jo ur nal ho me page: www.elsev ier .com/ locate /neule t
fast and simple differentiation protocol to study the pro-neurogenicctivity of soluble factors in neurospheres
asmine Schramm, Dorothea Schulte ∗
nstitute of Neurology (Edinger Institute), University Hospital, Goethe University, D-60528 Frankfurt, Germany
i g h l i g h t s
Neuronal differentiation in SVZ neurospheres occurs within hours.Assay to measure differentiation largely independent of cell death and cell proliferation.Neuronal differentiation in SVZ neurospheres drops with increasing passage number.Inhibition of EGFR-, Erk1/2-, Akt-, and Shh-signaling induces neurogenesis.
r t i c l e i n f o
rticle history:eceived 11 November 2013ccepted 2 January 2014
eywords:eurosphere assayGF receptorrk1/2kthh
a b s t r a c t
Sphere-forming assays are widely used for the propagation, characterization and manipulation of adultbrain-derived stem- and progenitor cells. However despite the broad application of this cell culturesystem in neural stem cell- and brain tumor research, no standardized protocols exist. Variations inexperimental procedures not only concern the use of media components but also cell density, the numberof passages the cells are propagated before analysis and, in cases where the neurogenic or gliogenicpotential of the cells is investigated, the duration that the cells are allowed to differentiate. The latterdeserves consideration because the proportion of differentiated cells obtained at the endpoint of theexperiment depends not only on the absolute number of cells that differentiate at a given time, but alsoon the number of cell divisions prior to differentiation and the rate of cell death in the cultures. In the
present study we describe a fast and simple differentiation protocol to investigate the pro-neurogenicpotential of soluble factors added to subventricular zone (SVZ)-derived neurospheres. The assay relieson the use of primary neurospheres and very short differentiation times, thereby largely excluding thecontribution of cell proliferation and cell death to the results. We use this modified assay to test theconsequence of pharmacological inhibition of the EGF receptor-, Erk1/2-, Protein Kinase B/AKT-, andSonic Hedgehog-pathways on neuronal differentiation of SVZ-neurosphere cultures.
. Introduction
The subventricular/subependymal zone (SVZ/SEZ), a thin layerf cells located between the lateral walls of the lateral ventriclend the striatum, is a major stem cell niche in the uninjured, adultammalian brain. SVZ neural stem cells, also known as type B-cells
ccording to the nomenclature proposed by Doetsch and Alvarez-
uylla [1], exhibit astroglial characteristics and retain the capacityo proliferate and self-renew over extended periods. They pro-uce an intermediate population of fast dividing progenitor cells
(transient amplifying progenitors, ‘TAPs’ or type C-cells), whichcan differentiate into neurons, astrocytes and oligodendrocytes, thethree primary neural lineages in the mammalian brain [2,3].
An important step toward reaching general acceptance of theidea of ongoing neurogenesis in the adult mammalian brain wasthe discovery that cells from the adult rodent forebrain can be cul-tured over extended periods in a serum-free culture system [4].Under non-adherent conditions and in the presence of definedgrowth factors, which included epidermal growth factor (EGF),these cells form free-floating spheres of actively proliferating cells,termed neurospheres. In principle, the neurosphere assay requiresthe mechanical or enzymatic dissociation of the tissue of choiceinto a single cell suspension and plating them under non-adherent,
serum-free conditions. Although widely used, the results obtainedwith the neurosphere assay have, however, to be interpretedwith caution [5–7]. In particular, sphere numbers and sphere sizeobtained in the neurosphere assay have been used as indicators
or the presence of operationally defined stem cells in a given tis-ue sample. Yet neurospheres are rather heterogeneous structuresnd contain activated stem cells and rapidly proliferating progeni-ors (TAPs) as well as differentiated cells. Both, activated stem cellsnd TAPs, are EGF-responsive. In addition, cell density in the cul-ure critically affects sphere formation, because neurosphere cellselease soluble factors into the culture medium, which affect cellroliferation and survival in an autocrine or paracrine manner.nother complication arises from the fact that spheres are highlyotile in culture and tend to fuse, which obscures the intrinsic
phere-forming capacity of individual cells.Despite these shortcomings, the neurosphere assay proved itself
s straightforward and easy-to-carry-out cell culture system totudy the biology of stem- and progenitor cells in acutely dissoci-ted brain samples. A particular strength of the neurosphere assays that it may allow to separate different physiological parametershat affect neurogenesis in vivo. Specifically, the absolute number oferminally differentiated cells that are generated over a particularime depends not only on the ability of the respective progeni-or cells initiate differentiation programs, but also on the rate ofell proliferation prior to differentiation and cell survival. This isarticularly important for in vivo approaches, such as intracra-ial injection of viruses or the delivery of pharmacological agentsy micro-osmotic pumps, where the results are usually analyzedeveral days after the manipulation.
