Salk Institute For Biological Studies, La Jolla CaliforniaJuan Carlos Izpisua Belmonte

Recapitulatingearlymammalianembryogenesisbyusingculturedcells

Recapitulatingearlymammalianembryogenesisbyusingculturedcells

Cell Symposia: Engineering Organoids and Organs

UCLA,1992

Yosiki Sasai

OpticalCup

CorticalStructureswere counted with FACSAria (BD), and the data were analyzed with the

FACSDiva software (BD). The sorted cells were collected in ice-cold N2

medium containing 10% FBS and quickly reaggregated using low cell-adhe-

sion 96-well culture plates (5000 cells/well). Recombinant mouse FGF8b

(50 ng/ml), mouse FGFR3-fc (500 ng/ml), human BMP4 (0.5 ng/ml), and

Figure 6. Self-Organized Formation of PolarizedCortical Tissues from SFEBq-Cultured mESCs(A and B) Cryosections of SFEBq-cultured mESC aggre-

gates on day 7 (A) and day 8 (B) were immunostained

with Reelin and Bf1.

(C) Reelin+/Tbr1+ neurons and Emx1+ rosettes on day 10.

Arrowheads, Reelin+/Tbr1+ neurons in the superficial zone

(facing to the aggregate surface) of the Emx1+ rosettes.

(D and E) Immunostaining of the cortical markers Pax6,

Tbr1 (D), and Tbr2 (E) on day 10.

(F) Schematic of the cellular distribution pattern of the

SFEBq-induced cortical rosette.

(G–I) The distribution of the early/lower CP marker Ctip2

(H and I) and the late/upper CP marker Cux1 (H) and

Satb2 (I) in Emx1+ clusters (G).

(J–R) Cryosections of SFEBq-cultured hESC aggregates

on day 46 were stained for cortical markers, as indicated

in each panel. In (J) and (L)–(N), confocal images of serial

sections at the same region are shown. (K) shows a high

power image of the perilumenal portion. The hESC-

derived Bf1+/N-cadherin+ neuroepithelial structure coex-

pressed Pax6 (M) and Emx1 (N). Tbr1 (N) and Ctip2 (Q)

were expressed in the TuJ1+ zone located superficially

to the Bf1+/Pax6+ neuroepithelium, while Tbr2+ cells

were mostly found in or adjacent to the Pax6+ neuroepi-

thelial zone.

Scale bars: 30 mm in (A) and (B); 50 mm in (C), (D), and (E);

60 mm in (G)–(I); 20 mm in (K); and 100 mm in (J), (L)–(O), (P),

and (R).

human Wnt3a (20 ng/ml) were added to the culture me-

dium. For forced neuronal differentiation, DAPT (10 mM)

was added on the next day of FACS sorting. For induc-

tion of Tbx21+ neurons, Bf1::Venus+ cells were sorted

by FACS and reaggregated on day 7, treated with

FGF8 (50 ng/ml), and DAPT (10 mM) from day 8 and cul-

tured for 2 weeks. Tbx21+ neurons were not induced by

DAPT treatment alone.

Birth-Date AnalysisFor in vitro birth-date analysis (Ajioka and Nakajima,

2005), aggregates were treated with BrdU (5 mg/ml) on

day 8, 9, 10, 12, or 14 and rinsed with medium to re-

move it on the next day. On day 16, cell aggregates

were fixed and cryosectioned. Sections were immuno-

stained for BrdU and each layer-specific marker as indi-

cated in Figure 5. The percentages of BrdU+ cells in the

Bf1+/Ctip2+ (most of the Ctip2+ cells were Emx1+), Bf1+/

Brn2+, Reelin+, or Tbr1+ cells were quantified. For the

quantification, 20–25 aggregates were examined for

each experiment, which was repeated at least three

times.

Brain Slice Coculture AssayFor coculture with forebrain slices, E14.5 or P1 mouse

brains were excised, and coronal sections (200 mm) were

prepared using a vibratome (F1000SL, Leica). Bf1::Venus+

neuronal masses were cut out in appropriate sizes from

the SFEBq cell aggregates under a fluorescent dissecting

microscope and cocultured with the forebrain slice (by in-

serting the masses into the ventricle space so that the inserted aggregates

were in contact with both pallial and subpallial walls) for 3 or 6 days on a Trans-

well culture insert (Corning) containing the slice-culture medium (DMEM/F12,

N2 supplement, 15% FBS, and penicillin-streptomycin) under 40% O2 and 5%

CO2 conditions.

