), and include seven key primates. Our analyses revealthat the rapid evolution of the testis is driven by accelerated evolutionary fixation rates of regulatory
mutations, amino acid altering substitutions, and new genes in late spermatogenic stages – likely
facilitated by reduced pleiotropic constraints, haploid selection, and a transcriptionally permissive
chromatin environment. We identify temporal expression differences of individual genes across species,
which probably contributed to species-specific phenotypes, but also conserved expression programs
underlying ancestral spermatogenic processes. Sex-related analyses show that genes predominantly
expressed in spermatogonia (i.e., germ cells fueling spermatogenesis) and Sertoli cells (i.e., somatic
support cells) independently accumulated on X chromosomes across mammals during evolution,
presumably due to male-beneficial selective forces. Further work uncovered that the process of meiotic
sex chromosome inactivation (MSCI) is common to mammalian sex chromosome systems, contrary toprevious suggestions. Thus, the general mechanism of meiotic silencing of unsynapsed chromatin, which
underlies MSCI, represents an ancestral mammalian feature. Together, our study illuminates the cellularand molecular basis of the evolution of mammalian spermatogenesis and associated selective forces, andprovides a resource for investigating the biology of the testis across mammals.

ASHBi Symposium 2021

S-2
The evolution of X-dosage compensation in mammals
James TurnerFrancis Crick institute, London, UK

Females and males have the same autosome complement but differ with respect to their sex
chromosomes: females are XX, while males are XY. The resulting disparity in X-gene dosage is solved
by X-inactivation, the silencing of one X chromosome in females. X-inactivation is essential fordevelopment, is implicated in disease, and is a paradigm for understanding epigenetic regulation. In thisseminar, I will describe how our work on an unusual model organism, the marsupial, is shedding light on
the evolution of X-inactivation, embryonic lineage formation and epigenetic reprogramming in placental
mammals.

ASHBi Symposium 2021

S-3
X chromosome inactivation in humans
Claire Rougeulle
Department of Epigenetics and Cell Fate; CNRS/Université de Paris, Paris, France.

X chromosome inactivation (XCI) in mammals is an essential developmental process and aparadigm of epigenetic regulations. X chromosome inactivation has been mainly studied inmouse, where the actors of the process, mainly long non-coding RNAs, and the kinetics of themolecular events leading to the transcriptional silencing of one of the two X chromosomes havebeen partly deciphered. However, it is now known that X-inactivation initiates throughremarkable diverse strategies in different species. Understanding the molecular basis of suchplasticity, and in particular how X-inactivation is regulated in human is critical, especially sinceatypical or abnormal X inactivation patterns are linked to human pathologies such as cancer or
autoimmune diseases. We are using human embryonic stem cells as a model system for early
human development, to characterize the early stages of X chromosome inactivation and to
identify regulators of the process in human. We are in particular exploring the extent to whichlong non-coding RNA contribute to the variation in XCI strategies between species.

ASHBi Symposium 2021

S-4
Single-cell enhancer identification reveals genetic and evolutionary mechanisms of humandiseases
Yasuhiro Murakawa
ASHBi, Kyoto University

Recent large-scale genome-wide association studies (GWAS) have yielded an increasing number of
disease-associated genomic loci. However, the mechanistic interpretation is still far from complete.
Recently, it has become apparent that disease-associated genetic variants are often found in cell-typespecific enhancers, suggesting the importance of sensitive identification of enhancers in disease-relevant
tissues and cell-types. Here we developed our original computational and experimental approach toidentify active enhancers using single-cell RNA sequencing. Using this approach, we comprehensively
detected active enhancers from ∼500,000 heterogeneous CD4+ T cells, and constructed a comprehensive
atlas of transcribed enhancers. Integration of transcribed enhancers with GWAS data allowed us to predict
putative disease-relevant specialized CD4+ T cell subsets as well as causal nucleotide variants withincell-type-specific enhancers. Furthermore, our conservation analysis highlighted the evolutionary
diversity of autoimmune diseases. Thus, we provide a general framework to investigate molecularmechanisms underlying human diseases.

ASHBi Symposium 2021

S-5
Human evolution: how humans managed to adapt to life around the globe.
Aida Andrés
UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, London,UK

Modern humans evolved in Africa, colonising the rest of the world in only 50-100,000 years. This rapid
colonization of vastly different habitats was marked by pressures to adapt to each novel environment. The
resulting local adaptations generated genetic differences among human populations. These populationdifferences matter because natural selection can only act on variants with important effects in phenotype
and fitness. Any population difference that emerged due to local adaptation is thus likely to haveimportant consequences even today.
Ambient temperature was undoubtedly among the strongest selective pressures, as it varies widely aroundthe world. Temperature perception is particularly important as the first essential step for thermoregulationand thus survival. In fact, protein changes in the TRP cation channel that mediates cold perception(TRPM8) has mediated adaptation to cold temperatures across mammals. We previously provided
evidence that TRPM8 also mediated adaptation to cold habitats in humans. rs10166942, a SNP upstreamof TRPM8, shows remarkable population differentiation (5% frequency in Nigeria, 88% in Finland), its
allele frequency correlate with latitude and temperature, and its patterns of linked variation are best
explained by strong, local positive selection. Now, novel analytical methods and growing numbers ofancient genomes allow us to decipher the evolution of this variant in further detail. We infer thatrs10166942’s derived allele was under strong positive selection very shortly after the out-of-Africa
migration, resulting in high frequency of the beneficial allele in early European groups. Nevertheless,preliminary analyses suggest that positive remained strong after subsequent population admixtures anduntil relatively recent times. This is interesting because the putatively selected allele has been associatedwith increased expression of the cold receptor, increased cold sensitivity and increased risk of migraine. It
is thus likely that local adaptation has shaped both temperature perception and the risk of migraine incertain human populations.

ASHBi Symposium 2021

S-6
The contribution of ancient humans to understanding modern human genetic variation
Janet Kelso
Max Planck Institute for Evolutionary Anthropology

The genomes of archaic and ancient modern humans offer a unique way to learn about their histories andto gain insights into their unique physiologies. However, the sequencing and analysis of DNA fromancient humans is complicated by DNA degradation, chemical modifications and contamination. Recent
technological advances have made it possible retrieve and sequence DNA from bones and other remains
found at archaeological excavations, and we have been able to reconstruct the genomes of several
Neandertals. We have also identified, based on their genome sequences, a previously unknown extinct
Asian hominin group related to Neandertals, who we call “Denisovans”. The genomes of our extinct
Neandertal and Denisovan relatives offer a unique opportunity to learn about the similarities anddifferences between us. We have used these archaic genome sequences to identify genetic changes thatare unique to modern humans and not shared with Neandertals or Denisovans, and ongoing work aims todetermine whether any of these genetic variants might underlie traits characteristic of modern humans.
Further, we have also shown that the ancestors of some of us interbred with both Neandertals andDenisovans such that all present-day people outside of Africa carry approximately 2% Neandertal DNA,and that some populations, largely in Oceania, also carry DNA inherited from Denisovans. This
introgressed DNA has been shown to have both positive and negative outcomes for present-day carriers:
underlying apparently adaptive phenotypes such as high altitude adaptation, as well as influencing
immunity and disease risk. In recent work we have identified Neandertal haplotypes that are likely ofarchaic origin and determined the likely functional consequences of these haplotypes using publicgenome, gene expression, and phenotype datasets.

