Identification of Small-Molecule Modulators of Mouse SVZ Progenitor Cell Proliferation and Differentiation Through High-Throughput Screening
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Original Articles
Identification of Small-Molecule Modulators of Mouse SVZ
Progenitor Cell Proliferation and Differentiation Through
High-Throughput Screening
YAPING LIU,1 RAUL LACSON,1 JASON CASSADAY,1 DAVID A. ROSS,1,3 ANTHONY KREAMER,1
EDWARD HUDAK,1 RICHARD PELTIER,1 DONNA McLAREN,2,4 IGNACIO MUÑOZ-SANJUAN,2,5
FRANCESCA SANTINI,1 BERTA STRULOVICI,1,6 and MARC FERRER1
Adult mouse subventricular zone (SVZ) neural stem/progenitor cells are multipotent self-renewing cells that retain the capac-
ity to generate the major cell types of the central nervous system in vitro and in vivo. The relative ease of expanding SVZ
cells in culture as neurospheres makes them an ideal model for carrying out large-scale screening to identify compounds that
regulate neural progenitor cell proliferation and differentiation. The authors have developed an adenosine triphosphate–based
cell proliferation assay using adult SVZ cells to identify small molecules that activate or inhibit progenitor cell proliferation.
This assay was miniaturized to a 1536-well format for high-throughput screening (HTS) of >1 million small-molecule com-
pounds, and 325 and 581 compounds were confirmed as potential inducers of SVZ cell proliferation and differentiation,
respectively. A number of these compounds were identified as having a selective proliferative and differentiation effect on
SVZ cells versus mouse Neuro2a neuroblastoma cells. These compounds can potentially be useful pharmacological tools to
modulate resident stem cells and neurogenesis in the adult brain. This study represents a novel application of primary somatic
stem cells in the HTS of a large-scale compound library. (Journal of Biomolecular Screening 2009:319-329)
Key words: subventricular zone (SVZ), neural progenitor cells, proliferation, differentiation, CellTiter-Glo, high-throughput
screening (HTS)
INTRODUCTION cognitive performance.4,5 Thus, the expansion of endogenous
T
stem cells/progenitors in the brain may be beneficial in various
he vast majority of cells in the central nervous system CNS disorders where neuronal injury occurs (ischemic stroke,
are born during the embryonic and early postnatal period. Parkinson’s disease) or hippocampal performance is compro-
However, the adult mammalian brain retains neural stem cells for mised (Alzheimer disease).6 It would be valuable to identify
ongoing neurogenesis in 2 restricted regions: the subventricular small-molecule compounds with direct effect on neural progeni-
zone (SVZ) of the lateral ventricles and the dentate gyrus sub- tors through high-throughput screening (HTS) to gain proof of
granular zone of the hippocampus.1 Although the main function concept that stem cell expansion strategies will be therapeutically
of neurogenesis in normal brain neurophysiology and in disease useful.
progression is not well understood, there is strong evidence that Neural stem cells isolated from adult mouse SVZ can be
increased neurogenesis is associated with injury response2,3 and propagated in vitro.7 These cells have the capacity of self-renewal
and retain the multipotent potential to generate all major classes
of CNS cell types (neurons, astrocytes, and oligodendrocytes).8,9
1
Department of Automated Biotechnology, Merck & Co., North Wales, SVZ cell culture contains a heterogeneous population of cells,
Pennsylvania including multipotent stem cells, rapidly amplifying progenitors,
2
Department of Neuroscience, Merck & Co., Terlings Park, UK.
3
Present address: GlaxoSmithKline, Inc., Research Triangle Park, North
and a small number of lineage-committed cells such as astrocytes
Carolina. and immature neurons.9,10 They are maintained in suspension
4
Present address: Stem Cell Sciences UK Ltd, Cambridge, UK. as spherical floating clusters (neurospheres) in serum-free medium
5
Present address: CHDI Foundation, Inc., Los Angeles, California. with growth supplements and growth factors such as epidermal
6
Present address: iZumi Bio, Inc., Mountain View, California. growth factor (EGF) and fibroblast growth factor 2 (FGF2) to
Received Oct 6, 2008, and in revised form Dec 19, 2008. Accepted for enrich for stem/progenitor cells. The relative ease of culturing and
publication Dec 23, 2008. expansion of SVZ cells makes them ideally suited for large-scale
Journal of Biomolecular Screening 14(4); 2009 screening, but their heterogeneity also poses certain challenges
DOI:10.1177/1087057109332596 for HTS applications. To date, SVZ cells have been used only in
© 2009 Society for Biomolecular Sciences www.sbsonline.org 319
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the screening of a very small-scale compound collection (1267 containing penicillin/streptomycin, B27 (Invitrogen), and EGF/
compounds) to identify inhibitors of neurosphere proliferation.11 FGF2 at 20 ng/mL (PeproTech, Rocky Hill, NJ). The resulting
In this report, we describe the development of a 1536-well multipotent neurospheres were passaged every 3 to 4 days to
cell proliferation assay using adult mouse SVZ progenitor cells single-cell suspensions using Accutase (Chemicon, Danvers,
that are grown clonally in serum-free media in the presence MA) and plated in culture flasks at a density of 2 × 105 cells per
of mitogen FGF2. This assay has been used in the HTS of mL for continued growth and enrichment of stem cells. Aliquots
1.4 × 106 small-molecule compounds to identify compounds with of SVZ cells were frozen stored at passage 13 and thawed to
proliferative effect on neural progenitor cells (activators of SVZ restart culture before HTS. Cells were used for HTS between
proliferation). As cells in a proliferative state frequently promote passages 16 and 18.