Cell Stem Cell

Corticogenesis in ESC Culture

530 Cell Stem Cell 3, 519–532, November 6, 2008 ª2008 Elsevier Inc.

2009

2008

2011

2012

2011

2009

KidneyorganoidTakasato etal.,Nature2015

tissues, which could survive indefinitely (currently up to 10 months)when maintained in a spinning bioreactor. Histological and grossmorphological analysis revealed regions reminiscent of cerebral cortex,choroid plexus, retina and meninges (Fig. 1c, d and Extended Data Fig. 1b).Notably, tissues typically reached a size limit, probably because of thelack of a circulatory system and limitations in oxygen and nutrientexchange. Consistent with this, extensive cell death was visible in thecore of these tissues (Extended Data Fig. 1c), whereas the various brainregions developed along the exterior. Furthermore, cerebral organoidscould be reproducibly generated with similar overall morphology andcomplexity from both human embryonic stem (ES) cells and iPS cells(Extended Data Fig. 1d, e).

Cerebral organoids display discrete brain regionsBrain development in vivo exhibits a striking degree of heterogeneityand regionalization as well as interdependency of various brain regions.Histological analysis suggested that human cerebral organoids mightsimilarly display heterogeneous brain regions. To examine this further,we first tested the efficiency of initial neural induction in these tissuesby performing reverse transcriptase PCR (RT–PCR) for several markersof pluripotency and neural identity (Extended Data Fig. 2a). As expected,pluripotency markers OCT4 (also known as POU5F1) and NANOGdiminished during the course of organoid differentiation, whereasneural identity markers SOX1 and PAX6 were upregulated, indicatingsuccessful neural induction.

To test for early brain regionalization in whole organoids, we per-formed RT–PCR for forebrain (FOXG1 and SIX3) and hindbrain(KROX20 (also known as EGR2) and ISL1) markers (Fig. 2a), reveal-ing the presence of both populations within the tissue. However, astissue development proceeded, forebrain markers remained highlyexpressed whereas hindbrain markers decreased, reminiscent of the

developmental expansion of forebrain tissue during human braindevelopment25.

In order to test whether cells with these brain region identities deve-loped as discrete regions within the organoids, as gross morphologywould suggest, or were randomly interspersed within the tissue, weperformed immunohistochemical staining for markers of forebrain,midbrain and hindbrain identities during early development of thesetissues (16 days; Fig. 2b and Extended Data Fig. 2b). PAX6 expressionrevealed several regions of forebrain identity, and OTX1 and OTX2expression marked forebrain/midbrain identity. These regions werelocated adjacent to regions that lacked these markers but that werepositive for hindbrain markers GBX2, KROX20 and PAX2, which wasreminiscent of the early mid–hindbrain boundary, suggesting similarregional communication and probably mutual repression.

In vivo brain development involves increasing refinement of regionalspecification. Therefore, we examined further-developed cerebral orga-noid tissues for regional subspecification. We performed staining forthe forebrain marker FOXG1 (Fig. 2c), which labelled regions display-ing typical cerebral cortical morphology. Many of these regions werealso positive for EMX1 (Fig. 2d), indicating dorsal cortical identity. Wealso tested for further subregionalization by staining for cortical lobemarkers, namely AUTS2, a marker of prefrontal cortex26 (Fig. 2e); TSHZ2,a marker of the occipital lobe26 (Extended Data Fig. 2c); and LMO4,a marker of frontal and occipital lobes but absent in parietal lobes26

(Extended Data Fig. 2c). These markers could be seen in neurons label-ling distinct regions of dorsal cortex, suggesting subspecification ofcortical lobes.

hPSCsEmbryoid

bodies Neuroectoderm Cerebral tissue

a

b c

d

Day 0 Day 6 Day 11 Day 15

hES media, low bFGF Neural induction media Differentiation media Differentiation media +RA

