Identification of Small-Molecule Modulators of Mouse SVZ Progenitor Cell Proliferation and Differentiation Through High-Throughput Screening

Original Articles

                  Identification of Small-Molecule Modulators of Mouse SVZ
                   Progenitor Cell Proliferation and Differentiation Through
                                  High-Throughput Screening

                                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

                                                                                               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
           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,
           Department of Neuroscience, Merck & Co., Terlings Park, UK.
           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
           Present address: Stem Cell Sciences UK Ltd, Cambridge, UK.                          as spherical floating clusters (neurospheres) in serum-free medium
           Present address: CHDI Foundation, Inc., Los Angeles, California.                    with growth supplements and growth factors such as epidermal
           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                                                                                           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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SVZ Progenitor Cell Proliferation and Differentiation

         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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                                                                                                                                                               Run CellTiter Glo
                        Dissociate                                       Dissociate and plate cells                                                           proliferation assay
                      neurospheres                                  add growth factors/test compounds
                                                                                                                                         No                        ATP
                                                                                                                                                              + Luciferin +Mg2+ + Luciferase

                                                                                                                                                                   ATP               Luminescence
                                                                                                    20 cells /ul                  +EGF & FGF2
                                                                                                  clonal density

                                                   Day1              Day2                                                                                                     Day4

                                                                                                               Kalypsys Robot

                  B                                                                                                                 C
                                               6                                                                                  1400

                                               5                                                                                  1200
                      Relative Proliferation



                                               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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SVZ Progenitor Cell Proliferation and Differentiation

         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

         Journal of Biomolecular Screening 14(4); 2009                                                                                   323

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                       A                                                                                               B
                                                                                        P 14                        6
                                                                                        P 16
                            1250                                                                                    5
                                                                                        P 18
                            1000                                                                                    4

                             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

                                      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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SVZ Progenitor Cell Proliferation and Differentiation

                                        A                                                                                         B
                                     PACAP control                                             EGF control

                                        600                                                                                  6                                                  50
                                                                                                                                                      S/N    %CV
                                        500                                                                                                                                     45
                                        400                                                                                                                                     40
                      % Activation

                                        300                                                                                                                                     35
                                                              Hit ?

                                        200                                                                                  3                                                  30
                                        100                                                                                                                                     25
                                            0                                                                                                                                   20
                                     –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
                                      200                                                                                    200
                                           150                                                                               150

                                           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
                                         C                                                                                      D
                                        150                                                                                  150

                                        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

         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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Liu et al.

                                              A                                                                  B

                                             150                                                                 20


                                                                                                % Activation
                             % Activation


                                             –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 had
SVZ Progenitor Cell Proliferation and Differentiation

                           A                                                                                     B
                                     130                                                                          20
                                                   Compd 1
                                     110                                                                           0
                                         90                                                                      –20

                                         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                                                                                          327

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Liu et al.

                      A                                                                                    C
                                                                                               1       2   4   7   325

                                                                                               0.1 3                 1


                          Primary HTS Hits   Confirmed Hits
                               4,984               325

                                             N2a Negative Hits

                                                                                                                                                                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

         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.

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