A simple way to separate differentiation–proliferation- andurvival-effects would be to shorten the time allowed for differ-ntiation to a minimum, which facilitates the onset of neuronalifferentiation in vitro while largely eliminating any contributionf cell division or cell death to the results. In the SVZ, acti-ated, proliferating stem cells (type B1-cells) divide with a cycle ofpproximately 17–18 h, whereas TAPs (type C-cells) divide every8-25 h [8]. These data correspond well to an estimated cell cycle
ength of 14 and 17 h for adult SVZ-derived stem-/progenitor cellultures growing in the presence of EGF and FGF2 [9]. By con-rast, only few studies have measured the time course of celleath in neurosphere cultures upon growth factor withdrawal.ne report observed activation of caspase-3 approximately 25 hfter growth factor withdrawal from free-floating neurosphere cul-ures obtained from the E14.5 mouse striatum [10]. Based on theseeports, we hypothesized that a differentiation time of well under0 h may be sufficient to quantify the onset of neuronal differenti-tion with minimal contribution of cell proliferation and cell deatho the results.
We here describe a variation of the commonly used neuro-phere assay protocol to separate the effect of pharmacologicalreatments on neuronal differentiation of adult SVZ derived stem-progenitor cells from any accompanying effects of these drugs onell proliferation and survival. The assay requires the use of primaryVZ-derived neurosphere cultures and a standardized treatmentrotocol. Under these conditions, differentiation times of few hoursre already sufficient to lead to robust and reproducible neuronalifferentiation. As a fist test of this assay, we show that inhibi-ion of the EGF receptor-, PKB/Akt-, Erk1/2- and Sonic Hedgehogathways leads to rapid induction of neuronal differentiation inVZ-neurosphere cultures in the presence of EGF and FGF2.
. Material and methods
.1. Neurosphere culture
Neurosphere cultures from the SVZ of 7–12-week old C57/BL6ice were prepared as described [11] and grown in the presence
f 2% v/v B-27 supplement (GIBCO), 10 ng/ml fibroblast growthactor-2 (FGF2, human recombinant; Peprotech, Rocky Hill, NJ,
ce Letters 562 (2014) 69–74
USA) and 20 ng/ml epidermal growth factor (EGF, human recom-binant; Peprotech) at 37 ◦C, 5% CO2. After 4 days, the neurosphereswere split in the presence of accutase and plated at a density of60,000 cells per cm2 on glass coverslips coated with 100 �g/mlpoly-d-lysine (in DPBS, 90 min at 37 ◦C; Sigma–Aldrich) followed by1 �g Laminine per cm2 (in DPBS; 90 min at 37 ◦C; Roche, Mannheim,Germany). After 16 h, different inhibitors were added as detailedbelow. For comparison of different passage neurospheres, sphereswere split every 4 days until the passages indicated. The firstspheres that formed in the cultures after tissue dissection were con-sidered ‘primary neurospheres’ or P1. ‘Tertiary’/P3 neurospheresaccordingly had been split twice and ‘quinary’/P5 neurospheresfour times prior to growth factor withdrawal and differentiation.Differentiation times varied between 4 h and 3 days.
2.2. Pharmacological inhibition of signaling pathways
Primary neurosphere cells growing over night as adher-ent monolayer on poly-d-lysine/Laminin coated coverslips weretreated with AG1478 (100 nM; LC- Laboratories, Woburn, MA; Cat#T-7310), AZD6244 (10 �M; Selleckchem, Munich, Germany; Cat#S1008), Akt Inhibitor VIII (2 �M; Merck Millipore, Billerica, MA;Cat# 124018), or Cyclopamine (10 �M; Calbiochem, San Diego, CA;Cat# 239803). Initially, different durations of treatment between2 h and 8 h and different inhibitor concentrations were tested.The minimal duration of treatment and minimal concentrationwas then used for further experiments. Cells treated with DMSO(vehicle) alone for identical times served as controls. All assayswere performed in triplicates. Standard deviation was calculatedbetween technical replicates, error bars represent S.E.M. Valueswere normalized to DMSO treated control cells obtained from thesame cell culture. Comparison between two groups was performedwith paired student’s t-test (Prism 5.01, Graph Pad). Statisticalsignificance was assumed when p < 0.05, indicated as *p < 0.05,**p < 0.01, ***p < 0.001.
