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�
Understanding of Shaping Principle? newt
ectoderm testicular Injection 9w
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)â€ƒ
Serum-free Floating culture of EB-like Aggregates in growth factor-minimized medium (SFEBq) day 0!
Mototsugu Â Eiraku Â
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)
hindbrain tel forebrain
Rostral-most neural by default
Default Direction of Neural Progenitors Derived from ES Cells
Recapitulation of layered cortex development in SFEBq culture hES cells
Layered cortical tissue (4 zones)
Eiraku et al Cell Stem Cell (2008)
SFEBq Day 46 secIon
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
Horizontal cells Bipolar cells Amacrine cells Ganglion cells
光 Lens Iris
No blood vessel invasion into the NR proper
Light reception Information process
Development of ReInal Anlage (OpIc Cup)
OV OS OC
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
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
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?
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
Generation of stratified neural retina ! from ESC-derived retinal epithelia!
Neural development still involves mysterious mechanisms: Presence of Latent Intrinsic Order