from es cells to neurons: a road map to neurogenesis in the embryo
DESCRIPTION
From ES cells to Neurons: A Road Map to Neurogenesis in the Embryo. Elsa Abranches , Domingos Henrique, Evguenia Bekman. Unidade de Biologia do Desenvolvimento Instituto de Medicina Molecular. Encontro Nacional de Ciência Lisboa, 29-07-2009. Embryonic stem cells & Neural Development. - PowerPoint PPT PresentationTRANSCRIPT
From ES cells to Neurons:
A Road Map to Neurogenesis in the Embryo
Elsa Abranches, Domingos Henrique, Evguenia BekmanUnidade de Biologia do DesenvolvimentoInstituto de Medicina Molecular
Encontro Nacional de CiênciaLisboa, 29-07-2009
Embryonic stem cells & Neural Development
in vitro generation of Neurons from ES cells
Promising approach to:
Gain a better knowledge of the cellular and molecular
events that are involved in neural development
Produce cells suitable for neural tissue repair and cell-
based replacement therapies of the nervous system
Embryonic stem cells & Neural Development
Question: How do cells go from one stage to the other?
ES cells Neural Progenitors Neurons
(i) Have proper apico-basal polarity (Divide apically & Produce neurons at the basal side)
(ii) Notch pathway is active
(iii) The timing of production of neurons and glia is correct
From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol
(iv) Cells show interkinetic nuclear movement
Abranches et al. PlosOne (2009)
ES cells Rosettes Neurons
in vitro model mimicks in vivo commitment to neural fate
Rosettes are Neural tube-like in vitro structures
Highdensity
Monolayer Replating
-1 0 1 32 4 5 76 8
Highdensity
Monolayer Replating
-1 0 1 32 4 5 76 8Time (days) Rosettes neural progenitors
Rosettes differentiating neurons
ZO1Sox1:GFP
ZO1Sox1:GFP
ß-Catenin
Sox1:GFP
Cluster of cells forming primitive epithelium and initiating neural commitment
Define the transcriptional profile of different neural progenitors populations (Microarray analysis)
ES cells Rosettes Neurons
Gain a better knowledge of the cellular and molecular events that are involved in neural development
Rapid & Reproducible process
Homogeneous populations
Large amounts of cells
From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol
Mouse Genome 430.2A Affymetrix45101 ProbeSets
Anova FDR <10-3 (p-value < 2.10-4)
From ES cells to Neurons Microarray analysis
6563 Differentially Expressed Genes
1750 Genes
Specific embryo-oriented criteria
5 Clusters
AIM: Identify different progenitor populations
0
1
3
3'
8
ES cells
0.000.100.200.300.400.500.60
Fre
qu
en
cy
Time point
Group I – ES cells
0
ES cell Gene Signature (188 genes)
“Stemness” character confirms the ES
cell identity of the starting population
Group II – Primitive Ectoderm (PE)
1
Primitive Ectoderm Gene Signature (66 genes)
0
1
3
3'
8
PE
0.000.100.200.300.400.500.60
Fre
qu
en
cy
Time point
Known PE-like signature
(FGF5+, Oct4+, Rex1-)
Calcium related genes
Group III – transient Neural Progenitors (tNPs)
Transient NPs Gene Signature (61 genes)
3
0
1
3
3'
8
tNPs
0.00
0.10
0.20
0.30
0.40
Fre
qu
en
cy
Time point
Genes important for neural progenitors specification that need to be switched off to allow progenitors to advance into the next stage
Group IV – neurogenic Neural Progenitors (nNPs)
3
0
1
3
3'
8
nNPs
0.00
0.10
0.20
0.30
0.40
Fre
qu
en
cy
Time point
Genes important for the next stage of NP development, when competence to enter neurogenesis is acquired
nNPs Gene Signature (763 genes)
Group V – Rosettes
8
Rosettes Gene Signature (673 genes)
0
1
3
3'
8
Rosettes
0.00
0.10
0.20
0.30
0.40
Fre
qu
en
cy
Time point
Genes coupled to the final stages of NP development and commitment to neural differentiation
Notch pathway
From ES cells to Neurons Microarray analysis
Time (days)
1. Delineate transient cellular states that occur during
neural development
( ES cells Primitive Ectoderm Neuroepithelial Progenitor populations)
From ES cells to Neurons Microarray analysis
2. Reveal signalling pathways associated with these
transitions
(e.g. Ca2+ signalling; Notch pathway)
Wild-type
Activated Notch
Notch inhibition
in vivo
Equilibrium between progenitors and differentiating neurons
Excess of progenitors at the expense of neurons
Excess of neurons at the expense of progenitors
From ES cells to Neurons Notch Pathway
From ES cells to Neurons Notch Pathway
A comprehensive analysis of the Notch pathway in
mammalian neural development has never been done
Problems
• Pleiotropic effects of the pathway in the embryo;
• Heterogeneity of embryonic cell stages and types that respond differently to Notch activity
Explore the simplicity of the rosette culture system to address
in detail how Notch operates to regulate neural development
From ES cells to Neurons Notch Pathway
Neural Progenitors
Differentiating Neurons
Neural Progenitors
Differentiating Neurons
Tuj1 Sox1
in vitro
Tuj1 Sox1
in vitroW
ild-t
yp
e (R
ose
ttes)
No
tch
inh
ibit
ion
From ES cells to Neurons Notch Pathway
K-Group3Down regulated after LY treatment
K-Group 0Down regulated after 12 h LY treatment
K-Group 1Up regulated after 6 h LY treatment
K-Group 2Up regulated by LY stronger effect after 12 h treatment
K-Group3Down regulated after LY treatment
K-Group 0Down regulated after 12 h LY treatment
K-Group 1Up regulated after 6 h LY treatment
K-Group 2Up regulated by LY stronger effect after 12 h treatment
Mouse Genome 430.2A Affymetrix45101 ProbeSets
Anova FDR <10-3 (p-value < 1.8410-5)
701 Genes
Notch-oriented criteria
4 Clusters
AIM: Identify Notch Pathway components
http://www.genepaint.org/Frameset.html
Novel potential Notch components
From ES cells to Neurons Notch Pathway
Notch synexpression group
Exp
ress
ion
Lev
el
FunGenES database: Different differentiation conditions
Dissect further the mechanisms underlying Notch activity during neural differentiation
Comprehensive resourse for studies aimed at elucidating the
genetic architecture underlying neural development
in vitro model mimicks in vivo
commitment to neural fate
delineate transient cellular states that
occur during neural development
reveal signalling pathways associated
with these transitions
Define more rational strategies to achieve controlled
production of specific neuronal cell types
From ES cells to Neurons Conclusions
Collaborations:• Herbert Schulz, Oliver Hummel (MDC Berlin, Germany)• Raivo Kolde, Jaak Vilo (EGeen, Tartu, Estonia)• Laurent Pradier (Sanofi-Aventis, France)
• Stem Cell Sciences (Edinburgh, UK)• Austin Smith lab (Cambridge, UK)