R E S E A R CH A R T I C L E

Multiple nanosecond pulsed electric fields stimulation withconductive poly(L-lactic acid)/carbon nanotubes films maintainsthe multipotency of mesenchymal stem cells during prolongedin vitro culture

Jiaqing Chen1 | Yiqian Huang2 | Jiabei Yang1 | Kejia Li1 | Yangzi Jiang3 |

Boon Chin Heng4 | Qing Cai2 | Jue Zhang5 | Zigang Ge1

1Department of Biomedical Engineering,

College of Engineering, Peking University,

Beijing, China

2State Key Laboratory of Organic-Inorganic

Composites, Beijing Laboratory of Biomedical

Materials, Beijing University of Chemical

Technology, Beijing, China

3Institute for Tissue Engineering and

Regenerative Medicine, School of Biomedical

Sciences, Faculty of Medicine, The Chinese

University of Hong Kong, Hong Kong, China

4Central Laboratory, Peking University School

of Stomatology Beijing, Beijing, China

5Academy for Advanced Interdisciplinary

Studies, Peking University, Beijing, China

Correspondence

Dr. Qing Cai, State Key Laboratory of Organic-

Inorganic Composites, Beijing Laboratory of

Biomedical Materials, Beijing University of

Chemical Technology, Beijing 100029, China.

Email: caiqing@mail.buct.edu.cn

Dr. Jue Zhang, Academy for Advanced

Interdisciplinary Studies, Peking University,

Beijing 100871, China.

Email: zhangjue@pku.edu.cn

Dr. Zigang Ge, Department of Biomedical

Engineering, College of Engineering, Peking

University, Beijing 100871, China.

Email: gez@pku.edu.cn

Funding information

Peking University Medicine Seed Fund for

Interdisciplinary Research, Grant/Award

Number: BMU2018ME001; National Natural

Science Foundation of China, Grant/Award

Number: 81772334

Abstract

Mesenchymal stem cells (MSCs) gradually lose multipotency when cultured for pro-

longed durations in vitro, which significantly hinders subsequent clinical applications.

Nanosecond pulsed electric fields (nsPEFs) have been recently investigated to over-

come this problem in our lab; however, the differentiation potency of MSCs could

only be partially and transiently recovered because the nsPEFs can only be delivered

to suspended cells once. Here, we develop a new strategy to apply multiple nsPEFs

to adherent MSCs with conductive films to mitigate the decreasing multipotency of

prolonged cultured MSCs. The poly(L-lactic acid)/graphitized-carboxylated

functionalized carbon nanotubes (PLLA/CNT) films were fabricated as conductive cell

culture platforms. Both single and multiple nsPEFs stimulation could significantly

increase the differentiation potential of MSCs, as evidenced by upregulated expres-

sion of chondrogenic, osteogenic, and adipogenic-related gene (SOX9, RUNX2, and

PPAR-γ), as well as increased production of proteoglycans, mineralized calcium, and

triglycerides. Multiple nsPEFs stimulation demonstrated significant efficacy in

upregulating expression of pluripotency genes of OCT4A (3.5- to 4.5-folds), NANOG

(3.5- to 4.0-folds), and SOX2 (1.5- to 2.0-folds) and stably maintaining high expres-

sion of these genes for nearly 23 days. Notably, nsPEFs stimulation did not signifi-

cantly shorten telomere length. In conclusion, multiple nsPEFs stimulation could

effectively mitigate decreasing multipotency of MSCs during prolonged in vitro

culture.

K E YWORD S

cell physical stimulus, differentiation, mesenchymal stem cells, multipotency, nanosecond

pulsed electric fields, senescence

Received: 14 April 2020 Revised: 3 June 2020 Accepted: 6 June 2020

DOI: 10.1002/term.3088

1136 © 2020 John Wiley & Sons, Ltd. J Tissue Eng Regen Med. 2020;14:1136–1148.wileyonlinelibrary.com/journal/term

1 | INTRODUCTION

Mesenchymal stem cells (MSCs) proliferate and differentiate into vari-

ous mesenchymal tissue lineages and play key roles in tissue homeo-

stasis and regeneration (Jiang et al., 2002; Pittenger et al., 1999).