ASHBi Symposium 2021

S-7
Dynamic transcriptional control of neural stem cells
Ryoichiro Kageyama1,2
1

Kyoto University, Institute for Frontier Life and Medical Sciences, Kyoto, JapanRIKEN Center for Brain Science, Wako, Japan

2

Neural stem cells (NSCs) actively proliferate and generate neurons and glial cells (active state) in theembryonic brain while they are mostly dormant (quiescent state) in the adult brain. The expression
dynamics of Hes1 are different between active and quiescent NSCs. In active NSCs, Hes1 expression
oscillates and periodically represses the expression of proneural genes such as Ascl1/Mash1, thereby
driving their oscillations. By contrast, in quiescent NSCs, Hes1 oscillations become higher levels even attrough phases (thus continuous), thereby continuously suppressing the proneural gene expression. By
using an optogenetic method, we found that high levels of Hes1 expression and the resultant suppressionof Ascl1 promote the quiescent state of NSCs, while oscillatory Hes1 expression and the resultant
oscillatory Ascl1 expression regulate their active state, suggesting that the expression dynamics of thesefactors are important for active versus quiescent states. Indeed, when oscillatory Ascl1 expression is
induced in quiescent NSCs in the aged mouse brain, these NSCs are efficiently activated, produce newneurons continuously and thereby ameliorate cognitive decline. We now plan to apply this method tomonkeys to see whether their endogenous NSCs can be activated to generate new neurons continuously inthe aged brain.

ASHBi Symposium 2021

S-8
Molecular genetics of human brain development and evolution.
Christopher A. Walsh, MD, PhD
Division of Genetics and Genomics and Howard Hughes Medical Institute, Boston Children’s Hospital,
Harvard Medical School, and Allen Discovery Center for Human Brain Evolution, Boston, MA 02115.

Although evolution must ultimately alter cognition and behavior at least in part through changes in the
genome, it has been remarkably difficult to ascertain those genomic changes that enable the uniquecognitive, cultural, and aesthetic capabilities of humans. Human Accelerate Regions (HARs) include≈3000 genomic sequences that are highly conserved across nonhuman species--suggesting they haveessential functions--but show accelerated divergence between humans and nonhumans, suggesting that
their functions changed during human evolution. However, very little is known about whether any, some,or all of them have essential functions in human brain. Analysis of transcriptional activity of >3100
HARs, using Capture MPRA, reveals that 49% of HARs show enhancer activity in neural cell lines, while
42% show greater activity for the human sequence than the chimp, versus 19% showing greater activity
for the chimp sequence. Analyzing DNA accessibility and chromatin marks in fetal human brain
identified HARs with open chromatin in fetal neurons, neural precursor cells, or both, including a few
hundred HARs with patterns suggestive of enhancer function in fetal human brain.
In order to identify HARs with essential neurological functions in humans, we sequenced HARs in >9600
individuals including thousands with social and cognitive disability. Confirming our previous work, wefind higher rates of rare homozygous HAR mutation in in patients with social and cognitive disabilities
than in matched controls, suggesting contribution of rare homozygous HAR mutations in ≈2% of autism
and other disabilities, and identifying HARs that are recurrently mutated in disease. These data identify
dozens of HARs, many with uncharacterized function, with key roles in human brain function.
Supported by the NIMH, NINDS, the Allen Frontiers Program through the Allen Discovery Center for
Human Brain Evolution, and the HHMI.

ASHBi Symposium 2021

S-9
Elucidating the genetic and evolutionary differences between promoters and enhancers at
high resolution
Shruti Bhagat1,2,3, Akiko Suga4, Akiko Oguchi2,5, Naoki Hirose1,2,6, Shuichiro Komatsu2, Shigeki
Hirabayashi7, Juha Kere3, Yoshihide Hayashizaki8, Hideya Kawaji2,6,8, Chikashi Terao2, Michael
P. Fautsch9, Takeshi Iwata4# and Yasuhiro Murakawa1,2,8,10#
1

Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, JapanRIKEN Center for Integrative Medical Sciences, Yokohama, Japan3
Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
4
Division of Molecular and Cellular Biology, National Institute of Sensory Organs, National Hospital Organization
Tokyo Medical Center, Tokyo, Japan
5
Department of Nephrology, Kyoto University, Kyoto, Japan
6
Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
7
Department of Hematology and Oncology, Kyoto University, Kyoto, Japan
8
RIKEN Preventive Medicine and Diagnosis Innovation Program, Wako, Japan
9
Department of Ophthalmology, Mayo Clinic, Rochester, MN, USA
10
IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy#
Co-corresponding authors
2

Promoters and enhancers are cis-regulatory elements that are instrumental in driving gene expression. We
sought to identify human retina-specific promoters and transcribed enhancers at nucleotide resolution to
elucidate which features differentiate these elements. We profiled transcription start sites of mRNAs andenhancer RNAs generated bidirectionally from functionally active enhancers in retina samples in humans,rhesus macaque, and mice at an unprecedented depth. We show that cell-type-specific transcription
factors such as the homeobox family are specifically enriched in retinal enhancers but not in promoters.
While sequences of many enhancers were conserved until mouse, a larger fraction of enhancers isconserved only in rhesus macaque (but not in mouse) when compared with promoters. Additionally,transcriptional events are also preserved for sequence conserved enhancers. We also identified SNPs
associated with macular thickness, which were located in transcribed enhancers potentially disrupting
transcription factor binding. Our study establishes a general framework for the functional interpretation ofenhancers providing insights into understanding the role of enhancers in genomic evolution and gene
regulation.
Keyword(s): promoters, transcribed enhancers, gene regulation, transcription factors, evolutionASHBi Symposium 2021

S-10
Analyzing human peri-implantation development using naïve pluripotent stem cells
Yasuhiro Takashima
CiRA, Kyoto University

We aim to understand early human development by focusing on the pre-implantation and postimplantation stages and their transition. Most knowledge about peri-implantation development isbased on mouse models. Recent single-cell RNA sequencing (scRNA-seq) analysis of preimplantation embryos, however, has revealed critical differences between humans and rodents. In
contrast, differences at the peri-implantation stage are obvious even morphologically whencomparing mouse and human embryos. For example, in humans, epiblast and primitive endoderm(hypoblast) make a bilaminar disc, but in mouse, one sees a cylindrical structure. In addition,
human embryos at this stage contain the amnion and extraembryonic mesenchyme, which do notexist in rodents before gastrulation. Therefore, to understand human development, human models
are ideal, but ethical limitations restrict access to human uteri during peri-implantation.
To overcome these issues and achieve our objective, our group has established a series ofmethods and human cells that correspond to human peri-implantation embryos. The blastocyst
contains three different cell types, epiblast, hypoblast, and trophectoderm. We successfully
induced hypoblast and trophectoderm from naive human pluripotent stem cells (PSCs) thatshare features with pre-implantation embryos. In this presentation, I will present the
specification in vitro of naive human PSCs to extraembryonic lineages and embryonicdevelopment during peri-implantation.

