Ion Channel Expression Patterns and Functional Responses

in Human Embryonic Stem Cell

Derived Neural Cell Lines 1 2 2, 2 1,2
D.W. Machacek , S. K. Dhara , C. Sturkie , M.C. Dodla , S.L. Stice
1 2
ArunA Biomedical Inc , and University of Georgia

k
++ ++
EXPRESSION OF NA AND CA
INTRODUCTION CHANNEL SUBUNITS
Human embryonic stem cells and their progeny can provide a A Calcium Channel
Subunit Expression
B Sodium Channel
Subunit Expression
novel tissue source for understanding developmental CACNA1A
CACNA1B *
NP cells NAV 1.1 * NP cells
differentiated NAV 1.2
** differentiated
pathways,pharmaceutical screening and tissue replacement

gene name

gene name
CACNA1C * NAV 1.3 * *
NAV 1.4 *
CACNA1H
**
therapies. Drug screening using human embryonic stem cell CACNA1S
*
NAV 1.5
NAV 1.6
*
**
CACNB1 *
(hESC) derived tissue uniquely offers several advantages CACNB2
NAV 1.7 * *

over traditional cell lines: Large quantities of cells can be CACNB3

CACNB4
*
**
NAV 1.8
NAV 1.9
NAV 2.3
**
*
CONCLUSIONS
produced due to the proliferative nature of the cells, repeated CACNG2 *
ENStem-A™ neural

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studies can be done on a common genetic background and

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80

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10

70

80

90

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0

-1

-5

0
fold change relative to fold change relative to progenitors derived from
signaling can be studied in endogenously expressed hESC expression hESC expression
human embryonic stem
receptors in non-transformed cells. We have isolated neural (A) Real-time PCR results showed that differentiated cells had significant up-regulation cells are:
progenitors which can be maintained in their proliferative ++
of CACNA1B,1C and1H consistent with N-type, L-type and T-type Ca currents.
state for multiple passages and differentiated by removal of (B) Real-time PCR results showed that both the progenitor and differentiated cells
TM expressed multiple sodium channel subunits. NAV 1.3 showed the highest up- Stable for multiple
bFGF. These cells are commercially available (ENStem-A , regulation. NAV1.1 was up-regulated in the differentiated cells. passages, highly
Millipore). Here we demonstrate that under basal Note: * denotes significantly different expression relative to hESCs (p<.05)
proliferative and can be
differentiation conditions these isolated hNP cells: maintained in an adherent
A
EXPRESSION OF GLUTAMATE monolayer.
j Up-regulate expression of genes consistent with a RECEPTOR SUBUNITS
diverse range of neural phenotypes Differentiate into neural
k
++ ++
Up-regulate multiple Na and Ca channel subunits A AMPA receptor B Kainate receptor C NMDA receptor
cultures containing
l
subunit expression subunit expression subunit expression
Up-regulate multiple glutamate receptor subunits GRIA 1 GRIK 1 GRIN 1
* diverse cellular
m Functional glutamatergic responses can be detected in * GRIN 2A
NP cells
gene name

gene name

gene name
GRIK 2 * differentiated
phenotypes.
++ GRIA 2 * GRIN 2B
**
differentiated cells using a FLIPR Ca assay. GRIK 3 GRIN 2C

GRIA 3 GRIN 2D *
GRIK 4
* GRIN 3A mRNA expression is up-
GRIA 4 *
*
GRIK 5
* GRIN 3B
regulated for a variety of