MSCs have been broadly used as seed cells in many tissue engineering

applications (Brown et al., 2019; Sadan, Melamed, & Offen, 2009;

Tang, Fan, Pei, Zeng, & Ge, 2015), and the immuno-regulatory activi-

ties of MSCs endow them with additional clinical applications

(Samsonraj et al., 2017; Shah et al., 2019). Due to being limited by rel-

atively low cell numbers, harvested primary MSCs are often required

to undergo prolonged in vitro expansion to attain adequate cell num-

bers for subsequent cell therapy or other clinical applications (Al-

Habib, Yu, & Huang, 2013; Kouroupis, Sanjurjo-Rodriguez, Jones, &

Correa, 2019). However, some significant changes occur during pro-

longed in vitro culture, such as genetic instability, decreased

pluripotency, and senescence of MSCs (B Gharibi & Hughes, 2012;

Izadpanah et al., 2008; Kim et al., 2015; Zhai et al., 2019). These defi-

ciencies limit the application of MSCs in regenerative medicine.

High expression levels of pluripotency genes (OCT4A, NANOG,

and SOX2) help to maintain the multipotency of MSCs and promote

trilineage differentiation potential in subsequent differentiation

medium (Juhee et al., 2015; Tsai, Su, Huang, Yew, & Hung, 2012).

Multiple exogenous stimuli, including hypoxia, physical factors and

materials, chemical agents, cytokine, trophic factors, and hormones,

have been used to enhance expression of pluripotency genes to

recover the decreased differentiation potential of MSCs after pro-

longed in vitro culture (C. X. Hu & Li, 2018; Kouroupis et al., 2019).

However, most methods could only enhance expression of

pluripotency genes (normally onefold to fourfold) with transient

effects of only a few days, usually less than a week. Various culture

conditions, such as calcium ions (Ca2+), hypoxia, or serum deprivation,

have been reported to enhance proliferation and pluripotency of

MSCs (hypoxia: 1.5-fold for NANOG; calcium and hypoxia: threefold

for OCT4A, fourfold for NANOG; Choi et al., 2017; Fehrer et al., 2007;

Kim et al., 2018a; Pochampally, Smith, Ylostalo, & Prockop, 2004). Cell

morphology (3D spheroids) induced by biomaterials has also been

reported to regulate the pluripotency of MSCs (1.2- to 1.4-folds for

OCT4A, 2- to 2.2-folds for NANOG, and 1- to 1.2-folds for SOX2;

Cheng, Wang, & Young, 2012; Huang, Dai, Yen, & Hsu, 2011; Yu

et al., 2012; D. Zhang & Kilian, 2013). Small molecules, such as

Pluripotin (SC1), PDGF receptor inhibitors, Akt, or mTOR inhibitors,

could enhance expression of pluripotency genes and promote differ-

entiation potential via activation or inhibition of biological processes

(Pluripotin [SC1]: fourfold for OCT4A and NANOG, twofold for SOX2;

PDGF receptor inhibitors: sixfold for OCT4A, fourfold for NANOG; Akt

or mTOR inhibitors: 1.5- and 1.2-folds for OCT4A, threefold for

NANOG of both; Al-Habib et al., 2013; Ball, Shuttleworth, &

Kielty, 2012; Ball, Worthington, Canfield, Merry, & Kielty, 2014;

B. Gharibi, Farzadi, Ghuman, & Hughes, 2014). Biological factors, such

as retinoic acid and combination of leukemia inhibitor factor with

basic fibroblast growth factor, were reported to enhanced expression

of pluripotency genes (retinoic acid: 20-fold for NANOG, threefold for

OCT4A; Cortez et al., 2018; W. L. Hu, Wu, Yin, Shi, & Yin, 2016). Gene

editing tools could directly enhance the expression of OCT4A and

NANOG, but there are regulatory hurdles to the clinical application of

genetically modified MSCs (Mendes et al., 2019; Sadan et al., 2009;

Tsai et al., 2012). However, due to the short half-life and short circula-

tion time of bioactive factors or other limitations (Halim, Ariyanti, Luo,

& Song, 2020), the aforementioned methods involve continuous stim-

ulation through the addition of fresh bioactive factors. Moreover, the

enhanced expression levels of pluripotency genes were transient, usu-

ally only for a few days (less than a week).

