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Joshue Leyva REǀNOUS Winter Meeting 1 01/27/14

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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Joshue Leyva REǀNOUS Winter Meeting 1 01/27/14

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  1. 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

  2. “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

  3. 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

  4. 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

  5. Types of Pluripotent Stem Cells Donovan and Gearhart Nature 2001

  6. 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

  7. Derivation of Neural Progenitor Cells 74% Glutamatergic; 20% GABA 18% Glut; 62% GABAergic

  8. 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

  9. Glutamatergic Neurons as a Therapeutic Model Progressive increase in glutamatergic neuron death with increasing Aβ globulomerconcentration

  10. 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

  11. 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

  12. Neuronal Phenotype Sensitivities to Aβ Glutamatergic neuronsGABAergic neurons Glutamatergic neurons exhibit progressively higher cell death with increasing concentrations of the globulomeric form of Aβ

  13. 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)

  14. Neuronal Phenotype Sensitivities to Aβ(Quantitative Data)

  15. 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

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