Neural Patterns and Structures in 3D ES cell culture:!Induction and self-organization!

RIKEN Center for Developmental Biology!

Yoshiki Sasai!

Shape  and  size  of  Organ

brain �liver� kidney �

Higher Functions in right shape �

Regenerative medicine �Drug discovery �Disease model �

Tissue-­‐engineered  Ear  

Understanding  of  Shaping  Principle? newt

mesoderm

endoderm

ectoderm

cartilage

神経

retinal

Bronchial epithel

neural

Gut epithel

Teratoma Human ES cells

hES cells

testicular Injection

9w

teratoma

Random differentiation but Some self-organization

Three major CNS regions with multiple-layered structures

Cerebral Cortex

Cerebellar Cortex

Neural Retina

Keiko Muguruma et al Nature Neurosci (2010)　

Serum-free Floating culture of EB-like Aggregates in growth factor-minimized medium (SFEBq)

3000 mES cell/well!

day 0!

quick aggregate formation (~5hr)!

low cell-adhesion plate!

culture in! serum-free !

GF-minimized !medium!

day 7!

uniform & reproducible!aggregate formation !

Mototsugu  Eiraku  

Neuroepithelial  Forma:on  from  ESC  aggregate

Sox1::GFP

SFEBq  culture  

Day  2-­‐  day  7

Why  is  neural  fate  default?    

Intrinsic activation of Zfp521 drives neural conversion

Kamiya et al, Nature (2011)

d3 d5 d0

E10.5 mouse

forebrain

midbrain

spinal cord

CNS Regionalization

hindbrain

tel

di

cbl

Default Direction of Neural Progenitors Derived from ES Cells

Rostral-most neural by default

Recapitulation of layered cortex development in SFEBq culture

SFEBq

Layered cortical tissue (4 zones)

3D culture

hES cells

6 weeks

Eiraku et al Cell Stem Cell (2008)

Self-­‐Organized  Layer  Forma:on  in  SFEBq-­‐Cultured  hES  Cells  

Ventricular  Zone    Early  CorIcal  Plate  

Cajal-­‐Retzius  Cells  

Basal  Progenitors  

SFEBq  Day  46  

Layer  markers  

Human  E50  cortex  

secIon  

Eiraku  et  al  Cell  Stem  Cell  (2008)  

mitosis  

pia  

ventricle  

No inside-out 4 zones instead of 6 layers

RETINA

Sclera ReIna

Cornea

Iris

Lens OpIc  Nerve

Light

RPE

Photoreceptor

Horizontal  cells Bipolar  cells Amacrine  cells Ganglion  cells

NR

外

内

光

視細胞

Light reception Information process

No blood vessel invasion into the NR proper

E10

　Optic Cup 　Optic vesicle

E10.5 E11

Development  of  ReInal  Anlage  (OpIc  Cup)

E9.5

マウス胎児　眼

Dien OV

OS

OC

間脳

Summary of self-organized optic-cup formation (CG) Culture in low KSR-containing medium supplemented with Laminin-containing ECM (Matrigel)

Evagination of Rx+ neuroepithelium!(retinal progenitors)!

Rx::GFP D3 - D7

Eiraku et al, Nature (2011)

Invagination of Neural Retina in SFEBq culture

D7-D9

Rx::GFP

ESC-derived Optic Cup!

Eiraku et al, Nature (2011)

Multi-Photon 3D Imaging for Long-Term Culture　（w/ Olympus）

3D  live  imaging  of  op:c-­‐cup  forma:on  by  MP  op:cs

Invaginating Optic cup (Rx::GFP)

Simultaneous observation of the “Whole” and “Parts” (in toto imaging)

Each cell (H2B-GFP)

Progressive morphologenetic change of ES-cell-derived retinal epithelium!

Spatio-temporal control of MLC2 phospholylation !During ES cell-derived eye cup morphogenesis!

Region-­‐specific  regula:on  of  Myosin  aci:vity

pMLC2: active Myosin

Low Myosin acitivity in invaginating NR (mechanically relaxed)

AFM-based measurement of relative tissue rigidity!

ESC-derived optic cup:!Cellular morphogenesis!

Mitotic inhibitor aphidicolin attenuates the invagination!

3D  abla:on  study  for  internal  force  direc:ons  (tension  vs  compression)

3D  Abla:on  for  Internal  Force  Direc:ons  (tension  vs  compression)

Phase 4 　RPE

Phase 4 　RPE + aphidicolin

Multi-photon laser-induced 3D cell ablation!

Relaxa:on-­‐Expansion  Model    for  Self-­‐Driven  OpIc-­‐Cup  Morphogenesis

神経網膜 （柔軟）

 Computer  simula:on  for  the  relaxa:on-­‐expansion  model  

Collaboration with T. Adachi (Kyoto Univ)

Does ESC-derived Neural Retina Develop into Postnatal-Type Stratified Tissue?

ES細胞由来の眼杯

Ph.R,

？ Multi-layered NR

(postnatal)

Bp

GC

Interkinetic nuclear migration in ESC-derived neural retina Multi-photon image

Generation of stratified neural retina tissues! from ES-cell-derived invaginated epithelia!

200-300µ 2 mm

Generation of stratified neural retina ! from ESC-derived retinal epithelia!

Neural development still involves mysterious mechanisms: Presence of Latent Intrinsic Order