2.3. Antibodies and immunhistochemical analysis
Cells were fixed in 2% PFA, washed three times in PBS andstained over night at 4 ◦C with a monoclonal antibody against neu-ronal �-tubulin, isotype III, (TuJ1; Covance, Princeton, NJ, USA)at 1:1000 in 5% v/v Chemiblock (Chemicon/Millipore BioscienceResearch Reagents, Billerica, MA, USA) and 0.5% Triton X-100(AppliChem, Gatersleben, Germany). Secondary antibody wasAlexa 488-conjugated anti-mouse (Dianova, Hamburg, Germany).Cells were counterstained with 4′-6-diamidino-2-phenylindole(DAPI) to visualize cell nuclei, embedded in Aqua Poly Mount (PolyScience, Inc.; Warrington, PA, USA) and analyzed with a 80i fluo-rescence microscope (Nikon, Tokyo, Japan).
3. Results
Adult stem- and progenitor cells are known to shift their charac-ter from neurogenic to gliogenic upon prolonged exposure to EGF[12,13]. To directly compare the proportion of neurons that could begenerated from a defined neurosphere culture upon extend cultureperiods, we serially passaged SVZ-derived neurosphere culturesfor up to five passages as free-floating spheres in the presence ofEGF and FGF2. From the primary (P1), tertiary (P3) and quinary(P5) spheres cells were dissociated and identical numbers of singlecells were plated in the absence of growth factors on poly-d-lysine/Laminine coated surfaces and allowed to differentiate for
three days. (Fig. 1A). A three day differentiation time was cho-sen, because after this time neuronal differentiation is occurringat a high rate (but is still far from complete), whereas cell death isstill rather low. Primary neurosphere cultures generated neuronal
J. Schramm, D. Schulte / Neuroscience Letters 562 (2014) 69–74 71
Fig. 1. SVZ-derived neurospheres rapidly lose their neurogenic potential in vitro. (A) Schematic outline of the experiment. Single cells prepared from the SVZ were propagateda ed cog oportc
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s free-floating neurospheres in the presence of EGF and FGF2. Spheres were passagenerate neurons upon growth factor withdrawal. See text for further details. (B) Prells after a 3-day differentiation period.
III tubulin-positive neurons at high frequency with an average of8.7% of all cells in the culture having adopted a neuronal fate by 3ays after growth factor withdrawal (Fig. 1B). By contrast, tertiaryeurosphere cultures contained only 6% �III tubulin-positive cellsfter the same differentiation period and the proportion of neuronsven dropped to 1% in P5 cultures (Fig. 1B). This result confirms thatVZ-derived neurospheres rapidly lose their neurogenic potentialn vitro.
.1. Primary but not tertiary neurospheres can be used toccurately determine the neurogenic potential of SVZeurospheres after very short differentiation times
As outlined above, the number of neurons that can be gener-ted from adult stem- and progenitor cells over a given period ofime depends not only on the cells’ abilities to adopt a neuronalate but also on the number of times they can divide before start-ng to differentiate (and hence on cell cycle kinetics) and theiresistance to cell death. Because the cell cycle length of activelyroliferating neurosphere cells and the time course of apoptosispon growth factor withdrawal represent over 14 h, we decided to
nvestigate if differentiation times of 8 h or less were sufficient tonduce neuronal differentiation in a robust, measurable and repro-ucible manner [8–10] (Fig. 2A). As a first step toward testing theeasibility of this approach, we blocked the EGF-receptor (EGFR)ignaling pathway by Tyrphostin AG1478 (N-(3-Chlorophenyl)-,7-dimethoxy-4-quinazolinamine). EGFR is expressed on activelyroliferating SVZ stem cells (type B1-cells) and TAPs (type C-cells)12]. In SVZ-derived stem- and progenitor cell cultures grown as
onolayers on Laminin in the presence of EGF and FGF2 and sup-lemented 0.1% v/v DMSO for 3 days as vehicle control, 1.92%±0.45%) of the cells spontaneously differentiated into neuronsFig. 2B). By contrast 3 days after EGF and FGF2 withdrawal, 28.7%±2.69%) of the cells were immunoreactive for neuronal �-III tubu-in. When the cultures were grown in the presence of EGF andGF2 but supplemented with 100 nM AG1478 (in DMSO) for 3ays, 17% (±2,2%) of the cells expressed �-III tubulin (Fig. 2B).lthough the net production of neurons was lower in AG1478-
reated cultures than after complete removal of EGF and FGF2,euronal differentiation was elevated under both conditions to atatistically significantly degree relative to the controls.
