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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

Higher Functions in right shape� Regenerative medicine� Drug discovery� Disease model�

liver�

kidney�

Understanding of  Shaping  Principle? newt

Tissue-­‐engineered Ear  

brain�


hES cells

çĽžçľŒ

ectoderm testicular Injection 9w

neural

cartilage

mesoderm retinal

endoderm

Gut epithel

teratoma

Bronchial epithel

Teratoma Human ES cells Random differentiation but Some self-organization


Three major CNS regions with multiple-layered structures Cerebral Cortex

Cerebellar Cortex Keiko Muguruma et al Nature Neurosci (2010) 

Neural Retina


Serum-free Floating culture of EB-like Aggregates in growth factor-minimized medium (SFEBq) day 0!

Mototsugu  Eiraku  

day 7!

3000 mES cell/well!

low cell-adhesion plate! culture in! serum-free ! GF-minimized ! medium!

quick aggregate formation (~5hr)!

uniform & reproducible! aggregate formation !


Neuroepithelial Forma:on  from   ESC  aggregate

SFEBq culture   Day  2-­‐  day  7

Sox1::GFP Why is  neural   fate  default?    


Intrinsic activation of Zfp521 drives neural conversion Kamiya et al, Nature (2011)

d0

d3

d5


CNS Regionalization

cbl

midbrain

hindbrain tel forebrain

di

Rostral-most neural by default

E10.5 mouse

Default Direction of Neural Progenitors Derived from ES Cells

spinal cord


Recapitulation of layered cortex development in SFEBq culture hES cells

Layered cortical tissue (4 zones)

SFEBq

3D culture

6 weeks

Eiraku et al Cell Stem Cell (2008)


Cajal-­‐Retzius Cells  

mitosis

SFEBq Day  46   secIon  

Layer markers  

Ventricular Zone     Early  CorIcal  Plate  

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

Basal Progenitors   pia  

No inside-out 4 zones instead of 6 layers Eiraku et  al  Cell  Stem  Cell  (2008)   ventricle  

Human E50  cortex  


RETINA RPE

外 Sclera

Photoreceptor

ReIna

Horizontal cells Bipolar  cells Amacrine  cells Ganglion  cells

Cornea

NR

Light

光 Lens Iris

OpIc Nerve

No blood vessel invasion into the NR proper

Light reception Information process

視細胞


Development of  ReInal  Anlage  (OpIc  Cup)

E9.5

Dien

E10

E10.5

E11

OV OS OC

 Optic Cup

 Optic vesicle

マウス胎児 眼

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

Rx::GFP

D7-D9


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

Each cell (H2B-GFP)

Invaginating Optic cup (Rx::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?

Ph.R, Bp

GC

ES細胞由来の眼杯

Multi-layered NR (postnatal)


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


Generation of stratified neural retina tissues! from ES-cell-derived invaginated epithelia! 200-300Âľ

2 mm


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

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

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