Electrical fields, which have long been utilized in clinical practice

(with better cost-effectiveness, longer lifetime, and higher reproduc-

ibility; Chen, Bai, Ding, & Lee, 2019), have been shown to enhance

the differentiation potential of MSCs into various lineages (Halim

et al., 2020; Hess et al., 2012; Leppik et al., 2018; Mooney

et al., 2012; Ning et al., 2019). Multiple mechanisms have been pro-

posed, such as calcium oscillation (Hanna, Andre, & Mir, 2017), tran-

sient receptor potential channels (Pattappa, Heywood, de Bruijn, &

Lee, 2011), and JNK signaling pathway (Ning, Guo, et al., 2019). Elec-

tric fields have been reported to induce chondrogenic differentiation

of MSCs via indirect activation of theTGF-β signaling pathway (Kwon,

Lee, & Chun, 2016). Nanosecond pulsed electric fields (nsPEFs) with

significantly short duration (nanosecond) and extremely high voltage

(tens or hundreds kV/cm) have shown much promise in overcoming

the limitation of conventional electrical stimulation (ES), reducing cell

damage caused by accumulation of Joule heating energy due to pro-

longed duration (hours to days) and voltage of conventional ES

(Voldman, 2006). Notably, with short duration (less than the charge

time of plasma membrane) and relatively high voltage, the effects of

Highlights

1. Stimulation with nanosecond pulsed electric fields

(nsPEFs: five pulses at 1-s interval) effectively maintains

the trilineage differentiation potential of mesenchymal

stem cells during prolonged in vitro culture.

2. The biocompatible conductive films fabricated from poly

(L-lactic acid)/graphitized-carboxylated functionalized

with multi-walled carbon nanotubes (PLLA/CNT) can

deliver nsPEFs to adherent 2D cell cultures, which

advances the earlier nsPEFs delivery method with single

cell suspensions.

3. Multiple nsPEFs stimulation (every 3 days during culture,

four times in total) demonstrates prolonged effects on

enhancing pluripotency gene expression after 23 days,

compared with single nsPEFs stimulation.

4. nsPEFs stimulation does not significantly accelerate cell

senescence.

CHEN ET AL. 1137

nsPEFs could reach deeper into the cell nuclei and organelles to affect

the intracellular membrane, rather than just the superficial plasma

membrane by conventional ES, which in turn could reduce broad non-

specific biological effects (Ning, Zhang, Heng, & Ge, 2019; Yao, Hu,

Mi, Li, & Sun, 2009).

In our previous research study, we have found that single

stimulation of nsPEFs enhanced the differentiation potential of

MSCs (Ning, Guo, et al., 2019; and data unpublished). However,

the method is quite limited due to the required pretreatment

before applying nsPEFs to MSCs. First, due to direct coupling stim-

ulation (Balint, Cassidy, & Cartmell, 2013), the conductive gap

cuvette used for nsPEFs only allows dissociated MSCs to get

proper ES. The trypsinization step may introduce inherent experi-

mental artifacts, and gravity may cause uneven distribution of

suspended cells within the gap cuvettes. Most notably, MSCs can

only be subjected to nsPEFs once in the conductive gap cuvettes;

