Download - Co-Action Publishing JOURNALS

Commitment of human pluripotent stem cells to a neural lineage is
induced by the pro-estrogenic flavonoid apigenin
Cleide S. Souza1*, Bruna S. Paulsen1*, Sylvie Devalle1, Silvia Lima Costa2, Helena L. Borges1 and
Stevens K. Rehen1,3
SUPPLEMENTAL DATA
METHODS
Pluripotent stem cells culture
H9 human embryonic stem cells (Wicell) (number of passages 44-50) and two human iPS
cell lines were used in this study (named here as iPS1 and iPS2, number of passages 33-39)
(Paulsen et al., 2012). Both H9 and iPS cells were cultured under feeder-free culture conditions
on matrigel-coated dishes in mTeSR™1 (STEMCELL Technologies). Passaging was performed
mechanically by splitting colonies in clumps every 5-6 days, cells were collected by gently
pipetting, and were re-plated on matrigel-coated dishes having their medium exchanged every
other day. All cells were maintained at 37°C in humidified air with 5% CO2.
Neural progenitor cells (NPCs)
NPCs were obtained after 18 days in culture using a previously described protocol with
slight modifications (49, 50). In this approach, retinoic acid was replaced by API in order to
better identify its effects in the early stages of differentiation. Differentiation efficiency was
comparable between API and RA treatments (Figure S7). When the colonies were almost
confluent, cells were exposed for 6 days to 10 μM API (Sigma) in DMEM/F12 medium
supplemented with 5% knockout serum replacement (KSR; Invitrogen), 2 mM L-glutamine
1
(Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1% nonessential amino acid solution
(Invitrogen), and 40 μg/mL gentamicin sulfate (Schering-Plough). Then, cells were cultured for
6 days in the same medium without API, followed by another 6 days of culture in the same
medium supplemented with 25 ng/mL of fibroblast growth factor (FGF)-2 (Invitrogen). During
the last 6-day period, neural rosettes were formed.
For neuronal differentiation, neural rosettes were morphologically identified in the NPC
culture and manually detached from the plate. They were replated in polyornithine/laminincoated dishes and allowed to proliferate and migrate for 8 additional days in Neurobasal
medium supplemented with 1× N2 (Invitrogen), 1× B27 (Invitrogen), 20 ng/mL FGF-2, and 40 μ
g/mL gentamicin sulfate. Afterward, 2×105 cells/cm2were plated in poly-ornithine/laminincoated dishes and cells were cultured for an additional 25 days in the same medium without
FGF-2 (Figure S1A). All experiments were performed in parallel with untreated controls in which
the cells were subjected to the same protocol, but only treated with vehicle (DMSO).
ER and RAR Antagonists Treatments during NPC Production
hES cells were treated with specific ER and RAR antagonists starting 2 hours before API
treatment through 6 days (during API treatment). Control cells were treated with DMSO. Over
the following 12 days, NPC were cultivated as previously described.
Antagonists (from Tocris) used in this study were: 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride; 10 nM), 4-[2Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; 1 µM), 4-[5-[8-(1Methylethyl)-4-phenyl-2-quinolinyl]-1H-pyrrolo-2-benzoic acid (ER 50891; 10 µM), 4-(7,8,9,10Tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic
acid (LE 135; 10 µM), 6-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3dithiolan-2-yl]-2-naphthalenecarboxylic acid (MM 11253; 10 µM) and 3-[4-Hydroxy-3-[5,6,7,8tetrahydro-5,5,8,8-tetramethyl-3-(pentyloxy)-2-naphthalenyl]phenyl]-2-propenoic
acid
(UV
3003 10 µM).
Immunostaining
2
For immunostaining, cells were fixed in 4% paraformaldehyde in phosphate-buffered
saline (PBS) for 15 min. Cells were then washed with PBS and incubated with 0.3% Triton X-100
in PBS (Sigma) for 5 min. Following, PBS containing 5% bovine serum albumin (BSA) (Invitrogen)
was added for 30 min. After blocking, samples were incubated with primary antibodies
overnight. Cells were washed with PBS three times. Then, secondary antibodies were added to
cells and incubated for 1 hour. The cells were washed with PBS three more times and incubated
with 1.0 μg/mL 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Staining was
visualized on a confocal microscope (TCS-SP5). Images were captured with either a 20×
objective or a 63× oil immersion objective.
