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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
Key Properties of Stem Cells
• Self-renew indefinitely
• Progeny generates different cell types
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
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
Tissue stem cells
• Hematopoeitic system
• Nervous system
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)
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)
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
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
The SVZ generates new neurons in the adult brain
Alvarez-Buylla and Garcia-Verdugo (2002) J. Neurosci. 22, 630
Organization and lineage in the SVZ
Alvarez-Buylla and Garcia-Verdugo (2002) J. Neurosci. 22, 632
New neurons are also incorporated in the adult dentate gyrus
Taupin and Gage (2002) J. Neurosci. Res. 69, 746
Stem cells in the adult brain are regionally specified
Merkle et al. (2007) Science 317, 381-384
Stem cell-derived neuronal phenotypes in the OB
Cells labeled at birth preserved their regional identity
Cultured cells remember where they come from
Alternative to stem cell transplantation
• Activation of endogenous neurogenesis
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)
Key to homogeneity: Maintaining pluripotency of all cells
Silva and Smith (2008) Cell 132, 532-536
• 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?
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
ES cells-derived neurons form functinal synaptic contacts
Neurotrophin signalling: 2 different receptors
tau egfp/rp75 (tau::p75NTR)tau egfp/egfp (control)
Controlled overexpression of p75NTR in ES cells
tau gfp/gfp (G30)tau promoter
GFP
GFP
tau gfp/rp75 (tau::p75)tau promoter
GFP
rat p75
tau::p75NTR neurons at d3 (βIII tubulin staining)
pH 4-7
200 kDa
10 kDa
WT cells labeled Cy5 (red)
Hans Vosshol, Dieter Müller and Sjouke Hoving (Novartis)
p75NTR::tau cells labeled Cy3 (green)
pH 4-7
200 kDa
10 kDa
Overlay
pH 4-7
200 kDa
10 kDa
Galectin-1 causes degeneration of neuronal
processes
Similar results with hES lines
Development according to Waddington 1957
Hochedlinger and Path (2009) Development 136 509-523
Differentiation commitment is reversible
A simple TF cocktail is sufficient
Takahashi, K. & Yamanaka, S. (2006) Cell 126, 663–676
Reprogramming achieved with somatic cells from the 3 germ layers
How does reprogramming work?
Methods: Summary
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