otherwise, additional short-term frequent cell dissociation and

adhesion are needed, which thus limit operation. In this study, we

hypothesize that multiple nsPEFs stimulation would exert persistent

enhancement on the multipotent differentiation potential of MSCs

during prolonged in vitro cultures. Here, we developed a poly(L-

lactic acid)/carboxylated carbon nanotubes (PLLA/CNT) films as a

conductive cell culture platform for multiple nsPEFs stimulation of

adherent cells. The effects of multiple nsPEFs on pluripotency gene

expression, trilineage differentiation potential, and senescence of

MSCs were thus evaluated (Figure 1a). Through multiple nsPEFs

stimulation, the trilineage differentiation potential of MSCs could

be maintained at a high level for a relatively long duration (at least

23 days), which could promote the application of MSCs in cell

therapy to facilitate tissue homeostasis and regeneration.

2 | MATERIALS AND METHODS

2.1 | Formulation of PLLA/CNT films

To fabricate PLLA/CNT films, 1.0 g of PLLA (MW = 100 kDa, Shan-

dong Pharmaceutical Sciences Pilot Plant, China) was dissolved in 5 ml

of trichloromethane, and 30 mg of graphitized-carboxylated

functionalized multiwalled carbon nanotubes (CNTs, Shenzhen Nano-

tech Port, China, outer diameter: 20 nm; inner diameter: 10 nm) were

dispersed in another 5 ml of trichloromethane under ultrasonication

for 30 min. Subsequently, the two solutions were mixed together and

ultrasonically treated for another 30 min to ensure the thorough dis-

persion of CNTs. The suspension obtained was then cast onto glass

plates and subjected to solvent evaporation overnight in air. The solid-

ified films were ready for use after being further vacuum-dried to con-

stant weight.

2.2 | Characterization of PLLA/CNT films

The morphology of PLLA/CNT films was evaluated by scanning elec-

tron microscopy (SEM; Quanta 200 FEG, FEI, USA). The samples were

mounted and sputter coated with gold–palladium, prior to being

examined with SEM at an accelerating voltage of 5 kV.

2.3 | Isolation and culture of MSCs

All animal experiments were approved by the Institutional Animal Care

and Use Committee of Peking University. MSCs were isolated from

the bone marrow of pigs (Yorkshire, boar, 10–12 months). The MSCs

F IGURE 1 Gene expression of OCT4A, NANOG, and SOX2 decreases with time when cultured for prolonged durations in vitro. (a) Schematicdiagram of the experimental design. (b) Pluripotency gene expression of OCT4A, NANOG, and SOX2 from Passage 3 to Passage 6 (P3 3d meansDay 3 at Passage 3, n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001) [Colour figure can be viewed at wileyonlinelibrary.com]

1138 CHEN ET AL.

were then washed with phosphate-buffered saline (PBS) for 5 min

before being cultured in growth medium composed of Dulbecco's

modified Eagle's medium (DMEM, Gibco-Invitrogen, China), 10% (v/v)

fetal bovine serum (FBS; Gibco-Invitrogen), 100 μg/ml of streptomy-

cin, and 100 μg/ml of penicillin at 37�C, within a humidified 5% CO2

atmosphere. Nonadherent cells were washed off after 72 h. When

cells reached 80% confluence, they were detached using 0.25% (w/v)

trypsin solution (Gibco-Invitrogen) for further passage. MSCs at the

first passage were frozen down in 90% (v/v) FBS (life) and 10% (v/v)

dimethylsulfoxide. When MSCs proliferated to P5, the cells were

seeded onto the PLLA/CNT films or were directly transferred to gap

cuvettes. After treated with nsPEFs, MSCs were still cultured in

growth medium. In pluripotency genes expression study after single

and multiple nsPEFs stimulation, cells were treated with mitomycin

(10 μg/ml, sigma) for 2 h, to inhibit cell proliferation, 24 h after seeding

on PLLA/CNT films or tissue culture polystyrene.