The following primary antibodies were used at the indicated dilutions: anti-Sox2 (mouse,
1:100; Chemicon, AB5603), anti-NESTIN (mouse, 1:100; Chemicon, MAB5326), anti-RARA
(rabbit, 1:100; Santa Cruz Biotechnology, SC-551) anti-β-tubulin III (mouse, 1:100; Chemicon,
MAB1637), anti-MAP2 (chicken, 1:500; Abcam, ab5392), anti-PSA-NCAM (mouse, 1:100;
Chemicon, MAB5324), anti-synapsin I (rabbit, 1:200; Chemicon, AB1543), anti-neurofilament
200 (mouse, 1:400; Sigma, N0142), anti-MBP (rabbit, 1:100; Sigma, M3821), anti-choline
acetyltransferase (CHAT) (rabbit, 1:200; Chemicon, AB143), anti-GAD1 (mouse, 1:100;
Chemicon, MAB5406), anti-calretinin (rabbit, 1:200; Chemicon, AB5054), anti-parvalbumin
(rabbit, 1:100; Chemicon, AB15736), anti-tau (mouse, 1:200; Chemicon), anti-DLG4 (rabbit,
1:100; Chemicon, 04-1066), anti-synaptophysin (mouse, 1:100; Chemicon, MAB368). The
following secondary antibodies were used at the indicated dilutions: Alexa Fluor 594-conjugated
goat anti-mouse IgG (1:400; Molecular Probes A11032), Alexa Fluor 633-conjugated goat antichicken IgG (1:400; Molecular Probe, A21103), Alexa Fluor 488-conjugated goat anti-rabbit IgG
conjugated (1:400; Molecular Probe, A11008), Alexa Fluor 488-conjugated goat anti-mouse IgG
conjugated (1:400; Molecular Probe, A11001), Alexa Fluor 594-conjugated goat anti-rabbit IgG
(1:400; Molecular ProbesA11037).
Western blotting
Total protein was extracted with RIPA buffer (1 mM Na 3VO4, 0.25% sodium
deoxycholate, 1% NP-40, 150 mM NaCl, Tris-HCl pH 7.4, and 2 mM EDTA,) supplemented with a
3
protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Samples were then centrifuged at
1200 rpm for 10 minutes, the supernatants were recovered and protein concentration was
measured using the Bradford method. Thirty micrograms of protein per lane were
eletrophoretically separated on a 12% polyacrylamide gel. After separation, proteins were
transferred into a polyvinylidenedifluoride (PVDF) membrane (Hybond, Amersham, Piscataway,
USA) in a semi-dry system (Bio-Rad, Hercules, USA) for 120 min at a constant current of 0.15
mA.
Membranes were blocked with 5% nonfat milk in tris-buffered saline containing 0.05%
Tween-20 (TBS-T) for 1 hour at room temperature under agitation. After blocking, membranes
were incubated with primary antibodies for either RARB (rabbit 1:1000, Santa Cruz
Biotechnology, SC-552), RARG1 (rabbit 1:2000, Thermo, PA3-813), RXRA (rabbit 1:1000, Santa
Cruz Biotechnology, SC-553), RXRB (mouse 1:2000, Thermo, MA3-812), RXRG (rabbit 1:1000,
Santa Cruz Biotechnology, SC-555), DLG4/PSD95 (rabbit 1:1000, Chemicon, 04-1066),
Synaptophysin (mouse 1:1000, Chemicon, MAB368), or α-tubulin (mouse 1:5000 Santa Cruz
Biotechnology, SC-23948) overnight at 4°C. Afterward, membranes were washed with TBS-T
three times under agitation at room temperature and then incubated for 1 hour at room
temperature under agitation with goat anti-rabbit peroxidase-conjugated (1:5000; Molecular
probes, G21234) or goat anti-mouse peroxidase-conjugated (1:5000; Molecular probes,
G21040) secondary antibodies diluted in 5% nonfat milk in TBS-T. Membranes were then
washed three times under agitation in TBS-T. Blots were developed using Amersham ECL Prime
(GE). After protein detection, densitometric analyses were performed using ImageJ software.
The values of each protein were normalized to the α-tubulin amount in the same lane.
Cell Counts
After antagonist treatments, the percentage of NESTIN-positive cells were calculated as
follows: cells were manually counted from microscopic images that were captured using a 63×
oil-immersion objective on a NIKON T300 connected to a confocal microscope (TCS-SP5) using
LASAS software (v. 2.4.1). NESTIN-positive cells and the total number of cells stained with DAPI
4
were counted in at least 10 fields-of-view per group. At least 3 independent experiments were
performed per group.