ASHBi Symposium 2021

S-11
A xenotransplantation model for studying the migration of human primordial germ cells
Diana J. Laird
Department of Obstetrics, Gynecology and Reproductive Science, Center for Reproductive Sciences, Eli and Edythe
Broad Center for Regeneration Medicine and Stem Cell Research, University of California, San Francisco, USA

Fertility hinges upon events early in development before organogenesis, beginning with the specificationof dedicated gametic precursors known as primordial germ cells (PGCs), which must traverse the growing
embryo to reach the nascent gonads. PGC migration and some of the underlying signaling pathways areconserved across the Metazoan kingdom, although specific migratory routes can be dramatically different.
Little is known about PGC migration in humans owing to the inaccessibility of embryos at the early
developmental window in which it occurs, and the barriers to emulating the complex embryonicenvironment in organoids. A useful model for migratory-stage PGCs is furnished by their in vitro
derivatives from pluripotent stem cells, known as PGC-like cells (PGCLCs), but their capacity to migrate
remains unknown. We took advantage of the accessibility and tractability of the chick embryo to establish
a system for studying human PGC migration. We developed methods for 3D quantitative imaging of
PGCs in chick embryos as well as detection of xenografted PGCs. Despite anatomical differences in the
migration routes, we found that mouse PGCs as well as human PGCLCs injected into the chickextraembryonic vasculature, which is the site of avian PGC specification, were capable of homing frombloodstream through the mesentery and colonizing the gonadal ridges. In addition to the remarkableconservation of critical navigation cues implied by these results, common signals for PGC proliferationand survival are suggested by an observed correlation in the rates of expansion between donor and host
PGCs in this system. Using single cell transcriptomics, we found discrete transcriptional signatures inhuman PGCs as compared to mice across developmental time and identified new candidate signaling
pathways between human PGCs and their niche. This xenotransplant system will serve as a platform forelucidating the mechanisms of human PGC migration as well as validating in vitro derived PGCLCs.

ASHBi Symposium 2021

S-12
Epigenetic Mechanisms Controlling Early Mammalian Development
Davor Solter
Max-Planck Society

Epigenetic state of each cell is crucial in determining its gene expression and consequently its phenotype.
Changes in the epigenetic state of the genome are essential for normal development. While gradualchanges in epigenetic states occur throughout development there are two crucial moments when massive
reprogramming of the genome takes place: transition from somatic cells to primordial germ cells and in
zygote, following fertilization, when the germ cell epigenome transits to somatic epigenome state. During
those transition a general DNA demethylation takes places, however, there are several components of the
genome e.g. imprinted genes that need to be protected. TRIM28 is one of the components of theprotective mechanism and its absence during oogenesis leads to post-fertilization failure. Variations in
failure phenotype suggest that, in absence of TRIM 28, epigenetic marks are randomly lost, and not
replaced, from individual genes resulting in epigenetic chimerism. These results emphasize theimportance of maintaining and protecting correct epigenetic state during mammalian development.

ASHBi Symposium 2021

S-13
Reconstituting human somitogenesis in vitro
Cantas Alev
Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan

Somitogenesis is a fundamental process during which the segmented vertebrate body plan is laid out via
the controlled emergence of epithelial somites from paraxial/presomitic mesoderm (PSM). It has been
extensively studied using model organisms such as mouse, zebrafish or chick, but remains largely elusive
and poorly understood when it comes to human and other primates. Using embryonic development-inspired
induction of human PSM from pluripotent stem cells (PSC), we recently succeeded to recapitulate featuresof human paraxial mesoderm development in vitro. We hereby visualized and quantified oscillatory activityof putative segmentation clock associated genes and identified core molecular and functional features ofhuman normal as well as abnormal spine development and disease. Building upon these findings we thenasked whether we could recapitulate the actual process of human segmentation in vitro. Utilizing pluripotent
stem cells as starting material and following the guiding principles of the embryo we were able to establish
a novel 3D model of human axial development, enabling us to observe and assess the sequential formationof human epithelial somites in vitro. Our newly established bottom-up in vitro model system provides a
promising approach to study various aspects of axial development & disease in human and othermammalian species.

ASHBi Symposium 2021

S-14
Charting human development using a multi-endodermal organ atlas and organoid models
J. Gray Camp1,2
1Roche Institute for Translational Bioengineering (ITB), Roche Innovation Center Basel, Switzerland,2University of Basel, Basel, Switzerland

Organs are composed of diverse cell types that traverse transient states during organogenesis. To
interrogate this diversity during human development, we generate a single-cell transcriptome atlas frommultiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cellstates, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions acrosslineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent
stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that
HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal
epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1
enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 isrequired for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cellatlases and organoid technologies to understand how human organ development is orchestrated.

ASHBi Symposium 2021

S-15
Kidney organoids for modeling human development and disease
Ryuichi Nishinakamura
Institute of Molecular Embryology and Genetics, Kumamoto University, Japan

Recapitulating the three-dimensional organ structure in vitro is a major challenge for developmental
biology and regenerative medicine. The kidney develops by the reciprocal interactions between thenephron progenitor and ureteric bud. We established an induction protocol toward the former frompluripotent stem cells (PSCs) (Taguchi et al. Cell Stem Cell, 2014). Induced nephron progenitors robustly
formed glomerular podocytes equipped with slit diaphragm (SD), a filtration barrier. Mutations in the
NEPHRIN are known to cause congenital nephrotic syndrome, resulting from impaired SD formation inglomerular podocytes. By establishing iPS cells from a patient with a NEPHRIN missense mutation, we
reproduced the initial phase of this congenital disease (Tanigawa et al. Stem Cell Reports 2018).
However, these nephron organoids lacked the collecting ducts that connect the nephrons and constitutethe lower part of the urinary tract. As the collecting ducts are derived from the second precursor of thekidney: the ureteric bud, we also established protocols to induce the ureteric bud from mouse and human
PSCs. These protocols are applicable to model autosomal dominant polycystic kidney disease (Kuraoka etal. J Am Soc Nephrol 2020). Furthermore, mouse organoids reassembled from the differentially induced
ureteric bud and nephron progenitors, together with embryo-derived stromal progenitors, developed theinherent architectures of the embryonic kidney (Taguchi et al. Cell Stem Cell 2017). Induction of stromalprogenitors from PSCs and generation of an organotypic structure in humans is under way.