2.4 | nsPEFS stimulation

The generator was applied as previously described (Wang et al., 2012;

K. Zhang, Guo, Ge, & Zhang, 2014). The voltage waveform (forward

monophasic pulse) was monitored by a digital phosphor oscilloscope

(DPO4054, Tektronix) containing a high voltage probe (P6015A,

Tektronix). As for the simulation of nsPEFs in 0.2-cm gap cuvettes

(720002-20, Biosmith), MSCs was first dissociated with trypsin and

then counted with a hemocytometer, and 2 × 105 MSCs was

suspended in 900-ul PBS and added into gap cuvettes to perform the

nanosecond pulsed electrical fields stimulation with a certain field

strength and duration. As for the nsPEFs stimulation of adherent cells

on PLLA/CNT film, films (2 × 0.5 cm) seeded with 2 × 104 MSCs were

transferred into 6-well plates containing 3-ml PBS, then two copper

plate, connected with nsPEFs generator, were placed on the both side

of the films to perform the nsPEFs simulation. Simulation was carried

out with five pulses of nsPEFs (10–25 kV/cm, 10–100 ns), and the

time interval between each pulse was 1 s. The five pulses with 1-s

time interval were marked as once nsPEFs stimulation in this study.

2.5 | Cell toxicity assay

The cytotoxic effect of nsPEFs stimulation with different parameters

was evaluated by cell counting kit-8 (CCK-8, Solarbio, CA1210-500) on

Day 3. After nsPEFs stimulation, cells were cultured in normal growth

medium. At each time point, 10% CCK-8 solution was added to each

well and the 6-well plates were incubated in cell culture incubator for

3 h. Optical density (OD) was measured with Microplate Reader

(680, Bio-Rad) at a wavelength of 450 nm to quantify the viable MSCs.

2.6 | Gene expression

Quantitative real-time polymerase chain reaction (qRT-PCR) was

used to quantify the messenger RNA (mRNA) expressions levels of

OCT4A, NANOG, and SOX2 relative to glyceraldehyde-3-phosphate

dehydrogenase (GAPDH). Cell samples (n = 6 for each group) were

lysed in TRIzol reagent (Tiangen), and the total RNAs were extracted

following the manufacturer's protocol. After determining the con-

centration of RNAs with an ND-1000 spectrophotometer

(Nanodrop Technologies), complementary DNA (cDNA) was synthe-

sized using the iScript™ cDNA synthesis kit (Bio-Rad, CA, USA).

qRT-PCR was performed utilizing a Theromocycler PCR system

(PikoReal 96, Thermo) with Real-time Master Mix SYBR Green

(FP202, Tiangen), following the manufacturer's instructions. The rel-

ative expression of each gene was expressed as fold-changes with

respect to GAPDH. Relative expression of each gene was expressed

as fold changes by the 2−ΔΔCt method. The sequences of gene-

specific primers are listed in Table S1.

2.7 | Telomere length detection

To analyze changes in telomere length, the total cellular DNA were

extracted following the manufacturer's protocol (Tiangen, DP304).

After determining the concentration of DNAs with a ND-1000 spec-

trophotometer, qRT-PCR was performed utilizing the thermocycler

PCR system with Real-time Master Mix SYBR Green (FP202, Tiangen)

following the manufacturer's instructions. The sequences of telomere-

specific primers and 36B4 are listed inTable S1.

2.8 | Multilineage differentiation of MSCs andstaining analysis

After nsPEFs stimulation, 2 × 104 cells per well of MSCs of two

groups (s1 3d and s4 3d) were plated in 6-well tissue culture plates

(TCPs) and cultured with chondrogenesis, osteogenesis, and

adipogenesis inducing differentiation medium.

