8/7/2019 Ion Channel Poster Resized 101708 Final for Print

1/1

Ion Channel Expression Patterns and Functional Responsesin Human Embryonic Stem Cell

Derived Neural Cell LinesD.W. Machacek , S. K. Dhara , C. Sturkie , M.C. Dodla , S.L. Stice

ArunA Biomedical Inc , and University of Georgia

1 2 2, 2 1,2

1 2

man embryonic stem cells and their progeny can provide avel tissue source for understanding developmentalhways,pharmaceutical screening and tissue replacementrapies. Drug screening using human embryonic stem cellSC) derived tissue uniquely offers several advantages

er traditional cell lines: Large quantities of cells can beduced due to the proliferative nature of the cells, repeateddies can be done on a common genetic background andnaling can be studied in endogenously expressedeptors in non-transformed cells. We have isolated neuralgenitors which can be maintained in their proliferativete for multiple passages and differentiated by removal ofGF. These cells are commercially available (ENStem-A ,ipore). Here we demonstrate that under basalerentiation conditions these isolated hNP cells:

U

Up-regulate multiple Na and Ca channel subunits

TM

++ ++

p-regulate expression of genes consistent with a

diverse range of neural phenotypes

Up-regulate multiple glutamate receptor subunitsFunctional glutamatergic responses can be detected indifferentiated cells using a FLIPR Ca assay.

++

INTRODUCTION

vation of neural progenitors:

unohistochemistry:

+ Imaging:

Neuroprogenitors were isolated from H9 humanryonic stem cells as previously described (Shin et al., Stem Cells Dev. 2005,):266-9).These cells are commercially available as Enstem neural progenitorspore).

Cells were fixed in 2% paraformaldehyde and stained usingdard immunofluorescence protocols. Antibodies against the following proteins used: nestin (1:200, Neuromics), tuj-1 (1:500, Neuromics).

Cells differentiated for 2-4 weeks and plated into 96 well plates.ys were run on a flexstation 3

Culture:

time PCR:

Cells were cultured in neurobasal media supplemented withcillin/streptomycin, L-glutamine, B27, bFGF and LIF. Differentiation occurred afterval of bFGF.

(Molecular Devices) plate reader using a FLIPRum 4 assay kit (Molecular devices).

Real time PCR was run on an Applied Biosystems 7900HT system.e expression data (3 replications) were acquired and SDS software was used to

mate relative fold change values using Ct quantification method. GAPDH was as an endogenous control and neural progenitors in their proliferative state or hESwere used as a normalizer sample.

DD

METHODS

anniclls

7 DAYS 3 DAYS

derivationmedia

isolate fromneural rosettes onto

lamanin coated plates

remove feederlayer

maintain and characterizemultiple lines

stable adherentcharacterized

neural progenitors

7 DAYS MONTHS

j DIFFERENTIATION OFMULTIPLE NEURAL PHENOTYPES

synaptophysinKCC2

DATVAChT

HERG

SERTMAP2

b3-tubulin

RAB5ACD146KIR4.1

syntaxin 1A

0 10 20 30 40 50 60

125 days35 days14 days

fold change relative to NP

0 100 200 300 400 500

GAD1

peripherin

A

CONCLUSIONS

kCalcium Channel

Subunit Expression

-20

0 20 40

CACNG2

CACNB4

CACNB3

CACNB2

CACNB1

CACNA1S

CACNA1H

CACNA1C

CACNA1B

CACNA1A

80 100

NP cells

Sodium Channel

Subunit Expression

-100

-50

0 50 100

NAV 2.3

NAV 1.9

NAV 1.8

NAV 1.7

NAV 1.6

NAV 1.5

NAV 1.4

NAV 1.3

NAV 1.2

NAV 1.1

700

800

900

1000

differentiated

fold change relative tohESC expression

fold change relative tohESC expression

genename

genename

A BNP cellsdifferentiated

***

***

***

* *

***

**

**

**

** *

*

jm DEVELOPMENT OF FUNCTIONALGLUTAMATERGIC RESPONSES

A

EXPRESSION OF NA AND CACHANNEL SUBUNITS

++ ++

(A) Removal of bFGF for two weeks results in TUJ1 positive cells with a neuronalmorphology.

(B)

(C)

(D)100 M AMPA and kainic acid produce increased [Ca ] that are potentiated in the

presence of cyclothiazide (50 M) in cultures differentiated for 4 weeks. Nodetectable changes in were recorded with the addition of 100 M NMDA .

[Ca ] increased in response to glutamatergic agonists using a FLIPR assay.

Response to AMPA was potentiated by the AMPA potentiator cyclothiazide.Pooled data from 3 separate experiments run in triplicate demonstrate a does-response relationship of [Ca ] with addition of AMPA in the presence of cyclothiazide

(EC =4.5 M).

[Ca ]

++

++

++

I

I

50

i

++

i

(A) Real-time PCR demonstrated up-regulation of AMPA receptor subunits GRIA1,2 and4.

(B) Real-time PCR demonstrated up-regulation of Kainate receptor subunits GRIK2,4and 5.

(C) Real-time PCR demonstrated up-regulation of NMDA subunits GRIN1,2A and 2D.(D) RT-PCR with independent primers confirmed our real-time results (lane 1 are hNP,

lane 2 are cells differentiated for 2 weeks).Note: * denotes significantly different expression relative to hESCs (p