ASHBi Symposium 2021

S-16
Modelling human pancreas development with organoids
Anne Grapin-Botton
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany

To attempt to understand human pancreas development, as a complement to investigations in mice andwith mouse pancreas organoids, we established 3D (three-dimensional) culture conditions that enable theefficient expansion, differentiation and morphogenesis of pancreatic progenitors isolated from humanfetuses or produced from human pluripotent stem cells (hPSCs). Using single-cell sequencing, we
compared the transcriptional profile of cells grown in vitro in 2D, and 3D from hPSC-derived, to thoseisolated from fetal pancreas. Our experiments show a good molecular stability of pancreas progenitors
over time, retention of the endocrine differentiation capacity and a molecular signature closer to the invivo counterparts when grown in 3D than in 2D. We used this system to perform a screen for molecules
controlling human pancreas progenitor expansion and to study differentiation dynamics towards theendocrine lineage. The systems developed enable to address the mechanisms of pancreas development, aprocess normally hidden from us in the womb, notably the function of genes controlling architectural
events and morphogenesis operating in 3D that would be difficult to address in 2D culture, for example
ductal lumen formation. The human organoids also open a way to investigate whether some genes
identified in genome-wide association studies (GWAS) control the development of beta cells thereby
predisposing to diabetes later in life. We initiated this process by studying the effect of GLIS3 in humandevelopment and started to clarify the mechanisms by which it promotes diabetes and pancreatic cysts.

ASHBi Symposium 2021

S-17
Design principles of multicellular systems
Prisca Liberali
Friedrich Miescher Institute for Biomedical Research (FMI) Basel, Switzerland

Multicellular organisms are composed of cells and tissues with identical genomes but different properties
and functions. They all develop from one cell to form multicellular structures of astounding complexity.
During development, in a series of spatio-temporal coordinated steps, cells differentiate into different cell
types and establish tissue-scale architectures and functions. Throughout life, continuous tissue renewal
and regeneration is required for tissue homeostasis, which also requires fine-tuned spatio-temporal
coordination of cells. I will discuss how cellular interactions generate the specific contexts and spatiotemporal coordination underlying development and regeneration and how we specifically investigate what
are the molecular and physical mechanisms that allow a cell, in a tissue, to sense its complex
environment, to take individual coordinated decisions. Moreover, I will discuss the molecularmechanisms of intestinal organoid self-organization and the role of cell-to-cell variability in populationsof differentiating cells during symmetry breaking.

ASHBi Symposium 2021

S-18
Modeling human gastrointestinal diseases using organoid technology
Toshiro Sato
Department of Organoid Medicine, Keio University School of Medicine

Recent advances in sequencing technology delineated the genetic abnormality of human cancers.
However, there remains a gap between genetic abnormalities and clinical cancer phenotypes due to apaucity of disease models that can recapitulate a variety of biological aspects of clinical cancers.
Organoid technology was originally developed to regenerate tissue-like structures from pluripotent stem
cells or tissue stem cells. Recent works revealed that organoids could be derived from patient cancer
tissues without losing their original biological traits, such as drug sensitivity, pathohistology, cancer stem
cell hierarchy, and metastatic potentials. Despite this progress, the limited spatiotemporal resolution has
precluded us from understanding the biological behavior of patient-derived cancers at a single-cell level.
We have recently developed a live genetic fate-mapping system that allows longitudinal tracking ofindividual cells in xenotransplanted human colorectal cancer organoids and identified a dormant LGR5+
cancer stem cell subpopulation that repopulates the tumors after chemotherapy. Organoid-based
biological analysis revealed the molecular mechanism of how cancer tissues maintain the dormancy ofcancer stem cells, providing a feasible therapeutic strategy targeting dormant cancer stem cells. In this
symposium, I would like to share our recent findings and discuss the approach to override the drug
resistance of cancer cells.

ASHBi Symposium 2021

S-19
TBA

ASHBi Symposium 2021

S-20
Self-organisation in mammalian development
Takashi Hiiragi
ASHBi Kyoto University, Hubrecht Institute

A defining feature of living systems is the capacity to break symmetry and generate well-defined forms
and patterns through self-organisation. Our group aims to understand the principle of multi-cellular selforganisation using early mouse embryos as a model system. Mammalian eggs lack polarity and symmetryis broken during early embryogenesis. This results in the formation of a blastocyst consisting of three cell
types, each distinct in its position and gene expression. Our studies revealed that morphogenesis and gene
expression are highly dynamic and stochastically variable during this process. Determining how theblastocyst establishes a reproducible shape and pattern despite the preceding variability remains
fundamental open questions in early mammalian development. We have recently developed anexperimental framework that integrates biology, physics and mathematics. We aim to understand how
molecular, cellular and physical signals are dynamically coupled across the scales for self-organisation.

ASHBi Symposium 2021

S-21
The Dynamics of Spinal Cord Development
James Briscoe
The Francis Crick Institute, London, UK

The generation of the correct cell types in the correct position, at the correct time is the firststep in the assembly of functional tissues. One well-studied example of this is the developmentof the vertebrate spinal cord. In this tissue, distinct classes of neurons are generated in a precise spatial
and temporal order from progenitor cells arrayed along the dorsal-ventral axis of the neural tube.
Underpinning this organization is a complex network of extrinsic and intrinsic factors. Particularly wellunderstood is the mechanism that determines the generation of different neuronal subtypes in ventral
regions of the spinal cord. In this region of the nervous system, the secreted protein Sonic Hedgehog(Shh) acts in graded fashion to organize the pattern of neurogenesis. This is a dynamic process in which
exposure to Shh generates progenitors with successively more ventral identities. At the same time tissue
growth alters the arrangement of cells and the proportions of cell types and contributes to the elaborationof pattern. A gene regulatory network composed of transcription factors regulated by Shh signaling play
an essential role in this process. Together, the mechanism determines pattern, pace, precision andproportions in the forming neural tube. Thus, accurate development of the neural tube
and the specification of neuronal subtype identity relies on the interplay of cellular and molecular
processes.