the self-renewal capacity and prevent terminal differentiation,
these compounds could be useful in studying neural stem cell SVZ cell proliferation assay
self-renewing and modulating endogenous stem cells for CNS
repair. From the screening, inhibitors of proliferation were also The SVZ cell proliferation assay was developed in 96- and
identified as potential compounds that induce neural progenitor 384-well microplates and miniaturized to a 1536-well plate
cell differentiation. The ability of stem cells to differentiate is format. The assay protocol was as follows: SVZ neurospheres
often inversely linked to proliferation, and thus cells that are were dissociated to single cells (“preplating,” day 1) and
induced to terminally differentiate, in vitro or in vivo, typically cultured for 24 h to enrich for the progenitor cell pool. The
become quiescent and no longer proliferate. However, the enriched cells were then dissociated and resuspended in basal
compounds inhibiting SVZ cell proliferation might have other media (DMEM.F12.B27 media + 10 ng/mL FGF2) and plated
mechanisms of action that are not coupled to cell differentiation at clonal density (20 cells/µL) in 1536-well black solid-bottom
(weak cell cycle inhibitors, inhibition of cell aggregation required assay plates (Greiner, Monroe, NC) in a final volume of 7.5 µl/
for neurosphere proliferation, etc.), and further characterization well, followed by the addition of 10 nL of growth factors or
in neural cell differentiation assays will be needed to identify compounds and incubation at 37 °C in 5% CO2, minimal 95%
compounds inducing SVZ differentiation. Compounds that induce humidity for 48 h (day 2). On day 4, the total cell proliferation
SVZ differentiation can be useful pharmacological tools to gain of each well was measured using CellTiter-Glo Luminescent
insight in the regulation of neural stem cell differentiation and cell viability reagent (Promega, Madison, WI) according to the
cell fate. The compound hits generated from SVZ cell screening manufacturer’s protocol with minor modifications: assay plates
were followed up in confirmatory screens under 2 different were equilibrated to room temperature for 10 min, 2.5 µl of 4×
conditions (with or without FGF2): to characterize whether a CellTiter-Glo substrate was added in a final volume of 10 µl/
proliferation hit acted synergistically with FGF2 and to well and incubated at room temperature for 15 min, and then
distinguish differentiation-inducing compounds from cytotoxic the luminescent signal was measured on a Viewlux reader
compounds. The confirmed compounds were then counter (PerkinElmer, Waltham, MA) with an integration time of 3 s
screened in a proliferation assay using mouse Neuro2a per plate.
neuroblastoma cells, to characterize hits selectively active on
SVZ progenitor cells but not on a non-precursor neuronal cell SVZ cells HTS
line. These hits were also followed up in a SVZ cell dose-
response assay and proliferation imaging assay for neurosphere Small-molecule HTS was carried out on the fully automated
formation. A number of compounds were identified that regulate Kalypsys robotic platform (Kalypsys, San Diego, CA) in a
SVZ cell proliferation/differentiation with high potency and 1536-well format using the assay protocol described above.
potentially could be useful in future studies in modulating SVZ cells and CellTiter-Glo reagents were dispensed using
resident stem cells and neurogenesis in the adult brain. Kalypsys Bottlevalve dispensers, and 10 nL of 2 mM test
compounds in DMSO were added to the assay plate using a
MATERIALS AND METHODS Kalypsys Pin Tool, giving the final screening concentration at
2.6 µM. In the screening plates, the sample field was located in
SVZ cell culture columns 5 to 44 (1280 compounds per plate), whereas columns
2-3 and 46-47 contained negative and positive controls (basal:
Mouse SVZ neural stem cell cultures were prepared using DMEM.F12.B27 media + 10 ng/mL FGF2 + 10 nL DMSO;
the method previously described by Johansson et al.12 Briefly, maximal response: DMEM.F12.B27 media + 20 ng/mL EGF +
the subventricle zone was dissected from adult C57Bl6/J mice 20 ng/mL FGF2; positive 1: DMEM.F12.B27 media + 10 ng/
after cervical dislocation and enzymatically and mechanically mL FGF2 + 300 nM pituitary adenylate cyclase activating
dissociated. Cell suspensions were purified by sucrose gradient polypeptide [PACAP]; positive 2: DMEM.F12.B27 media + 10
centrifugation and plated in suspension in Dulbecco’s modified ng/mL FGF2 + 10 µM forskolin). The wells on the plate edges
Eagle’s medium (DMEM)/F12 1:1 (Invitrogen, Carlsbad, CA) surrounding the controls were not used. The median value of
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the basal control and the maximal response control wells were imaging plates (Corning) in 2000 cells/50 µl/well, followed by
calculated to determine assay window: signal/noise (S/N) = the addition of 200 nL of growth factors or screening compo
median RLU (maximal response control wells)/median RLU unds (final concentration 2.56 µM) and incubation at 37 °C in
(basal wells). The percentage activation value (%Activation) 5% CO2, 95% humidity for 72 h. Cells were then fixed in 4%
of a sample was defined to calculate the percentage change paraformaldehyde and 1:1000 Hoechst 33342 stain for 15 min
of cell viability from the basal control: %Activation = 100 * at room temperature, washed 3 times with Tris-buffered saline
[RLU sample – median RLU (basal wells)]/[median RLU (TBS) buffer, and imaged on an iCyte imaging cytometer
(basal wells)]. (CompuCyte, Cambridge, MA) to assess the number and size
of formed neurospheres. We gated neurospheres into 4 subpop
Confirmatory screens in the presence and absence of FGF2 ulations (single cells, small object, medium object, and large
object) defined by nuclear area and calculated the number of
Small-molecule compounds selected from the primary neurospheres in each category.