For chondrogenic differentiation, cells were cultured for

14 days in high glucose DMEM (HyClone-Thermo Fisher, China),

supplemented with 10−7 M of dexamethasone (D4902, Sigma),

50 μg/ml of ascorbic acid (A5960, Sigma), 1 mM of sodium pyru-

vate (Sigma-Aldrich, China), 4 mM of proline (Sigma-Aldrich, China),

1% (v/v) ITS+ premix, 1% (v/v) Penicillin–Streptomycin, and

10 ng/ml TGF-β3 (100-36E, PeproTech). Chondrogenic differentia-

tion was evaluated by assaying the accumulation of proteoglycan,

through staining with Alcian blue 8GX in 3% (v/v) acetic acid,

pH 2.5 (G1565, Solarbio). Briefly, after fixation with 4% (w/v) para-

formaldehyde for 20 min at room temperature and washing with

PBS, 1 ml of Alcian blue solution was added to each well for

30 min. After washing with PBS for two times, the stained images

were captured. To quantify proteoglycan, the bound Alcian blue

dye was extracted with 6-M guanidine-HCl and measured spectro-

photometrically at 630 nm.

For osteogenic differentiation, cells were cultured for 14 days in

α-MEM (HyClone-Thermo Fisher, China), containing 16.6% (v/v) FBS,

10−8 M of dexamethasone (D4902, Sigma), 50 μg/ml of ascorbic acid

CHEN ET AL. 1139

(A5960, Sigma), 10-mM β-glycerophosphate, and 1% (v/v) Penicillin–

Streptomycin (Gibco). Osteogenic differentiation was assayed by

staining with Alizarin red S. Briefly, after fixation with 4% (w/v) para-

formaldehyde for 30 min at room temperature and washing with PBS,

1 ml of Alizarin red S solution (0.2%) was added to each well for

30 min. After washing two times with distilled water, the stained

images were captured. To quantify matrix mineralization, the dye was

extracted with 100-mM cetylpyridinium chloride for 30 min and mea-

sured at 570 nm.

For adipogenic differentiation, cells were cultured for 14 days in

α-MEM (HyClone-Thermo Fisher, China), containing 16.6% (v/v) FBS,

10−7 M of dexamethasone (D4902, Sigma), 50 μg/ml of ascorbic acid

(A5960, Sigma), 50 μM indomethacin, 0.45-mM 3-isobutyl-1-methyl-

xanthine, 10 μg/m insulin, and 1% (v/v) of Penicillin–Streptomycin

(Gibco).

The result of adipogenic differentiation was measured by staining

with Oil red O solution. Briefly, after fixation with 4% (w/v) parafor-

maldehyde for 30 min at room temperature, washing with PBS and

treatment with 60% isopropanol for 5 min, 1 ml of Oil red O (2 mg/ml)

was added to each well for 20 min. After washing with PBS for two

times, the stained images were captured. The dye was extracted with

isopropanol for 20 min and then measured spectrophotometrically at

490 nm to quantify the result.

2.9 | Statistical analysis

All experiments were conducted at least three times, and the results

are expressed as the mean values ± SD. Statistical analysis was per-

formed using the SPSS 24 software (one-way analysis of variance

[ANOVA] or Student's t test, [*] for P < 0.05, [**] for P < 0.01, and

[***] for P < 0.001).

3 | RESULTS

3.1 | nsPEFs stimulation transiently enhancespluripotency gene expression of dissociated MSCswithin gap cuvettes in vitro

The gene expression levels of pluripotency markers, such as OCT4A,

NANOG, and SOX2, decrease with time during prolonged in vitro cul-

ture. The relative gene expression level of OCT4A was observed to

decrease gradually from Day 3 to Day 28 (Passage 3 to Passage 6, P3

to P6, from 2.0- to 0.5-folds). The relative gene expression level of

NANOG decreased more quickly from 2.8- to 1.4-folds from Day 3 at

P3 to Day 1 at P6, whereas the relative gene expression level of SOX2

decreased from 3.4- to 1.2-folds. Subsequently, gene expression of

F IGURE 2 Fabrication and characterization of poly(L-lactic acid)/graphitized-carboxylated functionalized carbon nanotubes (PLLA/CNT) films.