Suspension Suspension Matrigel droplet Spinning bioreactor

Spinning droplet Stationary

N-c

adhe

rinH

oech

st

Expandedneuroepithelium

SOX2 TUJ1 Hoechst

Figure 1 | Description of cerebral organoid culture system. a, Schematicof the culture system described in detail in Methods. Example images of eachstage are shown. bFGF, basic fibroblast growth factor; hES, human embryonicstem cell; hPSCs, human pluripotent stem cells; RA, retinoic acid.b, Neuroepithelial tissues generated using this approach (left) exhibited largefluid-filled cavities and typical apical localization of the neural N-cadherin(arrow). These tissues were larger and more continuous than tissues grown instationary suspension without Matrigel (right). c, Sectioning andimmunohistochemistry revealed complex morphology with heterogeneousregions containing neural progenitors (SOX2, red) and neurons (TUJ1, green)(arrow). d, Low-magnification bright-field images revealing fluid-filled cavitiesreminiscent of ventricles (white arrow) and retina tissue, as indicated by retinalpigmented epithelium (black arrow). Scale bars, 200mm.

a b c

d

Cho

roid

ple

xus

Pre

front

al lo

be

Hip

poca

mpu

s

TTR DAPI

NRP2 Hoechst

16 d

ays

FOXG1 DAPIFOXG1

f

e NKX2-1 PAX6 DAPINKX2-1

TTR

gAUTS2 DAPIAUTS2

Fore

brai

n

Vent

ral c

orte

x

NRP2

Calretinin DAPI

12 d

ays

16 d

ays

FOXG1

KROX20

ACTB

Fetal

brai

n

20 d

ays

FZD9 PROX1 DAPIFZD9 PROX1

ISL1

SIX3

FOXG1 CalretininCalretinin

Cor

tical

inte

rneu

rons

h i

Ret

ina ONL

RPE

INL

EMX1 DAPIEMX1

Dor

sal c

orte

x

j

PAX6 KROX20 PAX2 DAPI

Figure 2 | Human cerebral organoids recapitulate various brain regionidentities. a, RT–PCR for forebrain markers (FOXG1 and SIX3) and hindbrainmarkers (KROX20 and ISL1) at 12, 16 and 20 days of differentiation. Humanfetal brain complementary DNA was used as positive control.b, Immunohistochemistry in serial sections for the forebrain marker PAX6(red, left) and the hindbrain markers KROX20 (green, left) and PAX2 (red,right) at 16 days of differentiation. Note the juxtaposition reminiscent of themid–hindbrain boundary (arrows). DAPI (49,6-diamidino-2-phenylindole)marks nuclei (blue). c–i, Staining for various brain region identities: forebrain,FOXG1 (c); dorsal cortex, EMX1 (d); prefrontal cortex (note the discreteboundary, arrow), AUTS2 (e); hippocampus, NRP2, FZD9, PROX1 (f); ventralforebrain, NKX2-1 (g) and choroid plexus, TTR (i). g, Staining for adjacentventral (arrow) and dorsal (PAX6, arrowhead) forebrain and for calretinin(green) in a serial section revealing cortical interneurons in the ventral region(arrow). Calretinin interneurons within dorsal cortex (h) exhibit typicalmorphology of tangential migration (arrows). j, Haematoxylin and eosinstaining of retinal tissue exhibiting stereotypical layering: retinal pigmentepithelium (RPE), outer nuclear layer (ONL) and inner nuclear layer (INL).Scale bars, 100mm.