screen were retested in triplicate using the same protocol as
used in the primary HTS in 2 different culture conditions: RESULTS AND DISCUSSION
DMEM.F12.B27 media supplemented with 10 ng/mL of FGF2
(+FGF2 mode) and DMEM.F12.B27 media without FGF2 SVZ cell culture for HTS
(–FGF2 mode). To minimize edge effect, we reformatted
compound plates to leave the 4 edge rows and columns empty A major challenge of using primary stem cells in the HTS of
and not populated with compounds. Similar to primary screen, a large-scale compound collection is to prepare a large quantity
the %Activation value was defined to calculate the percentage of stem cells in vitro and maintain them in a consistent stem cell
change of cell viability relative to the basal conditions, with the stereotype. SVZ cells isolated from adult mouse subventricular
basal controls in +FGF2 and –FGF2 modes corresponding to zone contained a heterogeneous population of cells, including
the control wells cultured in the respective assay media. multipotent stem cells, rapidly amplifying progenitor cells, and a
small number of lineage committed cells.9 When they were
Neuro2a cell counter screening maintained in suspension as neurospheres in serum-free medium
with growth supplements and growth factors such as EGF and
Mouse Neuro2a cells cultured in DMEM with 10% fetal FGF2, the neural stem cell population was enriched to retain its
bovine serum (FBS, Invitrogen) were dissociated and plated in self-renewal capacity and the potential to differentiate into
384-well plates at 500 cells/40 µL/well cell density in serum- multiple cell lineages of the CNS.7 Such properties of SVZ stem/
free media (– serum mode) or DMEM media containing 2% progenitor cells may change over time due to different culture
FBS (2% FBS mode). After 24 h, 200 nL of 2-mM compounds conditions and cell passages. To have a large quantity and
was added using the Cybi-Disk liquid handler, followed by consistent supply of SVZ cells for the HTS of ~1.4 million small
incubation at 37 °C in 5% CO2, minimal 95% humidity for 72 h. molecules, we prepared a large number of frozen SVZ cell aliq-
After the assay plates were equilibrated to room temperature uots at passage 13 by storing them in liquid nitrogen. Before
for 10 min, 40 µL of 2× CellTiter-Glo reagent was added and commencing HTS, 2 to 3 vials were thawed to prepare a fresh
incubated at room temperature for 15 min. Finally, the culture. The newly thawed SVZ cells took approximately 7 to 10
luminescent signal was measured with a ViewLux reader using days for full recovery and acceleration of the proliferation rate,
an integration time = 30 s per plate. In the screening plates, the during which time the cells were passaged 1 to 2 times, with
following controls were included as positive and negative media changes between passages. After recovery, the cells were
controls for monitoring assay performance and calculating passaged every 3 to 4 days to enrich for neural stem cells. All
sample activity: basal (– serum mode = DMEM media + 200 nL independent thaws of SVZ cell cultures grew consistently and
DMSO; 2% FBS mode = DMEM media + 2% FBS + 200 nL formed spherical, free-floating neurospheres between passages
DMSO), retinoic acid (final concentration at 2.6 and 20 µM), 13 and 18. On average, a T225 culture flask with 100 mL SVZ
and FBS (final 5% FBS). The %Activation calculation was def cells, seeded at 2 × 105 cells/mL, yielded 8 × 107 to 1.2 × 108 cells
ined similarly as the SVZ cell proliferation assay: %Activation= after 3 days in culture. After passage 19, the SVZ cells started to
100 * [RLU sample – median RLU (basal wells)]/[median RLU adopt an increasingly nonspherical appearance with adherence to
(basal wells)]. the culture flask and yielded a significantly lower number of
cells per culture. These are characteristic signs of SVZ cell
SVZ neurosphere formation imaging assay differentiation and therefore not usable for the HTS proliferation
assay. We thus limited the SVZ culture passage number to
SVZ cells preplated on day 1 were dissociated and maximum passage 18.