ASHBi Symposium 2021

S-22
Tracing and perturbing lineages during human brain organoid development
Barbara Treutlein
ETH Zurich, Department of Biosystems Science and Engineering, Basel Switzerland

Induced pluripotent stem cell (iPSC) derived organoids provide models to study human organ
development. Organoids are complex, containing numerous cell states and integrative, multi-modal
single-cell technologies are needed to understand the mechanisms underlying organoid development.
In my talk, I will present two efforts from our lab where we develop novel integrative single-cell methods
to understand human brain organoid development. First, I will present iTracer, a lineage recorder thatcombines reporter barcodes with inducible CRISPR/Cas9 scarring, and is compatible with single-cell andspatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral
organoid development, and identify a time window of fate restriction as well as variation in neurogenic
dynamics between progenitor-neuron families. We also establish long-term 4-D lightsheet microscopy for
spatial lineage recording in cerebral organoids and confirm regional clonality in the developing
neuroepithelium. We incorporate gene perturbation (iTracer-perturb), and assess the effect of mosaic
TSC2 mutations on cerebral organoid development.
Second, I will present a data set of paired single-cell transcriptome and accessible chromatin profilingdata over a dense time course of human brain organoid development, which we utilize to infer a gene
regulatory network of human brain organoid development. To this aim, we have developed Pando, aflexible computational framework that incorporates multi-omic data and transcription binding site
predictions to infer a global GRN describing organoid development. We use pooled genetic perturbation
with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state
regulation in organoid and show interesting alterations of abundance of cell fates.
Together, these techniques can be adapted in any iPSC-derived culture system to dissect lineage
relationships and regulomes during normal or perturbed development.

ASHBi Symposium 2021

S-23
Oogenesis in vitro: what we can and cannot reconstitute.
Katsuhiko Hayashi
Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University
Department of Germline Genetics, Graduate School of Medicine, Osaka University

Oocytes are highly specialized cells to confer totipotency on fertilized eggs. To create oocytes, the female
germ line, originating from primordial germ cells, undergoes a unique sequence of differentiation
processes that includes biologically important events such as epigenetic reprogramming, meiosis andoocyte growth associated with follicular development. Reconstitution of these processes using pluripotent
stem cells provides a unique platform for a comprehensive understanding of mechanisms underlying
oogenesis, as well as an alternative means of egg production.
Pioneering in vitro gametogenesis in mammals, reconstitution of oogenesis in mice has progressed
rapidly over the past decade, during which we developed a culture system to produce cells closely
resembling primordial germ cells, oocytes, and follicular somatic cells from mouse embryonic stem cells
and induced pluripotent stem cells. By combining these culture methods, it is now possible to produce arobust number of mouse oocytes from pluripotent stem cells without sacrificing mice and their embryos.
Is the role of the mouse as a pioneer in this field over? This seems highly unlikely, as we found several
important processes that cannot be reconstituted using pluripotent stem cells. In this symposium, I will
present our recent work on reconstitution of oogenesis in vitro and discuss further directions of thistechnology for a deeper understanding mammalian oogenesis.

ASHBi Symposium 2021

S-24
Genomic imprinting, human development and the ‘immortal’ germline
Azim Surani
Gurdon Institute, University of Cambridge

The germline generates the totipotent state at fertilization, where the parental genomes make an equal
genetic contribution. However, in therians (mammals and marsupials) but excluding monotremes, theparental genomes exhibit an epigenetic asymmetry due to genomic imprinting, which requires epigenetic
resetting starting soon after the specification of primordial germ cells (PGC), with the erasure of imprints
and their re-initiation in gametes. “Imprinted genes”, which show mono-allelic expression during
development and in adults depending on their parental origin, are thought to play an essential role inmammals, balancing embryonic growth and development, mammalian physiology, behaviour andmetabolism.
Investigations of early germline development are challenging in human embryos that are largely
inaccessible. Nonetheless, significant advances have been possible with the generation of in vitro
models using pluripotent stem cells and non-rodent animals as surrogates for human development.
Research on human germline can contribute widely to advances in human development and diseases
through, for example, advances in germline epigenome reprogramming that is critical for the ‘immortal’
germline and obligatory requiring comprehensive epigenetic erasure because of imprinting in mammals.

ASHBi Symposium 2021

S-25
The selfish testes of ageing men and de novo mutations
Anne Goriely
MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford,
OX39DS, UK.
E-mail: Anne.goriely@imm.ox.ac.uk

It is now well-established that our genomes mutate at a slow but constant rate of 30-100 new point
mutations per generation. Although most de novo mutations (>80%) originate from the father andincrease in frequency at the rate 1-2 mutation/paternal year, very little is known about the cellularmechanisms that allow the adult testis to reconcile the contradictory demands for abundant sperm
production - that takes place over many decades - and a low mutation rate across generations.
We have previously described a process where some pathogenic mutations hijack the homeostaticmechanisms of sperm production to their own advantage. This mechanism called ‘selfish selection’ wasoriginally proposed to explain the paternal age-effect and high birth prevalence observed for some
Mendelian disorders, such as Apert syndrome (FGFR2) or achondroplasia (FGFR3). It relies on
principles similar to oncogenesis to explain why these pathogenic mutations occur spontaneously at levels
up to 1000-fold higher than the background rate. Importantly, this process emphasizes the intimate linkthat exists between testicular homeostasis and germline mutation rate.
I will summarise our current understanding of de novo mutations in humans, the impact of paternal age
and their importance for human disease and for genome heterogeneity/interpretation. I will then describe
the data that have led to the discovery of the selfish selection process and the novel strategies we aredeveloping to study de novo mutations directly within human testes. Finally, I will speculate on the
broader implications of selfish selection and the importance of the regulation of spermatogenesis forhuman disease, genome diversity and evolution.

ASHBi Symposium 2021

S-26
Mechanism and In Vitro Reconstitution of Mammalian Germ-Cell Development
Mitinori Saitou1,2,3
1

Institute for the Advanced Study of Human Biology, Kyoto University,
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University,
3
Center for iPS Cell Research and Application, Kyoto University.
2

The germ-cell lineage ensures the creation of new individuals, perpetuating/diversifying the genetic andepigenetic information across the generations. We have been investigating the mechanism for germ-celldevelopment, and have shown that mouse embryonic stem cells (mESCs)/induced pluripotent stem cells(miPSCs) are induced into primordial germ cell-like cells (mPGCLCs) with a robust capacity both for
spermatogenesis and oogenesis and for contributing to offspring. These works have served as a basis for
exploring the mechanism of key events during germ-cell development such as epigenetic
reprogramming/programming, sex determination, and meiotic entry.
By investigating the development of cynomolgus monkeys, we have defined a developmental coordinateof the spectrum of pluripotency among mice, monkeys, and humans, and have identified the origin of thegerm-cell lineage in cynomolgus monkeys in the amnion. Accordingly, we have induced human iPSCs(hiPSCs) with a primed pluripotency into human PGCLCs (hPGCLCs) and then into oogonia and early
oocytes with appropriate epigenetic reprogramming. More recently, we have shown that hPGCLCs canbe propagated to ~106-fold over a period of 4 months under a defined condition. These studies havecreated a foundation for human in vitro gametogenesis.
Here, I would like to discuss our latest findings regarding the mechanism and in vitro reconstitution of
mammalian germ-cell development.

