Neural stem cells (NSCs) are pluripotent cells that give rise to neurons, astrocytes and oligodendrocytes in the nervous system. They hold promise for treatment of brain and spinal injuries and diseases. However, very little is known about their regulatory mechanisms. Here we used Next-Generation Sequencing (NGS) to define the temporal transcriptome signatures of NSCs. Cultured human embryonic stem cells (H9) were compared to induced NSCs at days 0, 7 and 14. Total RNAs were extracted and Ion AmpliSeq™ Transcriptome libraries were created for sequencing. The expression profiles of the same dataset were also evaluated by Whole transcriptome (WT) RNA-Seq and Affymetrix Gene 2.0 ST arrays. The transcriptome profiles of H9 cells differed little between days 0, 7, and 14 while NSCs induced from H9 cells showed remarkable differences from day 0 to days 7 and 14 during differentiation. Hierarchical clustering results also showed more robust sample classification for NSCs than H9 cells. Comparing the expression profiles of NSCs versus H9 cells, a total of 4001 and 4768 were differentially expressed at day 7 and day 14 respectively (p-value < 0.01, fold change > 2) . We further clustered their expressions into 24 groups by Self-organizing map. A total of ~250 genes showed similar expression patterns to known NSC markers including SOX1 and PAX6. These genes are enriched for neural differentiation related pathways and are potential candidates for novel NSC markers. Their expressions will be further verified by TaqMan® gene expression assays. In summary, we used NGS to construct a temporal transcriptome database of H9 cells and NSCs. We also developed an analysis pipeline to systematically identify potential novel NSC markers.

1. Yuan-Chieh Ku1, Yongming Sun1, Scott Dewell1, Colin Davidson1, David Piper2, and Stephen Jackson1
1Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080, 2Carlsbad, CA
Identification of biomarkers for neural differentiation with Ion AmpliSeq™ Transcriptome solution
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
ABSTRACT
Neural stem cells (NSCs) are pluripotent cells
that give rise to neurons, astrocytes and
oligodendrocytes in the nervous system. They
hold promise for treatment of brain and spinal
injuries and diseases. However, very little is
known about their regulatory mechanisms.
Here we used Next-Generation Sequencing
(NGS) to define the temporal transcriptome
signatures of NSCs. Cultured human
embryonic stem cells (H9) were compared to
induced NSCs at days 0, 7 and 14. Total RNAs
were extracted and Ion AmpliSeq™
Transcriptome libraries were created for
sequencing. The expression profiles of the
same dataset were also evaluated by Whole
transcriptome (WT) RNA-Seq and Affymetrix
Gene 2.0 ST arrays. The transcriptome profiles
of H9 cells differed little between days 0, 7, and
14 while NSCs induced from H9 cells showed
remarkable differences from day 0 to days 7
and 14 during differentiation. Hierarchical
clustering results also showed more robust
sample classification for NSCs than H9 cells.
Comparing the expression profiles of NSCs
versus H9 cells, a total of 4001 and 4768 were
differentially expressed at day 7 and day 14
respectively (p-value < 0.01, fold change > 2) .
We further clustered their expressions into 24
groups by Self-organizing map. A total of ~250
genes showed similar expression patterns to
known NSC markers including SOX1 and
PAX6. These genes are enriched for neural
differentiation related pathways and are
potential candidates for novel NSC markers.
Their expressions will be further verified by
TaqMan® gene expression assays. In
summary, we used NGS to construct a
temporal transcriptome database of H9 cells
and NSCs. We also developed an analysis
pipeline to systematically identify potential
novel NSC markers.
INTRODUCTION
Neural stem cells (NSCs) derived from
pluripotent stem cells hold the promise of
delivering therapies and drug screening tools in
the future. Current methods to characterize
differentiated cells rely on established cellular
markers and are not sufficient to discriminate
between many possible neural cell types. To
define the temporal transcriptional signature of
NSCs, cultured human embryonic stem cells
(H9) were compared to induced NSCs at d0, d7
and d14 (Figure 1). The expression profiles
were determined from whole transcriptome
RNA-Seq, and Ion AmpliSeq™ Transcriptome
Sequencing on the Ion ProtonTM platform. It was
also measured by Affymetrix Gene 2.0 ST
arrays. We are further evaluating our results
against known markers and developing new
molecular signatures for assay and product
development.
