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Derivation of g lutamatergic neurons from human pluripotent stem cells as a therapeutic intervention for Alzheimer’s disease. Joshue Leyva REǀNOUS Winter Meeting 1 01/27/14. “Efficient derivation of cortical glutamatergic neurons from human

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slide1

Derivation of glutamatergic neurons from human pluripotent stem cells as a therapeutic intervention for Alzheimer’s disease

Joshue Leyva

REǀNOUS

Winter Meeting 1

01/27/14

slide2

“Efficient derivation of cortical glutamatergic neurons from human

pluripotent stem cells: A model system to study neurotoxicity in

Alzheimer's disease”

Neurobiology of Disease (2014): 62-72

TandisVazin, K. Aurelia Ball, HuiLuc, HyungjuPark, YasamanAtaeijannati, Teresa Head-Gordon, Mu-ming Poo, David V. Schaffer

outline
Outline
  • Stem cells general review
    • Human embryonic stem cells (hESCs)
    • Human induced pluripotent stem cells (hiPSCs)
  • Creating neuronal progenitor cells
    • Glutamatergic neurons
    • GABAergic neurons
  • Glutamatergic neurons as a therapeutic model
  • Differential neurotoxic selectivity on neuronal populations
slide4

Stem cell types

• Embryonic (Ex. hESC or hiPSC)

– Can form all cell types

(pluripotent)

– immortal in culture

– Can form tumors

(teratomas)

– Methods to control

differentiation poorly

understood

• Adult (Ex. muscle stem cells, hematopoietic stem cells)

– Limited potential

(multipotent)

– Rare

– Some are difficult to

isolate

– Do not form teratomas

– In active clinical use

slide5

Types of Pluripotent Stem Cells

Donovan and Gearhart Nature 2001

derivation of neural progenitor cells
Derivation of Neural Progenitor Cells
  • Glutamatergic neurons
    • Induce hPSCs or hESCs into neuronal lineage by inhibiting SMAD signaling pathway
    • Inhibit Shh signaling pathway to form the dorsal telencephalon phenotype
  • GABAergic neurons
    • Induce hPSCs or hESCsinto neuronal lineage by inhibiting SMAD signaling pathway
    • Proceed with Shh signaling pathway to form the ventraltelencephalic neuronal phenotype
derivation of neural progenitor cells1
Derivation of Neural Progenitor Cells

74% Glutamatergic; 20% GABA 18% Glut; 62% GABAergic

glutamatergic neurons as a therapeutic model
Glutamatergic Neurons as a Therapeutic Model
  • Create Aβglobulomers (stable, soluble from of amyloid-beta)
  • Treat hESC-derived glutamatergic neurons with 2 μMglobulomers, get neuronal cell death after 72 h
glutamatergic neurons as a therapeutic model1
Glutamatergic Neurons as a Therapeutic Model

Progressive increase in glutamatergic neuron death with increasing Aβ globulomerconcentration

glutamatergic neurons as a therapeutic model2
Glutamatergic Neurons as a Therapeutic Model

Upon adding the pre-fibrillar from of Aβ to an increased cell culture maturation time, Aβ binding to cell membrane and dendritic spines significantly increased

25 days 58 days

neuronal phenotype sensitivities to a
Neuronal Phenotype Sensitivities to Aβ
  • Method:
    • give hESC-derived cultures (primarily comprised of glutamatergic or GABAergicneurons) Aβ globulomer and assess globulomer binding and neuronal apoptosis
neuronal phenotype sensitivities to a1
Neuronal Phenotype Sensitivities to Aβ

Glutamatergic neuronsGABAergic neurons

Glutamatergic neurons exhibit progressively higher cell death with increasing concentrations of the globulomeric form of Aβ

neuronal phenotype sensitivities to a2
Neuronal Phenotype Sensitivities to Aβ

Glutamatergic neuronsGABAergic neurons

Quantitative analysis demonstrate a concentration dependent onset of apoptosis in glutamatergic neurons treated with Aβ globulomers (as seen by caspase-3)

conclusions
Conclusions
  • Active manipulation with a Shhantagonist can leads to dorsal telencephalic NPCs, yielding glutamate expressing neurons
  • In the absence of Shh inhibition, NPCs adapted a ventral phenotype giving rise to GABAergicneurons
  • Glutamatergicneurons are more susceptible to Aβ toxicity and decrease in numbers with increasing Aβ concentrations