Treatment of chronic pain by designer cells controlled by spearmint aromatherapy

25 Pages·2017·0.93 MB·English

SUPPLEMENTARY INFOARMrtAiTcIlOeNs
https://doi.org/10.1038/s41551-018-0192-3
In the format provided by the authors and unedited.
Treatment of chronic pain by designer cells
controlled by spearmint aromatherapy
Hui Wang1, Mingqi Xie   1, Ghislaine Charpin-El Hamri2, Haifeng Ye3 and Martin Fussenegger   1,4*
1Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 2Département Génie Biologique, Institut Universitaire de Technologie,
IUT, Villeurbanne, France. 3Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal
University, Shanghai, China. 4Faculty of Science, University of Basel, Basel, Switzerland. *e-mail: [email protected]
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Supplementary Information

Plasmid Construction. pMOR256-17-EGFP and pRTP1s were kindly provided by
Thamani Dahoun and Horst Pick (Ecole Polytechnique Fédérale de Lausanne, Lausanne,
Switzerland). pNaEx8 was kindly provided by Norbert Klugbauer (Albert-Ludwigs-
University, Freiburg, Germany). The assembly of some plasmids required annealing of
complementary oligonucleotides: 50 pmol of each oligonucleotide was mixed in 50 µl
ddH O-diluted 1x NEB Buffer 4 (New England Biolabs, Ipswich, MA, USA), heated for 10
2
min at 95°C, cooled down over 4 h to 22°C, and incubated at 22°C for another 2 h prior to
cloning into the corresponding vector backbone. All relevant genetic components were
confirmed by sequencing (Microsynth, Balgach, Switzerland).
Cell culture and transfection. Human embryonic kidney cells (HEK-293T, ATCC:
CRL-11268), Hana3A cells1, human cervical adenocarcinoma cells (HeLa, ATCC: CCL-2),
telomerase-immortalized human mesenchymal stem cells (hMSC-TERT)2 and baby hamster
kidney cells (BHK-21, ATCC: CCL-10) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM; Invitrogen, Basel, Switzerland; cat. no. 52100–39) supplemented with 10%
(v/v) fetal bovine serum (FBS; Sigma-Aldrich, Buchs, Switzerland; cat. no. F7524, lot no.
022M3395) and 1% (v/v) penicillin/streptomycin solution (PenStrep; Biowest, Nuaillé,
France; cat. no. L0022-100). Chinese hamster ovary cells (CHO-K1, ATCC: CCL-61) were
cultured in ChoMaster® HTS (Cell Culture Technologies, Gravesano, Germany)
supplemented with 5% (v/v) FBS and 1% (v/v) PenStrep solution. All cell lines were cultured
at 37°C in a humidified atmosphere containing 5% CO . For passaging, cells of pre-confluent
2
cultures were detached by incubation in TrypLETM Express (ThermoFisher Scientific, cat. no.
12605) for 3 min at 37°C, collected in 10 ml cell culture medium, centrifuged for 3 min at
290 g and resuspended in fresh culture medium at standard cell densities (1.5 x 105 cells/mL),
before seeding into new tissue culture plates. Cell number and viability were quantified using
an electric field multichannel cell counting device (Casy Cell Counter and Analyzer Model
TT, Roche Diagnostics GmbH).
For transfection, 2 x 105 cells/ml cells were seeded into 48-well (225 µl medium per
well), 24-well (450 µl medium per well) or 6-well (1800 µl medium per well) plates. After 24
h, the cells were incubated for 8 h with 50 µl, 100 µl or 400 µl of a 3:1 PEI:DNA mixture
(w/w), containing 0.3 µg, 0.6 µg or 2.4 µg of total DNA, respectively. For experiments
performed in 24-well and 48-well plates, the culture medium was exchanged after 8 h for PEI-
1
free medium containing defined concentrations of control compounds, and analytical reporter
assays were performed at 48 h after medium exchange. For experiments performed in 6-well
plates, transfected cells were detached with TrypLETM Express (ThermoFisher Scientific, cat.
no. 12605) and re-seeded into 96-well plates (1.5 x 105 cells/mL, 120 µl) before control
compounds were added into or outside the wells containing the cell suspension. For SEAP-
based PainAssay, 15,000 transfected cells were seeded in 120 µL of culture medium
containing defined concentrations of control compounds. For GFP-based PainAssay, 24,000
transfected cells were re-suspended in 120 µL culture medium containing defined
concentrations of control compounds. Medium in each group was changed to medium
containing the same amount of chemicals or ssHwTx at 24 h. Analytical reporter assays were
performed after cultivation for 48 h in 96-well plates.
