b4: diseases of the nervous system embryonic and induced ... · translocations, lack of cell death,...
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Embryonic and inducedpluripotent stem cells:
A new tool to model diseases
of the nervous system
Y.-A. Barde May 19 2009
B4: Diseases of the nervous system
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Key Properties of Stem Cells
• Self-renew indefinitely
• Progeny generates different cell types
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Embryonic Stem Cells
Tissue Stem Cells
Both self-renew indefinitely and it is their
differentiation potential that distinguishes them
2 Different Types of Stem Cells
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Stem cells and diseases of the nervous system
• Tissue stem cells would be better suited for cellular therapy (c.f. hematopoietic system)
• Embryonic or induced pluripotentcells are a great resource for disease modeling
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Tissue stem cells
• Hematopoeitic system
• Nervous system
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Key findings
• Discovery of somatic stem cells: Till and McCulloch (1961)
• Isolation of mouse HSC (1988)• One defined cell can reconstitute the
hematopoietic system and generate more than109 cells per day
• Also generates a stable pool of HSCs(20’000-100’000)
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Hierarchy
• HSC: slow and rare division, resides in niches, does not senesce, can home when injected
• MPPs: Multipotent progenitors (first distinct progeny of HSCs)
• 2 oligopotent progenitors: CLP (common lymphoid progenitor) and CMP (common myeloid progenitor)
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Stem cells and tumor cells
• Tumor cells are also characterized by high telomerase activity
• May readily arise from dysregulatedprogenitors: mutations including translocations, lack of cell death, escape immune surveillance
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Nervous system
• Cell division noted in adult bird, rodent and cat brains in the 1960‘s
• Neurons in the olfactory bulb are constantly renewed in rodents
• Songbirds
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The SVZ generates new neurons in the adult brain
Alvarez-Buylla and Garcia-Verdugo (2002) J. Neurosci. 22, 630
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Organization and lineage in the SVZ
Alvarez-Buylla and Garcia-Verdugo (2002) J. Neurosci. 22, 632
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New neurons are also incorporated in the adult dentate gyrus
Taupin and Gage (2002) J. Neurosci. Res. 69, 746
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Stem cells in the adult brain are regionally specified
Merkle et al. (2007) Science 317, 381-384
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Stem cell-derived neuronal phenotypes in the OB
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Cells labeled at birth preserved their regional identity
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Cultured cells remember where they come from
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Alternative to stem cell transplantation
• Activation of endogenous neurogenesis
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Pluripotency of cultured ES cells
• An unstable state captured in vitro thanks to LIF
• The transcription factors Oct-4, Sox2 and nanogplay critical roles
• This role is now better understood: Blocking FGF signaling and GSK activity makes LIF and serum redundant (Ying et al. 2008, Nature 453,519-524)
• Isolation of RAT stem cells finally possible (see Buehr et al. and Li et al. Cell 2008, 135, 1287-1310)
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Key to homogeneity: Maintaining pluripotency of all cells
Silva and Smith (2008) Cell 132, 532-536
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• Unbiased phenotypic and molecular analyses with wild-type and genetically engineered cells
• Prerequisite: Homogenous cell populations
Why use cultured embryonic stem cellsto study the nervous system?
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ES cells can generate pure populations of defined neuronal progenitors
Neurons
Progenitors
ES cells: selection for mostrapidly dividing, i.e. undifferentiated ES cells
+RA
Pax6+ Radial Glial Cells
> 90% Glutamatergic neurons
Bibel et al. Nature Neuroscience (2004) 7, 1003-1009Bibel et al. Nature Protocols (2007) 2, 1034-1043
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ES cells-derived neurons form functinal synaptic contacts
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Neurotrophin signalling: 2 different receptors
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tau egfp/rp75 (tau::p75NTR)tau egfp/egfp (control)
Controlled overexpression of p75NTR in ES cells
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tau gfp/gfp (G30)tau promoter
GFP
GFP
tau gfp/rp75 (tau::p75)tau promoter
GFP
rat p75
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tau::p75NTR neurons at d3 (βIII tubulin staining)
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pH 4-7
200 kDa
10 kDa
WT cells labeled Cy5 (red)
Hans Vosshol, Dieter Müller and Sjouke Hoving (Novartis)
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p75NTR::tau cells labeled Cy3 (green)
pH 4-7
200 kDa
10 kDa
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Overlay
pH 4-7
200 kDa
10 kDa
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Galectin-1 causes degeneration of neuronal
processes
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Similar results with hES lines
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Development according to Waddington 1957
Hochedlinger and Path (2009) Development 136 509-523
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Differentiation commitment is reversible
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A simple TF cocktail is sufficient
Takahashi, K. & Yamanaka, S. (2006) Cell 126, 663–676
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Reprogramming achieved with somatic cells from the 3 germ layers
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How does reprogramming work?
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Methods: Summary