resuspended on day 2 in basal media (DMEM.F12.B27 media + We also observed that the number of days before preplating
10 ng/mL FGF2) and plated in 384-well black clear-bottom directly affected cell yield and assay performance; 3 days of
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A
Run CellTiter Glo
Dissociate Dissociate and plate cells proliferation assay
neurospheres add growth factors/test compounds
No ATP
Proliferation
+ Luciferin +Mg2+ + Luciferase
Proliferation
ATP Luminescence
20 cells /ul +EGF & FGF2
clonal density
Day1 Day2 Day4
48hr
Kalypsys Robot
B C
6 1400
5 1200
Relative Proliferation
1000
4
800
RLU
3
600
2
400
1 200
0 0
–12 –11 –10 –9 –8 –7 –6
Media FGF2 EGF+FGF2 PACAP Forskolin Log[EGF], g/ml
Growth Condition
FIG. 1. Mouse subventricular zone (SVZ) cell proliferation assay for small-molecule high-throughput screening. (A) Assay outline. (B)
Proliferation of SVZ cells measured by CellTiter-Glo assay in growth conditions indicated on the x-axis: media = DMEM.F12.B27 without
growth factors; fibroblast growth factor 2 (FGF2) = media + 10 ng/mL FGF2; epidermal growth factor (EGF) + FGF2 = media + 20 ng/mL
EGF + 20 ng/mL FGF2; pituitary adenylate cyclase activating polypeptide (PACAP) = media + 10 ng/mL FGF2 + 300 nM PACAP; forskolin =
media + 10 ng/mL FGF2 + 10 µM forskolin. The relative proliferation is shown compared to the basal control (FGF2), which is set to 1. Standard
deviation is shown by the error bars (n = 4). (C) Dose-response curve of EGF. EGF was diluted in basal control media (10 ng/ml FGF2). The
x-axis shows increasing EGF concentration versus raw fluorescent signal on the y-axis.
culture prior to preplating yielded an average of 1.5 × 107 cells clonal density (≤20 cells/µL)14 in 1536-well assay plates. After
per preplated T225 flask, compared to only ~7 × 106 cells per a 48-h culture in the presence of maximal growth factors (EGF/
T225 flask for 4 days of culture. In addition, cells cultured for FGF2 at 20 ng/mL) or compounds, the total cell viability was
3 days before preplating produced higher raw CellTiter-Glo measured using the CellTiter-Glo luminescent cell viability
signals and an EGF dose-response curve with smaller variation assay. The CellTiter-Glo assay determines cell viability
of replicate data points (data not shown). The cell passage based on quantitation of cellular adenosine triphosphate (ATP),
routine was thus maintained as a 3-day growth period to indicative of the presence of metabolically active cells. The cell
maintain a consistent supply of cells for HTS campaign. number per well correlates directly to the ATP content of the
cells and thereby to the luminescence output. Cell density at 20
Development of SVZ cell proliferation assay cells/µL produced a stable S/N assay window of ~5 (measured
by maximal growth condition vs. basal condition) and about
SVZ cell proliferation assay was first developed in 94- and 2-fold growth potentiation with positive control compounds
384-well microplates, then miniaturized to 1536-well plate PACAP or forskolin (Fig. 1B). EGF showed a robust dose-
format. The workflow of the assay is outlined in Figure 1A. response curve with an EC50 value of 4.2 × 10–10 g/mL (Fig.
SVZ cells were dissociated to single cells (preplating) and 1C). Because cells on the surface of a neurosphere react
cultured for 24 h to enrich for the progenitor cell pool.13 The differently to growth factors (and compounds) compared to
preplated cells were dissociated and resuspended as single cells cells in the center of neurosphere, and these cells have a
in basal medium containing 10 ng/mL of FGF2 and plated in potential to aggregate, it was necessary to run this assay using
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a very low cell density.14 Cell titration experiments also the screen and remained within acceptable screening parameters.
indicated that at cell densities greater than 40 cells/µL, the The median Z′ factor for the entire screen was 0.47. Assay
growth potentiation induced by weak control compounds such plates with a CV above 30% or a Z′ factor less than 0.3 were
as PACAP and forskolin were significantly reduced, whereas rescreened. The entire screening campaign was completed in 8
the S/N window was maintained at 5 (data not shown). Also, days with a reschedule rate of ~2%.