RESEARCH ARTICLE

3 7 4 | N A T U R E | V O L 5 0 1 | 1 9 S E P T E M B E R 2 0 1 3

Macmillan Publishers Limited. All rights reserved©2013

Lancasteretal.,Nature2013Cerebralorganoid

Fig. 2b, c and Supplementary Video 1). The presumed human iPSC-derived liver buds (iPSC-LBs) were mechanically stable and could bemanipulated physically. We visualized a formation of endothelial networkand homogenously distributed human iPSC-HEs by using fluorescent-protein-labelled cells (Fig. 1d). Although human iPSC-LBs are a tri-lineagemixed tissue and difficult to compare directly with human iPSC-HEs,quantitative polymerase chain reaction (PCR) analysis revealed that cellsin human iPSC-LB had significantly increased expression of early hepaticmarker genes such as alpha-fetoprotein (AFP), retinol binding protein 4(RBP4), transthyretin (TTR) and albumin (ALB) (Fig. 1e)4. Microarrayprofiling showed that FGF and BMP pathways were upregulated highly inhuman iPSC-HEs when co-cultured with stromal cells, suggesting theinvolvement of stromal-cell-dependent factors in liver-bud formation(Supplementary Fig. 3a). Consistent with this, loss- and gain-of-functionexperiments suggested that, in addition to the direct cell-to-cell interac-tions, stromal-cell-dependent paracrine support is essential for three-dimensional liver-bud formation through the activating FGF and BMPpathways (Supplementary Fig. 3b–i and Supplementary Discussion)9.

Human liver-bud formation is initiated on the third or fourth week ofgestation, and this corresponds to embryonic day 9.5 (E9.5) to E10.5 formouse liver-bud formation10. Similar to E10.5 mouse liver bud, immuno-histochemistry showed that human iPSC-LB is composed of proliferatingAFP-positive hepatoblasts11 as well as mesenchymal and endothelial

progenitors (Fig. 2a). Hepatic cells in human iPSC-LBs were as prolif-erative as E10.5 mouse liver buds (Fig. 2b). Flow cytometric character-ization revealed that 42.7 6 7.5% (n 5 6) of cells in human iPSC-LBswere identified as iPSC-HEs using fluorescence-labelled HUVECs andMSCs. Among these, approximately 71.9 6 7.3% (n 5 6) of human iPSC-HEs expressed ALB and 29.96 2.8% expressed AFP, and 19.3 6 4.5%were positive for both ALB and AFP (Fig. 2c).

To characterize the expression profiles of human iPSC-LBs and tocompare with those of a corresponding developmental stage, we carriedout microarray analysis of 83 selected genes that are serially upregulatedduring liver development. Hierarchical clustering analyses suggestedthat the expression profiles of human iPSC-LBs at day 4 of cultureresembled those of mouse E10.5 and E11.5 liver buds rather thanadvanced fetal or adult livers (Fig. 2d). These expression profiles wererelatively similar to those of human fetal liver cell-derived liver buds(FLC-LBs) (Fig. 2d), which also have an ability to form LBs (Sup-plementary Figs 2a and 4). The 83-gene expression profile of humaniPSC-LBs showed closer signatures to more advanced human liver

19.3 ± 4.5%(6.1 ± 1.4%)

46.1 ± 6.3%(14.5 ± 2.0%)

ALB Log APC intensity

AFP

Log

FlT

C in

tens

ity

a

d

Human iPSC-derived liver budCK8/18 AFP DAPI CK8/18 CD31 DAPI

CK8/18 Desmin DAPI AFP PCNA DAPI

CK8/18 AFP DAPI CK8/18 Flk1 DAPI

CK8/18 Desmin DAPI AFP BrdU DAPI

E10.5 murine liver bud

29.9 ± 2.8%(9.4 ± 1.9%)

AFP Log APC intensity

ALB Log APC intensityGFP-HUVEC

60

40

20

0 Kus

abira

Ora

nge

hum

an M

SC

46.4 ± 8.0%

10.9 ± 0.9%

42. 7 ± 7.5%

cb

Pro

lifer

atin

g he

pato

blas

t num

ber

(%)

iPSC-L

BE10

.5E15

.5E17

.5 8 w

Mouse E9.5 LT

Mouse E10.5 LT

Human iPSC-LB

Human FLC-LB

Mouse E11.5 LT

Mouse E13.5 LT

Mouse E15.5 LT

Mouse E17.5 LT

Mouse E19.5 LT

Mouse P0 LT

Mouse P3 LT

Mouse P8 w LT

Human ALT (30 yr)

71.9 ± 7.3%71.9 ± 7.3%(20.4 ± 1.9%)(20.4 ± 1.9%)71.9 ± 7.3%

(20.4 ± 1.9%)

28.3 ± 9.1%28.3 ± 9.1%(8.9 ± 2.9%)(8.9 ± 2.9%) 28.3 ± 9.1%(8.9 ± 2.9%)