Chemicals and food products. Acetylsalicylic acid (Aspirin; cat. no. A5376; stock
solution 1 M EtOH), amitriptyline hydrochloride (cat. no. A8404; stock solution 50 mM in in
ddH O), ethanol (EtOH; cat. no. 02860), buffered formalin solution (cat. no. HT5014),
2
dimethyl sulfoxide (DMSO; cat. no. D8418), Complete Freund's adjuvant (CFA; cat. no.
F5881), R-carvone (cat. no. 124931; stock solution 20 mM in 5% EtOH), tetracaine
hydrochloride (cat. no. T7508; stock solution 50 mM in ddH O), and Tramadol hydrochloride
2
(cat. no. 42965) were purchased from Sigma-Aldrich (Buchs, Switzerland). Blasticidin S HCl
(cat. no. A1113903), puromycin dihydrochloride (cat. no. A1113803), and ZeocinTM selection
reagent (cat. no. R25005) were purchased from ThermoFisher Scientific (Reinach,
Switzerland). Polyethyleneimine (PEI; cat. no. 24765-2; stock solution 1 mg/ml in ddH O)
2
was purchased from Polysciences (Eppelheim, Germany). Potassium chloride (KCl; stock
solution 4 M in ddH O; cat. no. A3582) and sodium chloride (NaCl; stock solution 2 M in
2
ddH O; cat. no. A2942) were purchased from AppliChem (Darmstadt, Germany).
2
Huwentoxin-IV (HwTx-IV; cat. no. STH-100) and Lidocaine (cat. no. L-105; stock solution 1
M in DMSO) were purchased from Alomone Labs (Jerusalem, Israel). XEN907 (cat. no.
2056; stock solution 10 mM in DMSO) was purchased from Axon Medchem (Groningen,
Netherlands). Morphine hydrochloride (cat. no. A1212-B27030-19) was purchased from
Coopération Pharmaceutique Française (Melun, France). Naloxone hydrochloride (cat. no.
HY-17417) was purchased from Lucerna-Chem AG (Lucerne, Switzerland). Primavera®
Spearmint Essential Oil BIO (Primavera Life GmbH, Oy-Mittelberg, Germany) was
purchased from local pharmacies. Wrigley® chewing gum (Wrigley Jr. Company, Chicago,
IL), including spearmint chewing gum and Hubba Bubba Apple, and olive oil (Filippo Berio®
Extra Virgin Olive Oil; Lucca, Italy) were purchased from local supermarkets. For animal
2
experiments, a chewing gum extract solution was prepared by soaking Wrigley® spearmint
chewing gum in ddH O (1:1 w/w), shaking overnight at 37°C and removing the chewing gum
2
by centrifugation (10000 g, 5 min).
Quantification of target gene expression. Expression levels of human placental
secreted alkaline phosphatase (SEAP) in culture supernatants was quantified in terms of
change in p-nitrophenyl phosphate-derived light absorbance3. SEAP levels in mouse serum
were profiled using a chemiluminescence-based assay (Roche Diagnostics GmbH,
Mannheim, Germany; cat. no. 11 779 842 001). Expression levels of hFc or ssHwTx in
culture supernatants were quantified with an ELISA protocol4. For conjugation, hFc specific
anti-human IgG alkaline phosphatase antibody (Sigma-Aldrich, Buchs, Switzerland; cat. no.