Next, we tested if a similar effect could already be observedfter 8 h of AG1478 treatment. The proportion of neurons in con-rol cultures that were kept in EGF/FGF2 containing culture mediumsupplemented 0.1% v/v DMSO for 8 h) was below 0.5%. Addition of
G1478 for 8 h to the cultures, however, significantly increased theroportion of �III-tubulin-positive cells (Fig. 2C). Motivated by our
nitial observation that adult neurosphere cultures lose their neuro-enic potential upon prolonged cultivation in EGF/FGF2-containing
nsecutively, cells of the first, third and fifth passage were scored for their ability toion of �III-tubulin-positive neurons generated from different passage neurosphere
medium, we tested if this effect could also be observed with neuro-sphere cultures of higher passage numbers. However, the increasein �III-tubulin expressing neurons in AG1478 treated tertiary (P3)cultures was much less pronounced than in P1 cultures and neverreached statistical significance (Fig. 3D). We therefore concludethat an 8-h differentiation protocol is in principle sufficient toassess the pro-neurogenic activity of soluble factors in vitro whenprimary neurospheres are been used in this assay.
3.2. The impact of Protein Kinase B/AKT-, Sonic Hedgehog-, andErk1/2-pathway inhibition on the onset of neuronal differentiation
We applied this protocol to the study of the Akt-, Shh- andErk1/2-pathways, signaling pathways that had previously beenimplicated in SVZ neurogenesis.
Akt, also known as protein kinase B, promotes cell prolifera-tion by modulating the function of multiple proteins involved incell cycle progression or apoptotic/anti-apoptotic signaling path-ways. In the context of neurogenesis, activation of Akt, throughconditional deletion of the tumor suppressor gene PTEN or throughstimulation by reactive oxygen species, enhanced neural stem cellself-renewal, which resulted medium term in a sustained increasein neurogenesis [14,15]. We blocked Akt signaling in primary neu-ropshere cultures by Akt Inhibitor VIII, a cell-permeable compoundthat selectively inhibits Akt1/Akt2 activity by a allosteric inhibition.Similar to the EGFR inhibitor AG1478, addition of Akt Inhibitor VIIIto P1 neurosphere cultures for 4 h lead to a significant increase inthe number of �III-tubulin expression neurons compared to vehicletreated controls (Fig. 3A and B).
Adult neural stem cells and transient amplifying progenitorcells respond to Sonic Hedgehog (Shh) signaling and Shh promotesself-renewal of adult SVZ stem-/progenitor cells in vivo and in neu-rosphere cultures in vitro and consequently leads to an increase inthe generation of neuroblasts [16,17]. Treatment of primary SVZneurospheres with the known Shh pathway inhibitor cyclopamineresulted in a significant increase in the number of neurons gener-ated within an 8-h treatment period compared to vehicle treatedcontrol cultures (Fig. 3A and C).
Extracellular signal-activated kinases, Erk1 and Erk2, are centralcomponents of several intracellular signaling pathways involvedin adult neurogenesis, such as the EGFR-pathway, nucleotidesignaling, or dopamine D3- and cannabinoid receptor (CB1R) acti-vation [18–21]. Chronic activation of Erk1/2-upstream pathwaysin the embryonic or adult brain stimulates neural progenitor cellproliferation and increased self-renewal of SVZ cells, which could
be attributed, at least in part, to enhanced transcript and proteinlevels of the cell cycle component cyclin D1 (ccnd1) [20,21]. Wetreated primary SVZ neurosphere cultures for 8 h with AZD6244(Selumetinib). Again, more �III-tubulin+ cells were observed in the
72 J. Schramm, D. Schulte / Neuroscience Letters 562 (2014) 69–74
Fig. 2. Primary but not tertiary neurospheres allow accurate determination of neuronal differentiation after very short differentiation times. (A) Schematic outline of theexperiment. (B) Percentage of neurons generated from P1 neurospheres in the presence of EGF and FGF2, 3 days after removal of both growth factors from the culturemedium, and 3 days after addition of AG1478 to EGF- and FGF2-containing culture medium. (C) Relative neuronal differentiation in P1 neurospheres growing in EGF/FGF2c rols. (E the r
Atarp
4
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ontaining medium after 8 h of AG1478 treatment compared to non-treated contGF/FGF2 containing culture medium in P1 and P3 neurosphere cultures, relative to
ZD6244-treated cultures compared to vehicle-treated cell cul-ures (Fig. 3A and D). Collectively, pharmacological inhibition ofll three intracellular signaling pathways markedly induced neu-onal differentiation of SVZ progenitor cells within a very short timeeriod.