6.3 ± 2.2%6.3 ± 2.2%(2.0 ± 0.7%)(2.0 ± 0.7%) 6.3 ± 2.2%(2.0 ± 0.7%)

Figure 2 | In vitro characterization of human iPSC-LBs. a, Immunostainingof CK8/18, AFP, PECAM1 (CD31), FLK1, desmin, PCNA and BrdU(5-bromodeoxyuridine). Scale bars, 100mm. b, Proportions of proliferatinghepatic cells, as assessed by dividing the number of PCNA-positive orBrdU-positive cells by the number of CK8/18-positive cells (shown as apercentage). Results represent means6 s.d., n 5 3. c, Representative flow cytometryprofile showing the average number of AFP-positive and/or ALB-positive humaniPSC-HEs at day 4 of culture in six independent differentiation experiments.Human iPSC-HEs were separated from stromal populations by the use offluorescence labelled cells. Average percentages of the total cells and s.e.m. are givenin brackets. d, Comparison of liver developmental gene signatures among humaniPSC-LB, human FLC-LB, human adult (30 years old) liver tissue (ALT) and mouseliver tissue (LT) of various developmental stages (from E9.5 to 8 weeks after birth).

3 d 7 d 14 d

Vas

cula

r ar

ea (%

)

g

b

a

0 d Human iPSC-HE HUVEC human MSC

ihDextran

Dextran

ed f

*

Func

tiona

l ves

sel l

engt

h (m

m)

Hum

an v

esse

l len

gth

(μm

)

5,000 60

HUVEC MSC

Human

iPSC-L

B Tx

HUVEC MSC

Human

iPSC-L

B Tx Adult l

iver

7

6

5

80

70

60

50

40

30

20

10

0

4

3

2

1

0

40

20

10

0

4,000

3,000

2,000

1,000

0 d 3 d 7 d 15 d

45 d

60 d

0

Branch points

~~

*

NS

0 d 2 d 3 d

Human iPSC-HEHUVEC

human MSC Dextran

c

Human iPSC-HEHUVEC human MSC

mouse CD31

Human iPSC-HEHUVEC

human MSC

Human iPSC-HEHUVEC

human MSC

Hum

aniP

SC

-LB

Tx

Mou

se a

dult

liver

Figure 3 | Generation of human liver with functional vascular networks invivo. a, Macroscopic observation of transplanted human iPSC-LBs, showingperfusion of human blood vessels. Dotted area indicates the transplanted humaniPSC-LBs. b, Intravital tracking of human iPSC-LBs, showing in vivo dynamics ofvascularization. c, Dextran infusion showing the functional human vesselformation at day 3. Scale bar, 500mm. d, Visualization of the connections (arrows)among HUVECs (green) and host vessels (blue). Scale bar, 250mm. e, f, Localizationof human MSCs or human iPSC-derived cells at day 15. Scale bars, 100 and 250mm.g, Quantification of human vessels over time (mean6 s.e.m., n 5 3). Error barsattached to the bars relate to the left axis (vessel length), error bars attached tosquares relate to the right axis (branch points). h, Comparison of functional vessellength between human iPSC-LB and HUVEC human MSC transplants (Tx)(mean6 s.e.m., n 5 5,*P , 0.01). i, Vascular networks of human iPSC-LB-derivedtissue is similar to that of mouse adult livers (mean6 s.e.m., n 5 5, *P , 0.01).

RESEARCH LETTER

4 8 2 | N A T U R E | V O L 4 9 9 | 2 5 J U L Y 2 0 1 3

Macmillan Publishers Limited. All rights reserved©2013

Takebeetal.,Nature2013Liverbud

DOI: 10.1126/science.1077857 , 2188 (2002); 298Science

et al.Kenneth D. Poss,Heart Regeneration in Zebrafish

www.sciencemag.org (this information is current as of November 29, 2006 ):The following resources related to this article are available online at

http://www.sciencemag.org/cgi/content/full/298/5601/2188version of this article at:

including high-resolution figures, can be found in the onlineUpdated information and services,

http://www.sciencemag.org/cgi/content/full/298/5601/2188/DC1 can be found at: Supporting Online Material

found at: can berelated to this articleA list of selected additional articles on the Science Web sites