A9544) was used. hFc or ssHwTx levels in mouse serum were detected with a human IgG
ELISA kit (Immunology Consultants Laboratory, Inc., Portland, USA; cat. no. E-80G). GFP
levels (excitation: 460/9 nm; emission: 502/20 nm) of HEK-293 cells grown in a black 96-
well plate with a transparent bottom (Greiner Bio-one; cat. no. 655090) using phenol red-free
DMEM (ThermoFisher Scientific, cat. no. 21063029) were quantified with a microplate
reader (Infinite M1000 PRO plate reader, Tecan Group Ltd). Semi-quantitative analysis of R-
carvone levels in animal blood was performed by adding 20 µl of mouse serum to 120 µl
serum-free DMEM containing 2.5 x 104 pCI-OR1A1/pRTP1s/pCK53-transgenic Hana3A
cells and profiling SEAP expression in culture supernatants after 48 h.
Construction and characterization of stable cell lines. According to in vivo (fig.
S3E) and in vitro observations (fig. S3A), stable cell clones should show near-maximal
performance when induced with 10 µM R-carvone in vitro (fig. S3A). The monoclonal
Hana3A cell line, stably transgenic for R-carvone-stimulated ssHwTx expression,
WH113/114/117
was constructed by sequential integration of OR1A1, RTP1s and P -ssHwTx into Hana3A
CRE3
cells using the sleeping beauty transposon system5. After co-transfection of 9500 ng pWH113
(ITR-P -CI-OR1A1-pA:P -dTomato-P2A-BlastR-pA-ITR) and 500 ng pCMV-T7-
hEF1α RPBSA
SB100 (P -SB100X-pA) into 3 x 106 Hana3A cells and selection with 15 µg/mL
hCMV
blasticidin for three passages, the top 2% of the surviving population (Hana3A ) showing
113red
the highest dTomato intensity was collected by FACS-sorting. After co-transfection of 9500
ng pWH114 (ITR-P -RTP1s-pA:P -BFP-P2A-PuroR-pA-ITR) and 500 ng pCMV-
hEF1α RPBSA
T7-SB100 (P -SB100X-pA) into 3 x 106 Hana3A cells and selection with 1 µg/mL
hCMV 113red
puromycin for two passages, the top 2% of the surviving population (Hana3A )
113red114blue
showing the highest BFP and dTomato intensity was collected by FACS-sorting. After co-
transfection of 9500 ng pWH117 (ITR-P -ssHwTx-pA:P -EGFP-P2A-ZeoR-pA-ITR)
CRE3 RPBSA
3
and 500 ng pCMV-T7-SB100 (P -SB100X-pA) into 3 x 106 Hana3A cells and
hCMV 113red114blue
selection with 3.5 μg/mL blasticidin, 1 µg/ml puromycin, and 300 µg/ml Zeocin for three
passages, the top 0.2% of the surviving population (Hana3A ) showing the highest
WH113/114/117
dTomato, BFP, and EGFP expression levels was subjected to FACS-mediated single-cell
cloning. 150 cell clones were randomly selected, checked for the absence of Mycoplasma and
bacterial contamination, and clone no. 93 (AromaCell) showing optimal R-carvone-inducible
ssHwTx expression performance was used for all follow-up studies.
FACS-mediated cell sorting. Hana3A cells expressing BFP (405 nm laser, 450/50
emission filter), EGFP (488 nm laser, 505 nm long-pass filter, 530/30 emission filter), or
dTomato (561 nm laser, 570 nm long-pass filter, 586/15 emission filter) were sorted using a
Becton Dickinson LSRII Fortessa flow cytometer (Becton Dickinson, Allschwil, Switzerland)
while excluding dead cells and cell doublets. Untreated Hana3A cells or parental polyclonal
populations were used as negative controls.
In vitro production and concentration of ssHwTx. ssHwTx was secreted by
pWH50-transgenic HEK-293 cells or AromaCells grown in DMEM/F-12 (ThermoFisher
Scientific, cat. no. 11320033) supplied with 10% (v/v) Panexin basic (Pan Biotech, cat. no.
P04-96900) and 1% (v/v) penicillin/streptomycin solution. The ssHwTx-containing culture
supernatant was purified using a Filtropur S 0.2µm syringe filter (Sarstedt, cat. no.