P-1
MODELING LIMB SKELETOGENESIS IN ORGANOID CULTURE DERIVED FROM
MOUSE EMBRYONIC LIMB BUD AND HUMAN ES CELLS
Rio Tsutsumi1, Mototsugu Eiraku1, 2
1. ASHBi, Kyoto University, Kyoto, Japan
2. Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto Japan

Approximately 1 in 2000 infants are bone with Congenital limb deficiencies. Although developmental
genetic studies have contributed to our detailed understanding on molecular mechanisms which control
limb morphogenesis, in vitro model of 3-dimensional limb morphogenesis has not yet been established.
Limb skeletons are derived from limb bud mesenchymal cells during embryonic development. Shapes
the limb skeletons are different depending on their positions in the limb. For example, stylopod (upperarm and thigh) has one bones, zeugopod (lower arm and shin) has two bones, and autopod (hand, foot)has many small bones including phalanges. After formation of limb skeletons, those bone growdifferently depending on their position. For example, human thigh and shins grow relatively longer thanarms. This pattern of differential bone growth is one of the specific features of bipedal human body as
extant non-human apes shows longer arms than legs.
In order to establish in vitro model of position-specific cartilage morphogenesis and growth, we isolated
limb bud mesenchyme from mouse embryo and differentiate those cells into limb cartilage in 3dimensional culture. Interestingly, after manipulation of positional identities of those cells by cytokines,
those aggregates showed different patterns of cartilage formation and its growth. Those differences mightreflect position-specific cellular properties which contribute to position-specific morphogenesis during
limb development.
Next, we aimed to establish a model of human limb morphogenesis in 3-dimensional culture. We drove
differentiation of human embryonic stem cells towards limb bud mesenchyme and derived cells which
express typical limb mesenchyme marker genes. Those cells were able to form cartilage in 3-dimensional
culture.
We expect our study would establish novel model system to assess causality of human congenital limb
deficiencies. Furthermore, we recently reported the derivation of limb bud cells from mouse embryonicstem cells. Establishment of self-organizing system of limb skeletal morphogenesis from pluripotent stem
cells of multiple species would open up venues to understand cellular mechanisms of species-specificlimb morphologies.

P-2
Data-driven and technical approaches to understand spatial gene regulation
Shinya Oki1
1

Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine

Multicellular organisms are composed of a variety of tissues and cell types, and their dynamic changes
are organized by spatiotemporal gene expression. Although numbers of genes have been characterized to
be expressed in specific tissues and time points, underlying mechanisms of the spatiotemporal gene
regulation are poorly understood. To elucidate the mechanisms with a data-driven framework, we havedeveloped ChIP-Atlas, an epigenomics database fully integrating public ChIP-seq, ATAC-seq, andBisulfite-seq data (> 240,000 experiments; Oki, S., et al. EMBO Rep 2018). By taking advantage of thehuge amount of data, we identified multiple transcription factors pivotally involved in cellulardifferentiation, genetic diseases, and drug actions. In addition, to understand spatial gene expression in ahigh-resolution manner, we established a transcriptome profiling method coupled with photo-isolation
chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiatedregions of interest (Honda, M., et al. Nat Commun 2021). By using this method, we identified areaspecific transcripts not only from microtissues in mouse embryos and brains but also from subcellular andsubnuclear microstructures (stress granules and nuclear speckles, respectively). In this symposium, I
would like to talk about the regulatory mechanism of spatial gene expression revealed by the combinationof data-driven and technical approaches, and further discuss the application for the research ofdevelopmental biology and pathological processes.
Keyword(s): gene regulation, epigenomics database, spatial transcriptomics

P-3
Modeling disease-specific gene regulation of facio-scapulo-humeral muscular dystrophy(FSHD)
Mitsuru Sasaki-Honda1,2, Tatsuya Jonouchi1, Takuya Yamamoto1,3, Hidetoshi Sakurai1
1CiRA, Kyoto University
JSPS research fellow (CPD)3
ASHBi, Kyoto University
2

Facio-scapula-humeral muscular dystrophy (FSHD) is a skeletal muscle genetic disorder caused bypatient-specific structural variants on 4q35 region characterized by shortened macrosatellite D4Z4 repeats(<10, FSHD type 1) or relatively short D4Z4 repeats (basically 10~20) with chromatin regulators’
mutations (FSHD type 2). These abnormal variants are supposed to relax otherwise heterochromatin
structure to allow toxic DUX4 gene expression in a sporadic manner in muscle cells. How DUX4’s
stochastic expression is regulated is critical to understand pathology and a key to drug targets for cures,
but still remains unclear. Combining our FSHD patient-iPSC muscle cells model with patient-specific
DUX4 activation, a DUX4-sensitive fluorescence reporter system and FACS sorting, we established amodel of the sporadic pattern of DUX4 gene expression in FSHD cells, which showed distinct
transcriptome and chromatin structure in DUX4 positive cells compared to negative population or
genetically modified cells, indicating the rare population with high DUX4 expression is the main sourceof DUX4 expression in FSHD muscle. Moreover, for treatment, we developed a method to achieve
prolonged DUX4 suppression by transient activity of dCas9-mediated DNA methylation editing, which
could work for both types of FSHD cell models beyond difference in genetic backgrounds and seemedsufficient to target rare DUX4 high population. This method hopefully will provide a clue to avoid
potential risks around typical gene therapy strategy and drug delivery system.
Keyword(s): skeletal muscle disease, FSHD, DUX4, epigenome editingP-4
Hydrogel for precise manipulation of human pluripotent stem cells microenvironment
Zhe Wang1,2, Akira Numada1,2, Mototsugu Eiraku1,3
1Institute for Frontier Life and Medical Sciences, Kyoto University
Graduate school of Engineering, Kyoto University
3
Institute for the Advanced Study of Human Biology, Kyoto University
2

Mammalian cells sense the cellular microenvironment and adjust their behavior based on the surrounding
chemical/mechanical/topological conditions. Culture substrates that allow precise manipulation of theseproperties would help us better recapitulate the in vivo environment and benefit tissue engineering.
Hydrogels have been used intensively in biomedical research as cell culture substrates due to theirmechanical and chemical similarity with the extracellular matrix3. However, current well-used hydrogels
whether do not allow human pluripotent to attach and growth or do not fully recapitulate in vivo
microenvironment, in terms of their physical, chemical, and topological features. To overcome thisproblem, we developed a polyvinyl alcohol/Polyethylene glycol composite hydrogel (PVA-PEG gel)which allows us to spatially control the stiffness and the geological pattern at the micrometer level.
Laminin 511 conjugated PVA-PEG gel supports human pluripotent stem cell maintenance anddifferentiation. Furthermore, by spatially control the stiffness of the patterned gel, we could differentiate
cells towards different fates on the same patterned gel, thus generate a tissue with complex structure.
Generated patterned gel with millimeters thickness allows cultured cells to form aggregate and achievecomplicated tissue transformation. Taken together, our hydrogel provided a culture platform with wellcontrolled microenvironment for human pluripotent stem cell research.