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ModifiedfromDressler2006andDavidson2009

Interactionoftheuretericbud(UB)withthemetanephric mesenchyme(MM)initiateskidneydevelopment

UBandMMinteractiondrivesthebranchingandmaturationofkidney

YunXia

KidneyDifferentiationfromProgenitors

Xiaetal.,NatCellBiol,2013

YunXiaNTU,Singapore

UB

Lietal.,CellStemCell,2016

Zhongwei LiUSC,US

MM

Emulatingtheinvivomicroenvironmenttoacceleratekidneyorganoiddifferentiationandmaturation

NuriaMontserratIBEC,Barcelona(Spain)

LTLWT1NPHS1

Garretaetal.,Nature Materials 2019

Hydrogeltuning

Lowetal.,CellStemCell2019

01

2

3

4

10

6

7

85

9

62,506cellsfromday10,12,and14ofdifferentiation

Endothelial cluster

820cellsoftheendothelial cluster

Proliferatingcells

Arterial

Immatureendothelial

0

1

2

Generationofhighlyvascularizedkidneyorganoidsinvitro

CD31 NPHS1 CDH1DAPI

500μm

ModelingARPKDusingisogeniciPSC-derivedkidneyorganoids

Lowetal.,CellStemCell2019

H9ESC Genecorrected-ARPKD ARPKD

TargetingmetabolicreprogramminginPKD

unpublished

Control Cystic Drug1 Drug2 Drug3

Extracellularlactate Intracellularlactate IntracellularATP

ModelSystemsforHumanBiologyResearch

DiseaseModeling

DrugDiscovery/Testing

RegenerativeMedicine

EngineeringOrganoids:MajorApplications

Recapitulatingearlymammalianembryogenesisbyusingculturedcells

Recapitulatingearlymammalianembryogenesisbyusingculturedcells

JunWuRonghui LiCuiquing Zhong.YangYu

E18.5

MouseRatChimera

InterspeciesChimeras:RatMouse

Wuetal,Cell,2017

InterspeciesChimeras:RatMouse

Heart

Pancreas Liver

KidneyBrain

Spleen

Intestine

Lung

Rat-MouseChimericOrgans

Wuetal,Cell,2017

HumanChimerasinEvolutionaryDistantHostAnimalModels

MouseRabbitPigSheep

Wu et al, Cell, 2017

Lowpost-implantationinterspecieschimerism

ChimericcontributionofHumanPSCstopigembryos

Somatic cellsBlastocyst

Pluripotent stem cells

Early Embryo Development of Mouse

E0 E2 E2.5 E3 E3.5 E4.5

Zygote 2 cells 4 cells Compacted 16 cells 32 cells8 cells >100 cells

Trophectoderm

ICMTotipotent EpiblastPE

Engineering a blastocyst ?TSCells

Totipotentcells

ESCells

XENcells

Rivron,Niejsen,andcolleagues,Nature,2018

EnhancingChimericCompetency

EnhancingChimericCompetency

Current Protocols Yang et al Cell, 2017

EnhancingChimericCompetency

Yang et al Cell, 2017

25

EPS cellsderivedblastoid (EPS-blastoid)Blastocysts

Blastoids

TdPh

ase

Day 5Day 1 Day 2 Day 3 Day 4

SingleEPS cellsderivedblastoid

mcherryPhase

Characterization ofEPS-blastoids

• 1.Segregationoftrophectoderm(TE)andinnercellmass(ICM)

TE

PEEPIICM

• 3.DerivationofstemcellsfromTE,ICM/EPI,andPE

• 2.SegregationofICMintoepiblast(EPI)andprimitiveendoderm(PE)

1.Segregationoftrophectoderm(TE)andinnercellmass(ICM)

Middle plane Max projection

CDX2 Merged w/ Ho CDX2

SOX2 Merged w/ Ho

TEmakerexpression

ICMmakerexpression

TE+, ICM+

TE+, ICM−

TE−, ICM+

TE or ICM Mislocated74.3%

15.0%

9.3%1.4%

AmajorityofEPS-blastoids haveboth TEandICM lineagesproperlyallocated.