(a) The schematic diagram of nanosecond pulsed electric fields (nsPEFs) stimulation with PLLA/CNT films. (b) PLLA/CNT film. (c) FDA-PI stainingof mesenchymal stem cells (MSCs) cultured on PLLA/CNT films on Day 3. (d) Cell viability of MSCs cultured on PLLA/CNT films for Days 1 and3, as quantified by MTT (n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). (e) Morphology of PLLA/CNT films (above) and cells seededon PLLA/CNT films (down), as observed by SEM. (f) Gene expression levels of OCT4A, NANOG, and SOX2 in MSCs cultured on PLLA/CNT filmsfrom Days 0 to 14 at Passage 6 (n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). (g) Absorbance readings at 450 nm (CCK-8 assay) ofMSCs on Day 3 after nsPEFs stimulation with different field strengths and duration (n = 10, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001)[Colour figure can be viewed at wileyonlinelibrary.com]

1140 CHEN ET AL.

both NANOG and SOX2 declined slightly, from Day 3 to Day 28 (P6;

Figure 1b).

After treating the suspended MSCs with nsPEFs (100 ns

10 kV/cm, five pulses, 1-s interval) in conductive gap cuvettes, cells

were then cultured in stem cell growth medium, and the gene expres-

sion level of OCT4A increased immediately at 2 h and peaked

(3.8-fold) on Day 3, then gradually declining by Day 7 (Figure S1A,B).

The relative gene expression levels of NANOG and SOX2 showed a

similar trend, peaking on Day 3 and then decreasing gradually and

mildly (Figure S1B).

3.2 | PLLA/CNT films demonstrate to be a suitablensPEFs stimulation platform with low cytotoxicity

To deliver nsPEFs to cultured cells but not only to the suspended

cells, we developed a culture system with biocompatible conductive

PLLA/CNT films with two electrodes (Figure 2a) that allowed repeat-

able nsPEFs stimulation to attached cells. The instantaneous electrical

resistivity of PLLA/CNT films was similar to PBS (24.61 ± 3.59 kΩ/cm

compared with PBS 17.78 ± 1.91 kΩ/cm, Table 1). The majority of

cells adhered to PLLA/CNT films and proliferated well at Day 3 (Fig-

ure 2c). MTT assay showed that more than 90% of cells were viable,

and there was no significant difference between cells cultured on

PLLA/CNT films and culture dishes at Days 1 and 3, thus indicating

the good biocompatibility of the PLLA/CNT films (Figure 2d). The sur-

face of the PLLA/CNT films was smooth even at the micrometer scale,

and cells seeded on PLLA/CNT films exhibited spindle-like morphol-

ogy (Figure 2e). PLLA/CNT films exerted negligible effects on the rela-

tive gene expression levels of OCT4A, NANOG, and SOX2, as

compared withTCPs (Figure 2f).

