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