the EC50 value of the EGF titration curve increased almost Figure 3A shows a sample scatterplot of a representative
10-fold when cell density was increased from 20 to 40 cells/µL, plate from the screen, with compounds tested at 2.6 µM. Most of
making the assay less sensitive (data not shown). the compounds on the plate had no significant effect on SVZ cell
To screen ~1.4 million compounds in a time- and cost- proliferation and clustered at around 0% activation, with very
efficient fashion, we needed to accomplish the project in a few compounds having activation values higher than the average
1536-well format. The main challenges for conducting lengthy samples. To maximize the probability of identifying all positive
assays in the miniaturized format are that of keeping cells alive hits that induce SVZ cell proliferation, we used 2 hit selection
for several days in a small volume of medium and keeping the strategies: (1) hits significantly above the baseline of a given
edge effects caused by the severe evaporation along the edges plate were selected based on a 3-sigma cutoff of each assay plate
of the high-density, small-volume assay plates to minimum (local selection) and (2) hits selected based on a global 2-sigma
level. This assay was particularly sensitive because the cell hit cutoff of the entire screen (approximately 50% activation). In
density was limited to 20 cells/µL (i.e., 150 cells/well in 7.5 µL total, 781 compounds were selected using local selection metric,
volume) and assayed in a suboptimal culture condition (basal and the cutoff value of individual plates varied from 44% to 81%
media without EGF). The challenge was to maintain cells at a activation. In addition, 4930 compounds were selected by the
very low density while having enough cells to counteract edge latter selection metric, of which 727 compounds overlapped with
effects. To minimize evaporation, we modified the CellTiter- hits identified by the first metric. After combining the 2 hit lists,
Glo assay to maximize the amount of cell culture media 4984 compounds were identified as primary screening hits that
volume in each well. Therefore, the assay was conducted with increase SVZ proliferation. To select compounds that potentially
7.5 µL of cells and 2.5 µL of 4× CellTiter-Glo reagent, a induce differentiation, we selected hits inhibiting cell prolifer
4-fold dilution instead of the recommended 2-fold. Using this ation using a global cutoff value of between –50% and -80%
basic protocol and optimizing incubator temperature and activation. This is based on the assumption that compounds inducing
humidity controls, assay plate type, and incubation length, the differentiation also block cell proliferation and thus cause reduced
final assay window was acceptable for ultra-HTS (uHTS). This viability but not cell death. A –80% activation cutoff was included
assay had an S/N of 5.2, a Z′ factor of 0.52, and a coefficient of to minimize the number of potentially toxic compounds. After
variation (CV) of 16.1%. removing compounds with unfavored structures, 5000 compounds
SVZ cells from passages 14 to 18 were tested for S/N assay were selected as potential differentiation hits for subsequent
window and EGF dose-response EC50 value to determine the confirmatory screens.
best cell passages for the screen. Cells at P16 and P17 produced
a slightly better S/N window than P14 and P18 cells, with SVZ cell confirmatory screens
similar EC50 value for EGF dose response (Fig. 2A, B). We thus
designed a cell culture plan to produce SVZ cells at passages The combined ~10K proliferation and differentiation hits
16 to 17 for the entire HTS screen. Using Kalypsys online cell from the primary screen were retested in triplicate, using 2
suspension stirring device and dispenser, we also confirmed different assay conditions: in the presence of 10 ng/mL of
that the cells were stable for up to 5.5 h after cell dissociation FGF2 (+FGF2 mode, same as primary screen) and in the
(Fig. 2C), and an EGF control stored in a 1536-well plate absence of FGF2 (–FGF2 mode). This was done to characterize
yielded a stable assay window up to 7.5 h at room temperature whether a proliferation hit acted synergistically with FGF2 and
(Fig. 2D). to distinguish whether hits inducing differentiation had toxic
effects. The confirmatory screens were performed on Kalypsys
HTS for SVZ cell proliferation/differentiation modulators platform using the same protocol as in the primary screen. To
minimize the evaporation effects on the edges of the plates, we
A total of approximately 1.4 million compounds were used a special plate map with no samples populating the 2 rows
screened on the Kalypsys automated robotic platform using the of edge wells. Figure 4 shows scatterplots of representative
SVZ proliferation assay. The assay was robust during the plates from the confirmatory screen in the +FGF2 condition
screen, with a steady S/N window of ~4.5 and an average CV and –FGF2 condition. PACAP control increased SVZ cell
of ~25% (Fig. 3B). The relatively high CV was, in part, caused proliferation in the presence of FGF2 but had no effect on cell
by the very small number of cells (150 cells/mL) used in this viability in the absence of FGF2, indicating that PACAP works
assay (in suboptimal culture condition) and the presence of in synergy with FGF.
significant numbers of toxic compounds in the screening A total of 325 compounds with >50% activation above basal
collection. The assay performed consistently over the course of condition in the +FGF2 screen were selected as confirmed
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A B
1750
P 14 6
1500
P 16
1250 5
P 18
1000 4
RLU
S/N
750 3
500 2
250 1
0 0
−12 −11 −10 −9 −8 −7 −6 P14 P16 P18
EGF(g/ml) Cell Passage Number
C D
6 6
5 5
4 4
S/N
S/N
3 3
2 2
1 1
0 0
T-0 T-2.5 hr T-4.5 hr T-5.5 hr T-0 T-2.5 hr T-4.5 hr T-6.5 hr T-7.5 hr
Time in Suspension Time EGF control plates were made
FIG. 2. Optimization of subventricular zone (SVZ) proliferation assay. (A) Epidermal growth factor (EGF) dose-response curves at different
SVZ cell passages. EGF was diluted in basal control media (DMEM.F12.B27 + 10 ng/mL fibroblast growth factor 2 [FGF2]). The x-axis shows
increasing EGF concentration versus raw fluorescent signal on the y-axis. The EC50 value of EGF at cell passages 14, 16, and 18 was 0.23, 0.38,
and 0.43 ng/mL, respectively. (B) Assay window (signal to noise [S/N]) of cells at passages 14, 16, and 18. (C) Stability of SVZ cells on Kalypsys
robot. SVZ cells (passage 16) were kept in a stir flask on a Kalypsys dispenser and assayed for up to 5.5 h. Assay window (S/N) is shown on
the y-axis. (D) Stability of the EGF control plate on the Kalypsys robot. EGF control plates were made at 0, 2.5, 4.5, 6.5, and 7.5 h before being
transferred to assay plates using the Kalypsys pin tool. Assay window (S/N) is shown on the y-axis.
