genetic manipulation of mouse es cells toward a specific neuronal fate anne chiaramello, ph.d...

Genetic Manipulation of Mouse ES Cells

toward a Specific Neuronal Fate

Anne Chiaramello, Ph.D

Associate Professor

George Washington University Medical Center

Department of Anatomy and Cell Biology

Tel: 202-994-2173

anaaec@gwumc.edu

NIH/NINDS R01NS041391 June 27, 2007

Neural Cell LineageNeural Cell Lineage

Neural Stem Cell

Progenitor Cell

Neuroblast

InhibitoryNeuron

(GABAergic)

ProjectionNeuron

(Glutamatergic)

Oligo Astrocytes

Glioblast

LIF

?

Objective

To direct neural fate of mouse ES Cells by genetic manipulation for cell replacement therapies in the

context of specific neurodegenerative disorders and CNS injuries.

Embryonic Stem Cell

bFGF

Mature Neurons

Neural Stem Cells

Immature Neurons

AstrocyticCommitment

AstrocyticDifferentiation

Astrocytes

Astrocyte Progenitor

Neural Progenitor

Cell Cycle Exit Terminal

Differentiation

Neural Commitment

Subtype Specification

Ngn1/2

Initiation of Differentiation

NeuroD6

Strategy to Direct Fate of Mouse ES Cells

NeuroD6-Mediated Glutamatergic Pyramidal Fate and Corticogenesis

Wu, Sheng-Xi et al. (2005) Proc. Natl. Acad. Sci. USA 102, 17172-17177

Committed Neural Progenitor Cells(proliferative & post-mitotic)

Neural Stem Cells

Glutamatergic Pyramidal Neurons(Upper Motor Neurons)

PC12-NeuroD6 Cells

CCE/GFP (Control)CCE ESCs CCE/ND6Low CCE/ND6High

MSinSB U3 R U5SD SA

CASD

U5U3

S BX

GFP

GFP

Co

un

tsF

luo

res

ce

nc

eB

rig

ht

Fie

ldA

B

U3 R U5SD SA

CASD

U5U3

S BX

ND6 GFPIRESMSin-ND6-IR-GFP-SB

MFI = 23,383 MFI = 1,148 MFI = 10,666

Generation of ND6-Expressing Mouse Embryonic Stem Cells

NeuroD6 Promotes Neuronal Differentiation of Mouse ES Cells upon LIF and Serum Withdrawal

Collaborators

•Dr. Robert Hawley

•Dr. Ali Ramezani

•Dr. Martine Uittenbogaard

•Teresa Hawley