Puncta Analysis
To analyze the effects of API on synapse formation, hES cells were differentiated into
neurons [19]. Briefly, hES cells were cultured in the presence of 4×10-6M retinoic acid (RA,
Sigma) for 6 days, and then kept for additional 6 days without RA, followed by 6 more days in
medium supplemented with 25 ng/mL of FGF-2. Neural rosettes were morphologically identified
in the NPC cultures and manually detached from the plate. Those structures were re-plated in
polyornithine/laminin-coated dishes allowing NPCs to proliferate and migrate for 8 additional
days in Neurobasal medium supplemented with 1× N2 (Invitrogen), 1× B27 (Invitrogen), 20
ng/mL FGF-2 (Invitrogen) and 40 μg/mL gentamicin sulfate. After that, 2×105 cells/cm2were
plated in poly-ornithine/laminin-coated dishes and then allowed to remain in culture for up to
30 days. After a total of 51 days in culture, neurons were treated with 1 μM API for 72 hours
(Figure S1E), after which immunocytochemistry for DLG4 and SYP was performed. Control cells
were treated with DMSO (drug vehicle). After capturing 10 images per experimental group using
a 63× oil-immersion objective (1.5 zoom) on a NIKON T300 connected to confocal microscope
(TCS-SP5), green and red channels were aligned and neurites of the similar sizes were selected
and quantified using the Puncta Analyzer plug-in for ImageJ. Experiments were performed in
triplicate.
RNA Isolation and PCR Analysis
Total RNA was isolated using Trizol reagent (Invitrogen), and subjected to DNase
treatment using DNA-freeTM Kit (Applied Biosystems) following manufacturer’s instructions.
Complementary DNA was generated from 1 μg total RNA using the High Capacity RNA-to-cDNA
Kit (Applied Biosystems) according to manufacturer recommendations. Reverse transcription
(RT)-PCR was performed using the following primers listed in Table S1. Each RT-PCR reaction
was carried out for 35 cycles in a reaction mixture containing 1 U Taq DNA Polymerase (NB
Science), 1× Taq DNA Polymerase Buffer containing 25 mM MgCl2 (NB Science), 500 nM of each
5
primer (forward and reverse), 200 μMdNTP mixture containing the four deoxyribonucleotides
(dATP, dCTP, DTTP, dGTP), and 1 μL of cDNA in a total reaction volume of 20 μL.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 5 and validated with Student's
t-tests or One-way analysis of variance (ANOVA) when more than 2 groups were compared.
Confidence intervals were defined at 95% confidence level (p< 0.05 was considered to be
statistically significant). Fold change was calculated by dividing the average (mean) value of the
experimental group to that of the control group, in all figures, error bars represent SEM, of at
least 3 independent experiments.
Figure S1: Neural differentiation of human pluripotent stem cells induced by API
(A) Schematic view of the protocol used for the differentiation of human pluripotent stem cells
by API. Nearly confluent hES cells cultured in mTeSR™1 medium (d0) were exposed to 10 μM
API in DMEM/F12 with 5% KSR for 6 days. Then, cells were cultivated in the same medium
without API for 6 days, followed by culture in medium supplemented with 25 ng/mL FGF-2 for
an additional 6 days (total of 18 days in culture). Neural rosettes were manually replated on
polyornithine/laminin-coated dishes and were allowed to proliferate and migrate for 8
additional days in Neurobasal medium supplemented with N2/B27 and 20 ng/mL FGF-2. After
that, 2×105 cells/cm2 were plated on coated dishes and cells differentiated for extra 25 days in
the same medium without FGF-2. (B) Representative images of colonies of hES cells at the prior
to the API treatment. (C) Morphology of cells after 18 days in culture. (D) Representative images
of cells after 51 days in culture, when neural processes could be observed. (E) Experimental
approach used to evaluate synaptogenesis. After 51 days in culture, neurons were treated with
1 μM API for 72 hours, when analysis of synaptogenesis was performed. Scale bars = 50 µm.
6
Figure S2: API enhances expression of human neural precursor cell markers in iPS cells.
Representative immunofluorescence images identifying neural precursor cell markers NES
(green, A, B, D and E) counterstained with DAPI (blue). Two human iPS cells treated with 10 μM
API for the 6 initial days in culture showed intense immunostaining for NES (B). Untreated cells
(cells without API) displayed few staining for NES (A). Scale bar = 50 μm. The values are
expressed as the mean ± SEM; n = 3
Figure S3: API does not alter proliferation but decreases apoptosis in hES cells.
