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

“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

Tandis Vazin, K. Aurelia Ball, Hui Luc, Hyungju Park, Yasaman Ataeijannati, Teresa Head-Gordon, Mu-ming Poo, David V. Schaffer

OUTLINE

Stem cells general reviewHuman embryonic stem cells (hESCs)Human induced pluripotent stem cells (hiPSCs)

Creating neuronal progenitor cellsGlutamatergic neuronsGABAergic neurons

Glutamatergic neurons as a therapeutic model

Differential neurotoxic selectivity on neuronal populations

• 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

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

Types of Pluripotent Stem Cells

Donovan and Gearhart Nature 2001

DERIVATION OF NEURAL PROGENITOR CELLS

Glutamatergic neurons1. Induce hPSCs or hESCs into neuronal lineage

by inhibiting SMAD signaling pathway2. Inhibit Shh signaling pathway to form the

dorsal telencephalon phenotype GABAergic neurons

1. Induce hPSCs or hESCsinto neuronal lineage by inhibiting SMAD signaling pathway

2. Proceed with Shh signaling pathway to form the ventral telencephalic neuronal phenotype

DERIVATION OF NEURAL PROGENITOR CELLS

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

GLUTAMATERGIC NEURONS AS A THERAPEUTIC MODEL

• Create Aβ globulomers (stable, soluble from of amyloid-beta)

• Treat hESC-derived glutamatergic neurons with 2 μM globulomers, get neuronal cell death after 72 h

GLUTAMATERGIC NEURONS AS A THERAPEUTIC MODEL

Progressive increase in glutamatergic neuron death with increasing Aβ globulomer concentration

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 AΒ

Method:

give hESC-derived cultures (primarily comprised of glutamatergic or GABAergic neurons) Aβ globulomer and assess globulomer binding and neuronal apoptosis

NEURONAL PHENOTYPE SENSITIVITIES TO AΒ

Glutamatergic neurons GABAergic neurons

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

NEURONAL PHENOTYPE SENSITIVITIES TO AΒ

Glutamatergic neurons GABAergic neurons

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

NEURONAL PHENOTYPE SENSITIVITIES TO AΒ(QUANTITATIVE DATA)

CONCLUSIONS

Active manipulation with a Shh antagonist can leads to dorsal telencephalic NPCs, yielding glutamate expressing neurons

In the absence of Shh inhibition, NPCs adapted a ventral phenotype giving rise to GABAergic neurons

Glutamatergic neurons are more susceptible to Aβ toxicity and decrease in numbers with increasing Aβ concentrations