Figure 1. Experimental time course
Day 14Day 7Day 0
H9
H9 H9
NSC NSC
Oct4 (POU5F1) SSEA4 Nestin Sox1 Pax6
Pluripotency Neural differentiation
RESULTS
Figure 2. Workflow
Total RNA was purified using the RiboPure™ kit. WT
RNA-Seq samples were prepared from 1µg total RNA
with the Ambion® ERCC RNA Spike-In control.
Samples were ribodepleted with the RiboMinus™
Eukaryote System v2 and Proton libraries were
prepared with the AB Library Builder™ System.
AmpliSeq™ Transcriptome samples were prepared from
10ng of total RNA. Total RNA was first reverse
transcribed to cDNA with SuperScript® VILOTM cDNA
Synthesis Kit and used as template to amplify targets
with Ion AmpliSeq™ Transcriptome Human Gene
Expression Kit. Libraries were built following
AmpliSeq™ library preparation protocol. Multiplexed
samples were sequenced on the Ion Proton™ System.
Affymetrix Gene 2.0 ST array samples were prepared
following manufacture’s protocol.
Figure 5. Gene ontology analysis
Gene ontology (GO) analysis of differentially expressed genes
(p-value < 0.01, fold change > 2) between NSC versus H9 at
day 7. The 10 most enriched GO annotations and the numbers
of genes within each annotation are listed. Red highlights GO
annotations related to neural differentiation. (A) Ion AmpliSeq™
Transcriptome, (B) WT RNA-Seq and (C) Affymetrix Gene 2.0
ST arrays.
B
A
Figure 3. Sample Clusters
Samples are clustered based on
expression levels of
(A)Ion AmpliSeq™ Transcriptome
(B) WT RNA-Seq and
(C) Affymetrix Gene 2.0 ST arrays.
CONCLUSIONS
1. Ion AmpliSeq™ Transcriptome and WT RNA-Seq
results are highly correlated and show significantly
higher detection sensitivity of differentially
expressed genes than array data.
2. Ion AmpliSeq™ Transcriptome recapitulates
enriched biological processes and identifies
potential novel markers during neural stem cell
differentiation.
3. Additional work is in progress to narrow the targets
for further evaluation with other technologies, such
as TaqMan® gene expression assays.
Figure 6. Self-organizing map analysis
Five comparisons (H9 d7/H9 d0, NSC d7/H9 d0, NSC d7/ H9 d7,
NSC d14/ H9 d14 and NSC d14/ NSC d7) were filtered (p-value <
0.01, fold change > 8) and used as input for self-organizing map.
These genes were grouped into 24 clusters according to their
expression patterns. Clusters included known NSC markers or
shown similar patterns were highlighted in red.
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo
Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of
Roche Molecular Systems, Inc., used under permission and license.
Figure 4. Fold change comparisons among
Ion AmpliSeq™ Transcriptome, WT RNA-Seq
and Affymetrix Gene 2.0 ST arrays.
Fold change of NSC versus H9 at d7 was calculated.
Differentially expressed genes (FDR <= 0.05) of Ion
AmpliSeq™ Transcriptome were compared to WT
RNA-Seq and Affymetrix Gene 2.0 ST arrays. (A) Ion
AmpliSeq™ Transcriptome versus WT RNA-Seq. The
correlation is 0.94. (B) Ion AmpliSeq™ Transcriptome
versus Affymetrix Gene 2.0 ST arrays. The correlation
is 0.82.
C
0 50 100 150 200
developmental protein
neuron differentiation
cell projection morphogenesis
neuron projection morphogenesis
neurogenesis
neuron development
axonogenesis
cell morphogenesis involved in neuron differentiation
neuron projection development
cell part morphogenesis
Number of Genes
CellularProcesses
A
0 50 100 150 200
developmental protein
neuron differentiation
cell projection morphogenesis
cell part morphogenesis
cell morphogenesis involved in differentiation
cell morphogenesis involved in neuron differentiation
neuron development
neuron projection morphogenesis
cell projection organization
neurogenesis
Number of Genes
CellularProcesses
B
0 50 100 150 200
neuron differentiation
pattern specification process
cell-cell adhesion
cell migration
embryonic morphogenesis
regulation of neurogenesis
sensory organ development
neuron projection
kidney development
protein tyrosine kinase activity
Number of Genes
CellularProcesses
C
Day 14Day 7Day 0
H9
H9 H9
NSC NSC
1
2
3 4
5
A B