Keyword(s): Hydrogel, Stem cell, Microenvironment, Culture substrate

P-5
TBA

P-6
Organoids Platform: Control and Design the microenvironments to achieve organoidarchitecture
Isabel Koh1, Kasinan Suthiwanich1, Atsushi Takano1, Masaya Hagiwara1
1

Cluster of Pioneering Research, RIKEN

In vitro 3D and organoid culture methods that mimic the rich complexity of cell population andextracellular matrix (ECM) components of in vivo tissues contribute greatly to furthering ourunderstanding of various biological phenomena. Nevertheless, it remains a challenge to exert control over
the repeatability, shape and architecture, as well as tissue-tissue interactions of cultured organoids.
Engineering principles, on the other hand, are great tools that enable us to tailor the design, composition,and construction of organoids according to the intended purpose of the study. On the other hand, thetechnologies tend to be too complicated to be used when the multiple-factors need to be controlled
simultaneously.
In this research, we will present how our simple cube culture device, which comprises a polycarbonate
frame with rigid agarose walls and an inner ECM hydrogel, can be used to (i) control the spatial
distribution of cells by employing 3D-printed moulds to create cell seeding pockets in the ECM hydrogel,(ii) design tissues with localized ECM by isolating ECM hydrogels of varying the composition or
stiffness in separate compartments, and (iii) facilitate easy integration with microfluidics for regulating
the concentration gradient of morphogens to direct cell growth and differentiation, applying shear stressto cells, as well as connecting organoids for tissue-tissue interactions. The simple organoid platform
attains easy integration with other technologies to achieve complex environmental control.
Thus, it is envisioned that increasingly sophisticated and interconnected organoids will be achievable in
the future.

Keyword(s): Organoid culture, Tissue engineering, ECM, Repeatability

P-7
CancelledP-8
Human Transposable Elements in Repbase: Genomic Traces of Human Evolution
Kenji K. Kojima1, Weidong Bao1, Noriko F. Kojima1, Oleksiy Kohany1
1

Genetic Information Research InstituteRepbase is a comprehensive database of eukaryotic transposable elements (TEs) and repeat sequences, andcontains over 1300 human repeat sequences. Recent analyses of repeat sequences have accumulated
evidences for the contribution of repeat sequences to the human evolution through becoming functional
elements such as protein-coding regions or binding sites of transcriptional regulators. However, resolving
the origins of these repeat sequences is a challenge because of their age, divergence and degradation.
Ancient repeats have been continuously classified as TEs by characterizing similar TEs from otherorganisms. Here the most comprehensive picture of human repeat sequences is presented. Human genome
contains traces of 10 clades (L1, CR1, L2, Crack, RTE, RTEX, R4, Vingi, Tx1 and Penelope) of non-longterminal repeat (non-LTR) retrotransposons (long interspersed elements, LINEs), 3 types (SINE1/7SL,
SINE2/tRNA, and SINE3/5S) of short interspersed elements (SINEs), 1 composite retrotransposon (SVA), 4
classes (ERV1, ERV2, ERV3 and Gypsy) of LTR retrotransposons, and 11 superfamilies (Crypton,
Harbinger, hAT, Helitron, Kolobok, Mariner, Merlin, MuDR, P, piggyBac and Transib) of DNA
transposons. These footprints of TEs reveal an evolutionary pass of the human genome.

Keyword(s): Transposable elements (TEs), retrotransposons, DNA transposons, Repbase

P-9
Brain Energy Dynamics with integration of functions
Anjani Kumar Tiwari†‡, Priya Singh†, Anil Kumar Mishra‡*†Department of Chemistry, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, INDIA. 226025‡Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences(INMAS), Delhi, INDIA.110054

Glucose is one of the most important substrate of human brain that is responsible for different functions at
cellular level. Glucose molecule is metabolized at different node points for neuronal/microglial/astrocytic
signaling. It has been assumed that the rate of glucose consumption is one Glucose/synapse/ms(millisecond) for approximate 170 biochemical/metabolic processes occurring in different brain region.
But the next question is……..Is this rate is similar to all region….and answer is no. The speed of glucose
metabolism is heterogeneous throughout the brain. One of the major flux consumption is Neuron–
astrocyte cycling of glutamate and glutamine in glutamatergic neurons (approximately 80 % of glucose
metabolism in brain). However, many important insights of the energetics and their functions remain to
be firmly established for in vitro/in-vivo conditions.
The next question is about the regulation of this metabolism which is complex due to involvement ofvarious enzymes/membrane transporters of the related energy dynamics. Lactate, is one of the mostimportant marker which is shuttled in between glycolytic/oxidative cells. Now its transportation throughmonocarboxylate transporters (MCTs) and its rapid equilibrium is equally important for astrocytes andcancer cells.
How much quantification of cellular glucose and other related substrate in resting, activated state studied
using [18F]FDG -positron-emission tomography (studying CMRglc) and [13C/31P]glucose/magnetic
resonance spectroscopy (13C-MRS: for neuroenergetics & neurotransmitter cycling &31P-MRS: for energy
induction & redox state) has helped to increase our insight.
Keyword(s): Metabolism, Brain, PET, MRS

P-10
Disease modelling of pulmonary fibrosis using human pluripotent stem cell-derived
alveolar organoids
Takahiro Suezawa1,2, Shuhei Kanagaki2, Keita Moriguchi2, Atsushi Masui1,2, Kazuhisa Nakao2,
Masayasu Toyomoto1,3, Koji Tamai4, Ryuta Mikawa4, Toyohiro Hirai4, Koji Murakami2,
Masatoshi Hagiwara3, Shimpei Gotoh1,41Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Watarase Research Center, Kyorin Pharmaceutical Co. Ltd., Shimotsuga-gun, Tochigi, Japan.
3
Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan.
4
Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
2

Epithelial-mesenchymal interactions have long been reported to play important roles in various fibrosisrelated disorders, and in site-specific tissue regeneration. Although alveolar epithelial cell injury followedby mesenchymal cell activation play a critical role in the pathogenesis of pulmonary fibrosis, there arefew practical in vitro models to study the alveolar epithelial-mesenchymal interaction, because of the
difficulty of culturing alveolar epithelial type 2 (AT2) cells which are lung epithelial stem cells that canself-renew and differentiate into alveolar epithelial type 1 (AT1) cells. Here, we established a novel invitro pulmonary fibrosis model using alveolar organoids consisting of human pluripotent stem cellderived alveolar epithelial cells and primary human lung fibroblasts. In this human model, bleomycin(BLM) treatment induced phenotypes such as epithelial cell-dependent organoid contraction, cellularsenescence of AT2 cells, and presence of abnormally differentiated alveolar epithelial cells, which havebeen observed in the pulmonary fibrosis lung. Chemical screening identified that inhibition of TGFβ
signaling could therapeutically ameliorate these epithelial cell phenotypes. Furthermore, treatment of
BLM-stimulated alveolar organoids with a TGFβ signaling inhibitor increased the expression of AT1
markers and induced thinner morphology of epithelial cells, suggesting that inhibition of TGFβ could betherapeutic for the pathogenic phenotypes, such as cellular senescence and abnormal differentiation states,with maintaining or protecting the differentiation states of alveolar epithelial cells. In this model, theinjured epithelial cells specifically expressed integrin αVβ6, a major activator of latent TGFβ, indicatingthat epithelial cell-dependent activation of TGFβ signaling occurred in the organoids. In addition,organoid contraction and extracellular matrix accumulation, which could depend on activated fibroblasts,
were also suppressed by inhibiting TGFβ signaling. This human model may therefore accelerate thediscovery of effective therapeutic agents for the otherwise incurable pulmonary fibrosis by targetingalveolar epithelial cells and epithelial-mesenchymal interactions.
Keyword(s): alveolar epithelial cell, pulmonary fibrosis, epithelial-mesenchymal interaction, pluripotent
stem cell, contraction