Photomicrographs of cells in culture for 18 days. Cells, either untreated or treated with API,
were immunostained for phospho-histone H3 (PH3, red, A-B) and counterstained with DAPI
(blue). (C) Quantification of the percentage of PH3-positive cells. Immunofluorescence for
cleaved caspase-3 (CASP3, green) showed that API decreases the amount of apoptotic cells (E)
when compared to untreated cells (D). (F) Quantification of the percentage of cells positive for
cleaved caspase-3. Results are plotted as the mean ± SEM of 3 independent experiments. Scale
bar = 50 m.
Figure S4: ERs and RARs activation is required for API-induced differentiation into neural
progenitors.
Representative immunofluorescence images staining for NES (green) and counterstained with
DAPI (blue). hES cells were treated with 10 μM API for the 6 initial days in culture in the
presence of ER or RAR antagonists applied 2 h prior to API treatment. The antagonists used
7
were MPP (ESR1) and PHTPP (ESR2). API-induced neural differentiation was inhibited by ER
antagonists (A). API-induced neural differentiation was inhibited by RAR antagonists (B).
Figure S5: API induces morphological changes in hES cells.
Representative bright-field and DAPI images showing that hES cells treated with 10 μM API for
the 6 initial days of culture present neural tube-like structures (arrows, B and D) after 18 days.
In contrast, untreated cells (cells treated with vehicle DMSO) are devoid of neural tube-like
structures (A and C).
Figure S6:API induced differentiation in hES cells.
RT-PCR showing that NPCs differentiated by API express neuronal marker genes such as Pax6,
Map2, Lim home box transcription factor 1, β (Lmx1B), Chat, and Gfap. Gapdh was used as a
marker of successful RNA extraction, cDNA synthesis and sample amplification.
Figure S7: Expression of human neural precursor cell markers in hES cells differentiated with
retinoic acid and apigenin.
Quantification of neural precursor cells markers, NES and SOX2. hES cells were exposed to
DMSO (untreated), 10 μM API or 4 μM RA for the 6 initial days in culture. The values are
expressed as the mean – SEM; n=3.
Table S1: RT-PCR primers used to detect gene expression levels during hES cells differentiation
induced by API.
8
FIGURE S1
9
FIGURE S2
10
FIGURE S3
11
FIGURE S4
12
FIGURE S5
13
FIGURE S6
14
FIGURE S7
15
TABLE S1
Primer
Sequence (5'-3')
Gene Category
A. T.
(°C)
hGAPDH-F
TTC GAC AGT CAG CCG CAT C
hGAPDH-R
GAC TCC ACG ACG TAC TCA GC
hGFAP-F
CCC TGG ACA TCG AGATCG C
hGFAP-R
TGT GCT CCT GCT TGG ACT C
hMAP2-F
CCT TTG AGA ACA CGA CAC AAC GAA
hMAP2-R
GGA ATC CAT TGG CGC TTC
hPAX6a-F
AGA AAG AGT TTG AGA GAA CCC AT
hPAX6a-R
TCA TGT GTG TCT GCA TAT GTG G
hCHAT-F
GAA ACC TAC CTG ATG AGC AAC CG
hCHAT-R
ACT GCA GAG GTC TCT CAT GTC A
hLMX1B-F
CTC CAG AGC AAA AGA GAA TGA GAG
hLMX1B-R
CCT CCC TGT GCG TTT CCC
Constitutive
58
Glia
58
Ectoderm
55
Ectoderm
58
Cholinergic Neurons
57
Serotoninergic Neurons
58
49. Baharvand H, NZ Mehrjardi, M Hatami, S Kiani, M Rao, MM Haghighi. Neural differentiation
from human embryonic stem cells in a defined adherent culture condition.Int J Dev Biol. 2007;
51:371-378.
50.Paulsen BS, MR de Moraes, A Galina, SM Souza, CS Souza, H Drummond, PE Nascimento, H Jr
Silva, L Chicaybam, R Massuda, P Setti-Perdigao, M Bonamino, PS Belmonte-de-Abreu, NG
Castro, H Brentani, SK Rehen. Altered oxygen metabolism associated to neurogenesis of induced
pluripotent stem cells derived from a schizophrenic patient. Cell Transplant. 2012; 21:15471559.
16