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Na+, Ca++ channel

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METHODS fold change relative to

hESC expression
fold change relative to
hESC expression
fold change relative to
hESC expression
subunits and glutamate
D receptor subunits.
isolate from
derivation remove feeder neural rosettes onto
media layer lamanin coated plates
maintain and characterize
multiple lines
Differentiate into cells
7 DAYS 7 DAYS 3 DAYS MONTHS
which are functionally
H9 human
stable adherent
responsive to
embryonic
stem cells characterized
neural progenitors
glutamatergic agonists
(A) Real-time PCR demonstrated up-regulation of AMPA receptor subunits GRIA1,2 and
4.
(B) Real-time PCR demonstrated up-regulation of Kainate receptor subunits GRIK2,4 These cells represent a
and 5.
Derivation of neural progenitors: Neuroprogenitors were isolated from H9 human
(C) Real-time PCR demonstrated up-regulation of NMDA subunits GRIN1,2A and 2D.
novel tissue source for
embryonic stem cells as previously described (Shin et al., Stem Cells Dev. 2005,
14(3):266-9).These cells are commercially available as Enstem™ neural progenitors (D) RT-PCR with independent primers confirmed our real-time results (lane 1 are hNP, academic and commercial
(Millipore). lane 2 are cells differentiated for 2 weeks). researchers interested in
Note: * denotes significantly different expression relative to hESCs (p<.05)
Cell Culture: Cells were cultured in neurobasal media supplemented with
studying human neural
disease and development.
m
j
penicillin/streptomycin, L-glutamine, B27, bFGF and LIF. Differentiation occurred after
removal of bFGF. DEVELOPMENT OF FUNCTIONAL
Immunohistochemistry: Cells were fixed in 2% paraformaldehyde and stained using
GLUTAMATERGIC RESPONSES Funded by NIH-R41NS053272-01

standard immunofluorescence protocols. Antibodies against the following proteins used

were: nestin (1:200, Neuromics), tuj-1 (1:500, Neuromics). A B AMPA +cyclo
Ca 2+ response

8000 AMPA only

Ca++ Imaging: Cells differentiated for 2-4 weeks and plated into 96 well plates. buffer + cyclo
Assays were run on a flexstation 3 (Molecular Devices) plate reader using a FLIPR 6000 buffer only
AUC

calcium 4 assay kit (Molecular devices). 4000

2000
Real time PCR: Real time PCR was run on an Applied Biosystems 7900HT system.
Gene expression data (3 replications) were acquired and SDS software was used to 0
cntrl -7 -6 -5 -4 -3 -2
estimate relative fold change values using DDCt quantification method. GAPDH was log [compound] M
used as an endogenous control and neural progenitors in their proliferative state or hES
cells were used as a normalizer sample.

C D
j DIFFERENTIATION OF
100
Ca 2+ response
normalized AUC
Ca 2+ response

30000

MULTIPLE NEURAL PHENOTYPES

AUC

20000
50

fold change relative to NP 10000

A 0 100 200 300 400 500 0
cntrl -6 -5 -4 -3 0
GAD1
M A lo

ac ic lo

o
cy y

+ id

N lo
A
cl
in ka + c A
c

ic in yc
r + nl

id ac

c
D

log [AMPA]
P

cy
ffe r o

cy
PA M

peripherin
bu uffe

-50
+
A
b

D
M
A

0 10 20 30 40 50 60
N
ka

KCC2
synaptophysin
syntaxin 1A 14 days
35 days
(A) Removal of bFGF for two weeks results in TUJ1 positive cells with a neuronal
DAT 125 days morphology.
VAChT ++
HERG
(B) [Ca ]I increased in response to glutamatergic agonists using a FLIPR assay.
SERT Response to AMPA was potentiated by the AMPA potentiator cyclothiazide.
MAP2 (C) Pooled data from 3 separate experiments run in triplicate demonstrate a does-
b3-tubulin ++
response relationship of [Ca ]I with addition of AMPA in the presence of cyclothiazide
RAB5A
CD146 (EC50=4.5 µM).
KIR4.1 (D) 100 µM AMPA and kainic acid produce increased [Ca++]i that are potentiated in the
presence of cyclothiazide (50 µM) in cultures differentiated for 4 weeks. No
++
(A) Relative fold changes in mRNA expression comparing proliferative neural detectable changes in [Ca ]i were recorded with the addition of 100 µM NMDA .
progenitors to cells 14, 35 and 125 days following removal of bFGF. Genes shown
had a more than 5 fold up-regulation.