proliferation hits. Of these compounds, only 8 compounds also Mouse Neuro2a cell counter screening for progenitor
produced >50% activation in the –FGF2 screen, suggesting that selective modulators
most compounds worked synergistically with FGF2 (Fig. 4A,
B). Using the same %Activation cutoff value defined in primary To characterize whether a compound had selective prolifer
HTS, we were able to confirm the activity of ~1200 differentiation ation or differentiation activity on neural stem/progenitor cells,
hits with –80% to –50% activation in the +FGF2 screen (Fig. we developed a cell proliferation assay in mouse neuroblastoma
4C). To filter compounds with nonspecific cytotoxicity, we set cells (Neuro2a), a transformed neuronal cell line, as a counter
the cutoff value to > –50% activation in the –FGF2 screen, screen of all confirmed SVZ hits. Any compounds inducing
assuming that compounds inducing SVZ differentiation in the cell proliferation or differentiation in SVZ cells, but not in
presence of FGF2 are not inherently toxic and therefore should Neuro2a cells, will likely be selective for neural stem/progenitor
not decrease cell viability in the absence of FGF2 (Fig. 4D). In cells. We developed a cell viability assay to measure Neuro2a
total, 581 compounds satisfied both criteria and were chosen for cell proliferation in a 384-well format. Initial assay results using
further studies. 500 cells/well in a 384-well plate and 72-h incubation time
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A B
PACAP control EGF control
600 6 50
S/N %CV
500 45
5
400 40
4
% Activation
300 35
Hit ?
%CV
S/N
200 3 30
100 25
2
0 20
1
–100 15
–200 0 10
1 7 13 19 25 31 37 43 1 11 21 31 41 51 61 71 81 91 101111121131141
Plate Column Number Plate Number
FIG. 3. Screening for compounds inducing subventricular zone (SVZ) cell proliferation. (A) Scatterplot of a sample plate from primary high-
throughput screening (HTS). %Activation of samples in 1536-well plate are shown along the plate column number. Each dot represents a well
in the 1536-well plate. The sample marked by the circle represents a possible hit that increases SVZ cell proliferation and was selected for con-
firmatory screening. (B) HTS statistics. Signal to noise (S/N) of the maximal growth condition (DMEM.F12.B27 + 20 ng/mL epidermal growth
factor [EGF] + 20 ng/mL fibroblast growth factor 2 [FGF2]) compared to the basal condition (DMEM.F12.B27 + 10 ng/mL FGF2) is shown as
a blue circle across 145 plates in the primary screen (partial screen). S/N values are indicated on the left y-axis. The coefficients of variation
(CVs) of the sample fields across assay plates are shown as red triangles. The values are shown on the right y-axis.
A SVZ + FGF2 B SVZ- FGF2
PACAP
200 200
150 150
%Activation
%Activation
100 100
50 50
0 0
–50 –50
–100 –100
1 5 9 13 17 21 25 29 33 37 41 45 1 5 9 13 17 21 25 29 33 37 41 45
Plate Column Number Plate Column Number
PACAP
C D
150 150
100 100
%Activation
%Activation
50 50
0 0
–50 –50
–100 –100
1 5 9 13 17 21 25 29 33 37 41 45 1 5 9 13 17 21 25 29 33 37 41 45
Plate Column Number Plate Column Number
FIG. 4. Scatterplots of representative compound plates from confirmatory screens in the (A, C) + fibroblast growth factor 2 (FGF2) condition
and (B, D) –FGF2 condition. The graphs were scaled to show sample distributions (epidermal growth factor [EGF] and FGF2 controls were not
shown in the graphs due to much higher percent activity, and the pituitary adenylate cyclase activating polypeptide (PACAP) control was not
active in the –FGF2 condition). Panels A and B represent a compound plate enriched in proliferation compounds selected from primary screening.
The horizontal red lines indicate a cutoff value used for hit selections. Panels C and D represent a compound plate enriched in differentiation
compounds from primary screening. Compounds inducing differentiation are presumed to block cell proliferation and thus cause reduced viabil-
ity in the presence of FGF2; these are indicated by the red boxed area in panel C. The same compounds were tested in the absence of FGF2 and
did not decrease viability, which means they are not inherently toxic; these are found in the area indicated in red (D). Differentiator compounds
chosen for follow-up were found in both these regions.
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A B
150 20
0
100
% Activation
% Activation
–20
50
–40
0
–60
–50 –80
–100 –100
COMPOUND ID COMPOUND ID
FIG. 5. Data distribution of confirmed subventricular zone (SVZ) proliferation/differentiation hits in Neuro2a cell counter screens. (A) %Activation
values of 325 confirmed SVZ proliferation compounds in a Neuro2a proliferation assay carried out in a serum-free condition. Highlighted in the gray
area are hits that are negative in the Neuro2a assay and potentially selective for SVZ. (B) Data distribution of 581 SVZ differentiation hits in the Neuro2a
counter screen in the presence of 2% fetal bovine serum (FBS). Highlighted in gray area are hits that are potential selective differentiators for SVZ.