83.1826.001) before concentration and buffer-exchange into phosphate-buffered saline (PBS;
ThermoFisher Scientific, cat. no. 21600069) using a 10K Pierce™ Protein Concentrator
according to the manufacturer’s protocol (ThermoFisher Scientific, cat. no. 88527). ssHwTx
levels were determined with the human IgG ELISA kit and supplied with 0.1% bovine serum
albumin (BSA; New England Biolabs, cat. no. B9000S) for storage at -20 °C.
Patch-Clamp electrophysiology. Heterologous hNa 1.7 and hNa 1.5 were stably
V V
expressed in CHO-K1 and HEK-293 cells, respectively. Voltage-gated potassium channel
11.1 (hK 11.1, also known as hERG) was stably expressed in CHO-K1 cells (Sophion
V
Bioscience, Denmark). Whole-cell hNa 1.7 and hNa 1.5 channel currents were recorded with
V V
a MultiClamp700B amplifier connected to a Digidata 1550A interface and controlled by the
Clampex10.5 software (Molecular Devices, Sunnyvale, CA). The acquisition rate was set to
20 kHz and signals were filtered at 5 kHz. hERG-based whole-cell recording was conducted
using the HEKA EPC 10 USB patch clamp amplifier (HEKA Elektronik, Germany).
Concentrated ssHwTx solutions (46.3 ± 1.7 µM) and control compounds (tetrodotoxin,
amitriptyline and cisapride) were diluted in extracellular buffer solutions (for sodium currents:
2mM CaCl , 2mM MgCl , 10mM HEPES, 3mM KCl, 140mM NaCl, 10mM glucose at pH
2 2
4
7.4 and 305 mOsm; for potassium currents: 2mM CaCl , 1mM MgCl , 10mM HEPES, 4mM
2 2
KCl, 145mM NaCl, 10mM glucose at pH 7.4 and 305 mOsm) and perfused by gravity.
Channel inhibition was assessed after an incubation time of 15 min. Whole-cell patch-clamp
experiments, including the preparation of control compounds and buffer solutions, were
carried out by ChemPartner (Shanghai, People’s Republic of China; contract no. Biology17-
001358).
Drug addiction test. To assess the potential of developing drug addiction, mice
received one intraperitoneal injection of naloxone (4mg/kg, in 0.9% NaCl) after long-term
exposure to each medication (i.e. AromaCell: 3 weeks; Tramadol: 12 days of 3x50mg/kg i.p.
injections per day; morphine: 3 days of 3x50mg/kg i.p. injections per day). Following
naloxone injection 2h after the last administration of analgesics, the effects of drug
withdrawal resulted in addictive jumps by the animals which were counted over 30min6 by
investigators blinded to the treatment condition.
Locomotor activity and blood pressure. The impact of different pain treatment
strategies on locomotor activity, serpentine tail movements, paw tremor and whole-body
shaking7,8 were assessed by video-capture of all mice and analysis with the idTracker
software9,10. Systolic blood pressure was measured by tail-cuff plethysmography using the
NIBP LE-5002 blood pressure meter (Harvard Apparatus; Holliston, MA).
Immune response and infection. Plasma IgG-levels of treated mice were measured
using a mouse IgG ELISA kit (Immunology Consultants Laboratory, Inc., Portland, USA; cat.
no. E-90G). Plasma C-reactive protein (CRP)-levels were measured using a mouse C-
Reactive Protein ELISA (R&D Systems; cat. no.MCRP00; lot no. P132103).

5
Supplementary Figures
Figure S1. Control experiments related to Fig. 1 and 2. (A) Synthetic mammalian
excitation-transcription coupling systems. Synthetic excitation-transcription coupling systems
are human cells reprogrammed for depolarization-inducible transcription of a targeted set of
transgenes. Membrane depolarization triggers cytosolic calcium increase, calcineurin-
6
dependent nuclear translocation of nuclear factor of activated T-cells (NFAT) and
transcriptional activation of NFAT-regulated promoters (P ). In conventional excitation-
NFAT
transcription coupling systems (left), P regulates the expression of a reporter protein to
NFAT
enable depolarization-inducible reporter gene expression. In PainAssay (right), P
NFAT
regulates the expression of the mammalian paraben-mediated transsilencer (PMS), which
represses reporter gene expression from cognate P -specific promoters. Voltage-gated ion
PMS
channel blockers attenuate membrane depolarization and enable blocker-dependent reporter
gene activation. (B) Depolarization-inducible SEAP expression. pMX57-transgenic HEK-293
cells (pMX57, P -SEAP-pA) were transfected with pcDNA3.1(+) (no ion channels),
NFAT3
pNaEx8 (Na 1.7; P -hNa 1.7-pA), pCa 1.2 (Ca 1.2; P -Cacna1c-pA) or both
V hCMV V V V hCMV
pNaEx8 and pCa 1.2, and cultivated for 48 h in culture medium containing different amounts
V
of potassium chloride (KCl) and sodium chloride (NaCl), then the SEAP levels in culture
supernatants were scored. (C) Depolarization-repressible SEAP expression (PainAssay).