2.SegregationofICMintoepiblast(EPI)andprimitiveendoderm(PE)

GATA4 Merged w/ Ho NANOG

EPI PEMarker:

3.DerivationofstemcellsfromTE,ICM/EPI,andPE

Blastoid-ESCs

Blastoid-TSCsBlastoid-XENcellsTd

CK8 GFP Ho

dec

gc

sp

laby

Placenta

Yolksac

Chimera

EPS-blastoidformationmimicsearlydevelopment:Compaction, Polarization,andHippo/YAPpathway

Nance, JCB,2014

Compaction:E-cadherin,Par1/EMK1

Polarization:Par3,Par6B,aPKC

Hippo/YAP:YAP

EPS-blastoidformationmimicsearlydevelopment:

Compaction

Polarization

E-ca

d

EPS aggregateDay 1 Day 2

8-cell embryoE2.0

Day 3

PA

R6

SO

X2

EPS aggregate

Day 1 Day 2

16-cell embryo

E2.5

EPS-blastoidformationmimicsearlydevelopment:Hippo/YAP signaling

Day 3EPS aggregate

Day 1 Day 2 EPS-blastoid Blastocyst

Day 5 E4.5

YAP

BulkRNA-Seq scRNA-seq

TranscriptomefeaturesofEPS-blastoids

TranscriptomefeaturesofEPS-blastoids:scRNA-seq

DifferentialgeneexpressionbetweenEPS-blastoids andblastocysts

Invitrocultureofblastocystsbeyondimplantationstage

Bedzhov etal.,NatureProtocol,2014Shahbazi et al, Science, 2019

InvitrocultureofEPS-blastoid beyondimplantationstage

SOX2

TFA

P2C

Ho

SOX2

GAT

A6

Ho

SOX2

TFA

P2C

Ho

OC

T4 G

ATA

4 H

o

Blastocyst IVC EPS-blastoid IVC

EXE:TFAP2C

EPI:SOX2,OCT4

VE:GATA6,GATA4

Modelingpost-implantationmorphogeneticeventsusingEPS-blastoids

Blastoid- Day3 Blastoid- Day5

Epiblastpolarization

Pro-amnioticcavityformation

Merged F-actin Ho NANOG Ho

PCX SOX2 Ho

Blastocyst- Day5

aPKC SOX2 Merged w/ Ho

PCX OCT4 Merged w/ Ho

Bedzhov and Zernicka-Goetz,Cell,2014

InuterodevelopmentofEPS-blastoids

EPS-blastoid-induced deciduae

Controldeciduae at 7.5dpc

Control7.5dpc EPS-blastoid 7.5dpc

100 100

tdTomato tdTomatoOCT4 Merged w/ Ho EOMES Merged w/ Ho GATA4 Merged w/ Ho

EXEEPI VE

iPS-EPScellsderivedblastoidsFibroblasts

iPS-EPSblastoids

CDX2 NANOG CDX2NANOG Merged w/ Ho

Max proj.Middle plane

PCX OCT4 Merged w/ Ho

1.ExpressionofTEandICMmarker

2.Post-implantationmorphogenesis 3.Inuterodevelopment

42

Recapitulatingearlymammalianembryogenesisbyusingculturedcells

Li,Zhong etalCellInpress

PossibleApplications•Treatinginfertility• ImprovingIVF•Designingnewcontraceptives•SpeciesConservation•ModelingDisease•Preventingdisease•CreatingTissueandOrganProgenitors

43

TheSalkInstituteforBiologicalStudies

YunXiaNanyangTechnologicalUniversity

RonghuiLiSalkInstitute

CuiqingZhongSalkInstitute

JunWuUTSouthwestern

WeizhiJiYunnanKeyLaboratoryforPrimateResearch

Twitter:@JCIB_Lab_Salkbelmonte@salk.edu

EstrellaNunezDelicoda-UCAMEmilioMartinez-UniversityofMurcia

Josep M.Campistol-HospitalClinicBarcelonaPedroGuillen-linica CemtroMadridHongkui Deng-PekingUniversity

YangYu-PekingUniversityThirdHospitalNuria Montserrat-IBEC

YunXia-NanyangTechnologicalUniversityZhongwei Li-UniversityofSouth California

AlllabmembersattheSalkInstitute