. Discussion
Sphere-forming assays are widely used to assess the lineageommitment and differentiation potential of adult brain-derived
tem- and progenitor cells. Because the assays are particularlytraightforward, they are frequently combined with viral trans-uction to overexpress or knock-down particular gene productsr with the addition of pharmacological agents into the culture
D) Percentage of �III-tubulin expressing neurons 8 h after addition of AG1478 toespective, non-treated control cultures.
medium. Standard protocols to investigate the neurogenic or glio-genic potential of the cells after these treatments usually involvegrowth factor withdrawal followed by differentiation periods ofseveral days. During this time cells will exit the cell cycle at vari-ous times after the initial growth factor depletion. Consequently,the number of postmitotic cells that are generated during thedifferentiation period depends not only on the cells’ abilities toleave the cell cycle and differentiate but also on cell cycle kinet-ics and cell survival. To separate these parameters, we employed avariation of the classical differentiation protocol used in the neuro-
sphere assay. We show that, when primary neurosphere cells areused, a 4–8 h differentiation period is sufficient to induce neuronaldifferentiation in a reproducible and statistically significant man-ner. In addition, we provide evidence that inhibition of the EGFR-,
J. Schramm, D. Schulte / Neuroscience Letters 562 (2014) 69–74 73
F al diffi (B) Re
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ig. 3. Inhibition of the Akt-, Sonic Hedgehog-, and Erk1/2- pathways induce neuronn the presence of EGF/FGF2, supplemented with the indicated pathway inhibitors.
kt-, Erk1/2- and Shh-signaling pathways leads to a rapid induc-ion of neuronal differentiation in primary SVZ neurosphereultures.
As we show here, a small but substantial fraction of neurosphereells starts to express the neuronal marker protein �III-tubulinlmost immediately after growth factor removal from the cultureedium. It is tempting to hypothesize that these cells may already
ossess the complete molecular signature necessary for neuronalifferentiation, including transcript- and protein expression or epi-enetic modification of relevant genes. Growth factor removal mayllow for rapid progression toward neuronal differentiation andIII-tubulin expression in these cells, whereas the bulk of neu-
osphere cells still requires additional time to reach this state.reatment with the selective EGFR inhibitor AG1478 had a simi-ar neurogenesis-inducing effect as removal of EGF and FGF2 fromhe culture medium. This finding supports the notion that it is theGF signaling pathway that plays the primary role in preventingellular differentiation in neurosphere cultures. Interestingly, theroportion of cells that can initiate rapid neuronal differentiationrops when spheres of higher passages are used. This observation
s consistent with the known shift from a more neurogenic to aliogenic character of adult neurosphere cultures.
Rapid neuronal differentiation in primary neurosphere culturesas not only seen after inhibition of EGFR-signaling, but also after
nhibition of the Akt-, Shh and Erk1/2-pathways. Notably, theseignaling pathways have not only been implicated in SVZ neu-ogenesis before, but were also shown to induce proliferation ofdult or embryonic neural progenitor cells [14–21]. For instance,dding of Shh to SVZ cells prepared from P15 mice enhanced cellroliferation of neural progenitor cells, which ultimately led to an
ncrease in the production of young �III-tubulin+ neurons [17].ikewise, Erk1/2 influence cell cycle dynamics of cortical progenitorells by direct transcriptional control over ccnd1 and p27/Kip1, twoentral components of the G1/S checkpoint control [22]. Finally,kt promotes cell proliferation in many biological contexts includ-
ng embryonic cortical neurogenesis [23]. Interestingly, differentialegulation of cell cycle dynamics plays an important role in the reg-lation of cortical neurogenesis [24]. Specifically, shortening the1-phase though experimental overexpression of ccnd1 and cdk4,hich promote progression through G1 and entry into S-phase,
nhances progenitor cell proliferation and delays neurogenesis inhe embryonic cortex, whereas siRNA mediated knock-down ofcnd1/cdk4 had opposite effects [25]. The increase in neurogen-
sis after pharmacological inhibition of the EGFR-, Akt-, Shh-, andrk1/2-pathways we observed here may therefore be a direct con-equence of altered cell cycle dynamics in response to the drugreatments.
[
erentiation. (A) Relative frequency of �III-tubulin+ cells in P1 neurospheres growingpresentative examples of the cell cultures.
In summary, the results described here demonstrate that pri-mary neurosphere cells express neuronal markers as early as 4–8 hafter induction of cellular differentiation. Such short differentia-tion times may be useful when soluble factors are been screenedfor a potential pro-neurogenic activity on adult neural stem- andprogenitor cells.
Acknowledgments
The work was supported by the Deutsche Forschungsgemein-schaft SCHU1218/3-1 and the August Scheidel Foundation of theGoethe University Hospital Frankfurt.
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