pWH05/pMX109-transgenic HEK-293 cells (pWH05, P -SEAP-pA; pMX109, P -
PMS NFAT3
PMS-pA) were transfected with pcDNA3.1(+) (no ion channels), pNaEx8 (Na 1.7; P -
V hCMV
hNa 1.7-pA), pCa 1.2 (Ca 1.2; P -Cacna1c-pA) or both pNaEx8 and pCa 1.2, and
V V V hCMV V
cultivated for 48 h in culture medium containing different amounts of potassium chloride
(KCl) and sodium chloride (NaCl), then the SEAP levels in culture supernatants were scored.
Data in (B, C) are presented as mean ± SD, n=3 independent experiments. (D, E) SEAP-based
PainAssay. HEK-293 cells were co-transfected with pWH05 (P -SEAP-pA), pMX109
PMS
(P -PMS-pA) and pNaEx8 (P -hNa 1.7-pA) and depolarized with KCl (20 mM) and
NFAT3 hCMV V
NaCl (20 mM), and then control compounds such as analgesics or DMSO were added. (F)
Reporter assay-based cytotoxicity control. HEK-293 cells transfected with pSEAP2-Control
(P -SEAP-pA) were used as a positive control for the highest capacity of constitutive
SV40
protein production. SEAP levels in culture supernatants were scored at 48 h after addition of
compounds. Data presented are mean ± SD, n=3 independent experiments.

7
Figure S2. Functional validation of ssHwTx in vitro and in vivo. (A, B) Patch-clamp
experiments showing the effects of ssHwTx on (A) inward current kinetics and (B) voltage-
dependent hNa 1.5 inhibition. (A) Current amplitudes generated by a 20 ms, -10 mV
V
depolarizing potential from a holding potential of -120 mV at 0.2 Hz in the presence of 1 µM
ssHwTx. Insets show representative current traces inhibited by 1µM ssHwTx or 1µM
amitriptyline (positive control). (B) hNa 1.5 current-voltage relationships in the absence or
V
presence of 1µM ssHwTx were generated using 50 ms-depolarization steps between -80 to
+60 mV with 5mV increments from a holding potential of -120 mV. (C, D) Patch-clamp
experiments showing the effects of ssHwTx on (C) current kinetics and (D) voltage-
dependent hERG channel inhibition. (C) hERG currents were elicited by 5 s depolarizing
8
pulses at +20 mV from a holding potential of -80 mV in the presence of 1µM ssHwTx. (C,
insert) The representative hERG current traces and (D) current-voltage relationships were
generated by depolarizing from -80 to +40 mV with 10 mV-increments for 5 s from a holding
potential of -80 mV, and repolarizing to -50 mV for 5 s in the presence or absence of 1 µM
ssHwTx or 0.1µM cisapride (positive control). All data in (A-D) were normalized to the
maximum current amplitude. (B) and (D) are presented as mean ± SD, n=2 (hNa 1.5) and
V
n=3 (hERG) independent cells. (E) Impact of ssHwTx on animal blood pressure. Systolic
blood pressure of mice were scored before starting the formalin-test described in Fig. 2E. (F)
Impact of ssHwTx on animal locomotor activity. The formalin-test described in Fig. 2E was
video-recorded and the movement of mice were analysed using idTracker software. Data of
(E, F) are shown as the mean ± SEM, statistics by two-tailed t test, n = 8 mice per group.
9