produced a robust window (signal to background [S/B] ~3.2, SVZ cell dose-response assay
media with 10% FBS vs. serum-free media). The S/B window
increased to 4.4 if Neuro2a cells were plated in serum-free After the primary and confirmatory screens, the hits available
medium and cultured for 24 h prior to the addition of compounds/ at a high stock concentration (257 proliferation hits and 491
controls. We believe the 24-h serum starvation period helps differentiation hits) were tested in the SVZ cell proliferation
synchronize the cell population, making them more responsive assay to determine the 50% effective concentration (EC50).
to compound treatment. This serum-free condition was chosen These compounds were prepared as 4-fold serial dilutions in
for the screening of proliferation compounds. 1536-well plates and tested in triplicate to generate 8-point
To test for the effects of potential SVZ cell differentiators in dose-response curves. The curves were fitted using PRISM
mouse Neuro2a cells, we tested varying concentrations of FBS software (GraphPad, San Diego, CA) to determine EC50 values.
(from 1%-5%) with 20 µM of retinoid acid (a known compound Many compounds gave good response curves and exhibited
that induces differentiation in Neuro2a) as a control compound. EC50 values in the 0.01 to 1 µM range, showing a variety of Hill
Higher concentrations of FBS resulted in lower level of inhibition slopes and maximum activation values. Two representative sets
of cell growth from retinoid acid (data not shown). Reduction of of dose-response curves from proliferation and differentiation
the cell proliferation mediated by the retinoid acid control compounds with increased or decreased cell viability are
compound was found to be optimal with 2% FBS in cell culture shown in Figure 6. As indicated in Figure 6A, compound 1
media (~75% decrease in proliferation) and was chosen as the had a very clear dose-response curve with EC50 = 0.35 µM and
screening condition for assessing the potential differentiation maximum activation value at ~100% (highest level of activation
compounds. In addition, the + 2% FBS condition also provided in screened compounds), close to the maximum level of
useful information on proliferation hits (whether serum is required activation observed with the control PACAP compound. No
for their proliferation effect) and was used for counter screening compounds were identified giving a response similar to the
of both proliferation and differentiation compounds. EGF control in SVZ cells.
From the 325 confirmed proliferation hits tested in Neuro2a
proliferation assay, only 42 compounds showed higher than Image assay of SVZ neurosphere formation
50% activation in serum-free condition (Fig. 5A), and no
compounds had greater than 40% activation in the +2% FBS To further verify that the compounds identified from the ATP-
condition. The 42 hits may represent compounds acting to based viability assays affect SVZ cell proliferation and induce
stimulate cell proliferation generically and are not SVZ stem neurosphere formation, we performed an image-based assay
cell selective. For the 581 differentiation compounds, only 76 using SVZ cells in 384-well plates. The iCyte scanning cytometer
hits hadSVZ Progenitor Cell Proliferation and Differentiation
A B
130 20
Compd 1
110 0
90 –20
%Activation
%Activation
70 –40
50 –60
30 –80
10 –100
–10 –120
–10 –9 –8 –7 –6 –5 –4 –10 –9 –8 –7 –6 –5 –4
Log[M] Log[M]
FIG. 6. Dose-response curves of selected compound hits in the subventricular zone (SVZ) proliferation assay. (A) Proliferation compounds
from high-throughput screening (HTS). (B) Differentiation compounds from HTS. Compounds were prepared in 1:4 serial dilutions in DMSO
from 10-mM stocks and tested in the SVZ proliferation assay using same protocol as primary HTS. The x-axis shows increasing compound
concentration versus activation values on the y-axis. Data shown on the graphs are the average of triplicate points.
FIG. 7. Examples images of subventricular zone (SVZ) neurosphere cultures. (A) SVZ cells cultured in media alone. (B) SVZ cells cultured in
media supplemented with 10 ng/mL fibroblast growth factor 2 (FGF2; i.e., basal media). (C) SVZ cells cultured in media containing 20 ng/mL
FGF2 and 20 ng/mL epidermal growth factor (EGF). (D) SVZ cells cultured in basal media supplemented with 300 nM pituitary adenylate cyclase
activating polypeptide (PACAP). (E-G) SVZ cells cultured in basal media supplemented with representative compounds inducing SVZ prolif-
eration. (H) SVZ cells cultured in basal media supplemented with a hit compound inducing SVZ differentiation. Scale bar = 100 µm.
software, we analyzed the number and size of neurospheres compounds showed a significant increase of neurosphere formation.
formed in the presence of test compounds. We screened the 257 Example images of a few compounds found to increase SVZ cell
proliferation hits and 491 differentiation hits in the imaging proliferation and neurosphere formation are shown in Figure 7,
assay to assess their affect on SVZ neurosphere formation. Forty along with images of control compounds and a compound
Journal of Biomolecular Screening 14(4); 2009 www.sbsonline.org 327
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JBSc332596.indd 327 3/18/2009 12:25:48 PMLiu et al.
