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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
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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

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ES cells
Neural Progenitors
Neurons
Question How do cells go from one stage to the
other?
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ES cells
Rosettes
Neurons
Time (days)
(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
(iv) Cells show interkinetic nuclear movement
in vitro model mimicks in vivo commitment to
neural fate ? Rosettes are Neural tube-like in
vitro structures
Abranches et al. PlosOne (2009)
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? Rapid Reproducible process
ES cells
Rosettes
Neurons
? Large amounts of cells
? Homogeneous populations
Define the transcriptional profile of different
neural progenitors populations (Microarray
analysis)
Gain a better knowledge of the cellular and
molecular events that are involved in neural
development
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Mouse Genome 430.2A Affymetrix 45101 ProbeSets
Anova FDR lt10-3 (p-value lt 2.10-4)
6563 Differentially Expressed Genes
Specific embryo-oriented criteria
1750 Genes
5 Clusters
AIM Identify different progenitor populations
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• Stemness character confirms the ES cell
  identity of the starting population

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• Known PE-like signature
• (FGF5, Oct4, Rex1-)

• Calcium related genes

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• Genes important for neural progenitors
  specification that need to be switched off to
  allow progenitors to advance into the next stage

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• Genes important for the next stage of NP
  development, when competence to enter
  neurogenesis is acquired

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• Genes coupled to the final stages of NP
  development and commitment to neural
  differentiation
• Notch pathway

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Time (days)
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• Delineate transient cellular states that occur
  during neural development
• ( ES cells ? Primitive Ectoderm ?
  Neuroepithelial Progenitor populations)

2. Reveal signalling pathways associated with
these transitions (e.g. Ca2 signalling
Notch pathway)
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Wild-type
Equilibrium between progenitors and
differentiating neurons
Activated Notch
Excess of progenitors at the expense of neurons
Notch inhibition
Excess of neurons at the expense of progenitors
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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
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Wild-type (Rosettes)
Notch inhibition
?? Neural Progenitors
?? Differentiating Neurons
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Mouse Genome 430.2A Affymetrix 45101 ProbeSets
Anova FDR lt10-3 (p-value lt 1.84?10-5)
Notch-oriented criteria
701 Genes
4 Clusters
AIM Identify Notch Pathway components
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Notch synexpression group
Novel potential Notch components
Dissect further the mechanisms underlying Notch
activity during neural differentiation
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? 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
Comprehensive resourse for studies aimed at
elucidating the genetic architecture underlying
neural development
Define more rational strategies to achieve
controlled production of specific neuronal cell
types
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• 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)