P-11
Elucidating dynamic gene regulatory networks in human skeletal development using single
cell analyses on a human pluripotent stem cell-based model of endochondral ossification
Shoichiro Tani1,2, Hiroyuki Okada1,2, Masahide Seki3, Yutaka Suzuki3, Taku Saito2, Sakae
Tanaka2, Ung-il Chung1,4, Hironori Hojo1,4 and Shinsuke Ohba5
1

Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo,
Tokyo, Japan
2
Sensory & Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan3
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The Universityof Tokyo, Chiba, Japan
4
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan
5
Department of Cell Biology, Institute of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

Elucidating dynamics of gene regulatory networks is an essential step to understand the physiology andpathology in target organs. Although human pluripotent stem cells (hPSCs) can differentiate into any
embryonic cell type, it is challenging to recapitulate a whole process of skeletal development, from hPSCs
to 3D bone tissues. To recapitulate human bone formation, we induced the in vitro differentiation of hPSCs
into sclerotome and implanted them beneath the renal capsules of immunodeficient mice. RNA-seq analysis
demonstrated cell type-specific gene expression, indicating a stepwise differentiation of hPSCs into thesclerotome. In vivo micro-CT analysis after implantation showed the growth of mineralized tissues overtime. Histological analyses revealed structures similar to endochondral bones with specific marker
expression patterns: columnar structures of chondrocytes, bone collar, and bone marrow. The induced bone
tissues were then analyzed by single-cell RNA-seq, and the obtained data were integrated with the publicly
available dataset of human embryonic long bones at 8 weeks post conception. Clustering analysis revealed
that multiple skeletal cell types with distinct gene expression signatures, including embryonic skeletal
progenitors, were commonly present in both datasets. Pseudotime analysis predicted a bifurcating trajectoryfrom skeletal progenitors to osteoblasts or chondrocytes. By integrating differential gene expression
analysis, gene regulatory network analysis, and ligand-receptor analysis, we extracted novel transcriptional
factors that may regulate human osteogenesis. In situ hybridization of the hPSC-derived bone tissues
showed a partial co-expression of the identified regulators with RUNX2 and SP7, master transcriptional
regulators of osteoblasts. The knockdown of the identified regulators downregulated osteoblast marker
genes in a human osteosarcoma cell line and human mesenchymal stromal cells, indicating the involvementof these regulators in human osteogenesis. Collectively, our research may provide a valuable framework
based on a well-recapitulated human skeletal development to investigate detailed mechanisms underlying
its dynamic processes.
Keyword(s): pluripotent stem cells, human skeletal development, single-cell analysis, gene regulatory
networks

P-12
Elucidating the genetic and evolutionary differences between promoters and enhancers at
high resolution
Shruti Bhagat1,2,3, Akiko Suga4, Akiko Oguchi2,5, Naoki Hirose1,2,6, Shuichiro Komatsu2, Shigeki
Hirabayashi7, Juha Kere3, Yoshihide Hayashizaki8, Hideya Kawaji2,6,8, Chikashi Terao2, Michael
P. Fautsch9, Takeshi Iwata4# and Yasuhiro Murakawa1,2,8,10#
1

Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, JapanRIKEN Center for Integrative Medical Sciences, Yokohama, Japan3
Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
4
Division of Molecular and Cellular Biology, National Institute of Sensory Organs, National Hospital Organization
Tokyo Medical Center, Tokyo, Japan
5
Department of Nephrology, Kyoto University, Kyoto, Japan
6
Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
7
Department of Hematology and Oncology, Kyoto University, Kyoto, Japan
8
RIKEN Preventive Medicine and Diagnosis Innovation Program, Wako, Japan
9
Department of Ophthalmology, Mayo Clinic, Rochester, MN, USA
10
IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy#
Co-corresponding authors
2

Promoters and enhancers are cis-regulatory elements that are instrumental in driving gene expression. We
sought to identify human retina-specific promoters and transcribed enhancers at nucleotide resolution to
elucidate which features differentiate these elements. We profiled transcription start sites of mRNAs andenhancer RNAs generated bidirectionally from functionally active enhancers in retina samples in humans,rhesus macaque, and mice at an unprecedented depth. We show that cell-type-specific transcription
factors such as the homeobox family are specifically enriched in retinal enhancers but not in promoters.
While sequences of many enhancers were conserved until mouse, a larger fraction of enhancers isconserved only in rhesus macaque (but not in mouse) when compared with promoters. Additionally,transcriptional events are also preserved for sequence conserved enhancers. We also identified SNPs
associated with macular thickness, which were located in transcribed enhancers potentially disrupting
transcription factor binding. Our study establishes a general framework for the functional interpretation ofenhancers providing insights into understanding the role of enhancers in genomic evolution and gene
regulation.

P-13
An ex vivo system to study cellular dynamics underlying mouse peri-implantation
development
Takafumi Ichikawa1,2, Hui Ting Zhang1, Laura Panavaite1, Anna Erzberger1, Dimitri Fabrèges1,
Rene Snajder1, Adrian Wolny1, Ekaterina Korotkevich1, Nobuko Tsuchida-Straeten1, Lars
Hufnagel1, Anna Kreshuk1, and Takashi Hiiragi1,2
12European Molecular Biology Laboratory (EMBL)
Institute for the Advanced Study of Human Biology, Kyoto University

Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes
such as body axis specification and gastrulation. However, limited accessibility obscures study of these
crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse periimplantation development from E4.5 to 6.0. Our system not only recapitulates embryonic growth, axis
initiation, and overall 3D architecture in 49% of cases, its compatibility with light-sheet microscopy enables
study of cellular dynamics through automatic cell membrane segmentation. We find that upon implantation,
release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination.
The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis and patterning of theneighbouring epiblast, which subsequently gives rise to all embryonic tissues. This 3D-ex vivo system thus
offers an unprecedented access to peri-implantation development for in toto monitoring, measurement andspatio-temporally controlled perturbation, revealing a mechano-chemical interplay between extraembryonic and embryonic tissues.
Keyword(s): Peri-implantation development, Egg cylinder formation, Epiblast morphogenesis, Lumenformation, In toto live-imaging