A C
1 2 4 7 325
0.1 3 1
“Proliferator”
Primary HTS Hits Confirmed Hits
4,984 325
N2a Negative Hits
283
Compd 1
B SVZ +FGF SVZ–FGF N2a-serum N2a+serum
“Differentiator” Screen Condition
Primary HTS Hits Confirmed Hits
5,000 581
N2a Negative Hits
505
FIG. 8. Summary of compound hits identified from SVZ progenitor cell screen and follow-up screens. (A) Compound hits inducing SVZ cell
proliferation were selected from primary screen and funneled down in confirmatory screens. The resulting 325 hits were then characterized in a
Neuro2a cell proliferation assay, dose-response assay, and neurosphere formation imaging assay. (B) Compound hits inducing SVZ cell differ-
entiation were selected from the primary screen and funneled down in confirmatory screens. The resulting 581 hits were characterized in a
Neuro2a cell proliferation assay, dose-response assay, and neurosphere formation image assay. (C) Cluster analysis of 325 proliferation com-
pounds based on their activities in 4 different screen assays/conditions. Compounds with similar activity profiles are clustered together. Marked
compound 1 had the highest activation of SVZ proliferation in CellTiter-Glo and neurosphere formation image assays.
inducing SVZ differentiation (Fig. 7H). The strongest proli including a mouse Neuro2a cell proliferation assay and a SVZ
feration hit was again compound 1, increasing the size of the neurosphere formation imaging assay, were performed to
cell clusters (or neurospheres) relative to the basal condition characterize these confirmed hits (Fig. 8A,B). Compounds with
by 2-fold. Further analysis is ongoing to characterize the number a preferred activity profile could be identified by cluster analysis
and size of neurospheres of all screened compounds. We are also of various assay results, as shown in Figure 8C. We have
conducting cell lineage analysis using specific markers to identified a number of compounds inducing SVZ cell proliferation
characterize compounds that might drive differentiation of SVZ or differentiation with high potency. Further characterization of
cells into various cell types such as neurons, astrocytes, or these compounds could provide great insight into the mechanism
oligodendrocytes. involved in the regulation of neural stem cells. They could
In conclusion, we have developed a robust cell proliferation potentially be useful in future studies modulating resident stem
assay and applied it in the screening of >1.4 million small cells and neurogenesis in the adult brain. This work represents a
molecules to identify compounds regulating SVZ progenitor cell novel application of primary somatic stem cells in the HTS of a
proliferation and differentiation. Several follow-up assays, large-scale compound library.
328 www.sbsonline.org Journal of Biomolecular Screening 14(4); 2009
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JBSc332596.indd 328 3/18/2009 12:25:49 PMSVZ Progenitor Cell Proliferation and Differentiation
References 10. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A:
Subventricular zone astrocytes are neural stem cells in the adult mam-
1. Alvarez-Buylla A, Lim DA: For the long run: Maintaining germinal malian brain. Cell 1999;97:703-716.
niches in the adult brain. Neuron 2004;41:683-686. 11. Diamandis P, Wildenhain J, Clarke ID, Sacher AG, Graham J, Bellows
2. Magavi SS, Leavitt BR, Macklis JD: Induction of neurogenesis in the DS, et al: Chemical genetics reveals a complex functional ground state of
neocortex of adult mice. Nature 2000;405:951-955. neural stem cells. Nat Chem Biol 2007;3:268-273.
3. Zhang RL, Zhang ZG, Zhang CL, Zhang L, Robin A, Wang Y, et al: 12. Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J:
Stroke transiently increases subventricular zone cell division from asym- Identification of a neural stem cell in the adult mammalian central nerv-
metric to symmetric and increases neuronal differentiation in the adult ous system. Cell 1999;96:25-34.
rat. J Neurosci 2004;24:5810-5815. 13. Bonnert TP, Bilsland JG, Guest PC, Heavens R, McLaren D, Dale C,
4. Kempermann G, Wiskott L, Gage FH: Functional significance of adult et al: Molecular characterization of adult mouse subventricular zone
neurogenesis. Curr Opin Neurobiol 2004;14:186-191. progenitor cells during the onset of differentiation. Eur J Neurosci 2006;
5. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, et al: 24:661-675.
Requirement of hippocampal neurogenesis for the behavioral effects of 14. Engstrom CM, Demers D, Dooner M, McAuliffe C, Benoit BO,
antidepressants. Science 2003;301:805-809. Stencel K, et al: A method for clonal analysis of epidermal growth factor-
6. Lie DC, Song HJ, Colamarino SA, Ming GL, Gage FH: Neurogenesis in responsive neural progenitors. J Neurosci Methods 2002;117:111-121.
the adult brain: new strategies for central nervous system diseases. Annu
Rev Pharmacol Toxicol 2004;44:399-421.
7. Reynolds BA, Weiss S: Generation of neurons and astrocytes from iso- Address correspondence to:
lated cells of the adult mammalian central-nervous-system. Science 1992; Yaping Liu
255:1707-1710. Merck Research Labs
8. Mckay R: Stem cells in the central nervous system. Science 1997; Department of Automated Biotechnology
276:66-71. 502 Louise Lane, NW-1
9. Reynolds BA, Weiss S: Clonal and population analyses demonstrate that North Wales, PA 19454
an EGF-responsive mammalian embryonic CNS precursor is a stem cell.
Dev Biol 1996;175:1-13. E-mail: yaping_liu@merck.com
Journal of Biomolecular Screening 14(4); 2009 www.sbsonline.org 329
Downloaded from jbx.sagepub.com by guest on August 17, 2015
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