We optimized the platform based on the observed gene expres-

sion levels of OCT4A and NANOG. First, we studied the effective elec-

trical stimuli direction (electric current along the PLLA/CNT film,

Figure S2A,B). Mitomycin was used to avoid the proliferation effects of

cultured cells and exerted little influence on the gene expression levels

of OCT4A, NANOG, and SOX2 (treated with 10 μg/ml for 2 h, 24 h after

cells seeded, Figure S2C,D). Then field strength and duration of nsPEFs

stimulation were optimized (Figure S3A). We also narrowed down the

safe and effective range of the nsPEFs stimulation. For instance, cells

subjected to high-energy nsPEFs stimulation with an energy level of

25 kV/cm at 60 ns and 25 kV/cm at 100 ns, exhibited relatively low

absorbance (88% and 93%) based on the CCK-8 assay on Day 3, which

indicated the cytotoxicity and the influence to subsequent cell viability

of nsPEFs (Figure 2g). All other parameters had no significant effect on

absorbance (Figure 2g). The gene expression levels of OCT4A and

NANOG were highest in the treatment groups of 10 ns at 20 kV/cm

and 100 ns at 10 kV/cm (nearly fourfold), with variable degrees of

increase in all groups (Figure S2B). The gene expression level of SOX2

increased in most groups within a certain range, but, it was down-

regulated in three groups (10 ns at 10 kV/cm, 60 ns at 10 kV/cm, and

100 ns at 20 kV/cm, Figure S2B). It was observed that nsPEFs stimula-

tion at 10 ns at 20 kV/cm and 100 ns at 10 kV/cm significantly

enhanced gene expressions of OCT4A, NANOG, and SOX2, out-

performing other groups (Figure S2B). Thus, as there were no differ-

ences between these two sets of parameters, nsPEFs stimulation of

10 kV/cm at 100 ns was utilized for subsequent single and multiple

stimulation, as the data of 20 kV/cm at 10 ns could be found in the

supplementary results.

3.3 | Single nsPEFs stimulation of MSCs cultured onPLLA/CNT films enhances pluripotency geneexpression and trilineage differentiation potential

Similar to results in gap cuvettes, single nsPEFs stimulation (100 ns at

10 kV/cm) on cells cultured on PLLA/CNT films gave transiently

effects. It enhanced gene expression levels of OCT4A to 2.7-fold at

2 h and 3.4-fold at Day 3, before gradually declining (Figure 3a). The

gene expression profile of NANOG and SOX2 showed the same

trends, increasing and peaking at Day 3, then subsequently declining

(Figure 3a).

After single nsPEFs stimulation, the trilineage differentiation

potential of MSCs was assayed by culturing in differentiation media

for 14 days (Figure 3b). Single nsPEFs stimulation enhanced the differ-

entiation ability of prolong cultured MSCs, as evidenced by 1.2-fold

increased Oil red O staining for adipogenic differentiation, 1.3-fold

increased Alizarin Red Staining for osteogenic differentiation, and

1.2-fold increased Alcian blue staining for chondrogenic differentia-

tion (Figure 3c,d). Trilineage differentiation related genes were

increased too. Adipogenic related genes increased about 4.3-fold for

PPAR-γ, 2.9-fold for LPL, and 2.5-fold for AP2 (Figure 3e) at Day 14.

The gene expression levels of osteogenic genes increased about

2.8-fold for RUNX2, eightfold for OC, and 7.1-fold for ALP (Figure 3f),

and chondrogenic genes increased about 2.5-fold for SOX9, 11.2-fold

for COL II, and 6.5-fold for ACAN (Figure 3g).

In addition, single nsPEFs at 10 ns at 20 kV/cm yielded similar

results as nsPEFs at 100 ns at 10 kV/cm, in the case of both

pluripotency- and differentiation-related gene expression, as well as

ECM deposition (Figure S4A–F).

3.4 | Multiple nsPEFs stimulation mitigates declinein pluripotency gene expression and differentiationpotential of MSCs during extended in vitro culture

To investigate whether multiple nsPEFs stimulation could exert a

longer-term effect on MSCs, serial nsPEFs stimulation with 3-day

TABLE 1 The instantaneous electrical resistivity of PBS solutionand PLLA/CNT film

Mean ± std (kΩ/cm)

PBS solution 17.78 ± 1.91

PLLA/CNT film 24.16 ± 3.59

Abbreviations: PBS, phosphate-buffered saline; PLLA/CNT, poly(L-lactic

acid)/graphitized-carboxylated functionalized carbon nanotubes.

CHEN ET AL. 1141

interval was applied. Experimental conditions were labeled according

to actual treatment of the MSCs (Figure 4a). With increasing culture

duration in vitro, the expression levels of OCT4A, NANOG, and SOX2

were observed to gradually decrease in the control (routine cell cul-

ture, without nsPEFs stimulation, Figure 4b). However, after multiple

nsPEFs stimulation, gene expression levels of OCT4A, NANOG, and

SOX2 were enhanced, with nearly fourfold increase being observed

for OCT4A and NANOG, around twofold for SOX2 (Figure 4b). Nota-

bly, after 23 days of persistent four times of nsPEFs stimulation

(s4 14d, as shown in Figure 4a), the expression of multipotency gene

markers were maintained at a high level, of up to 40% of peak values,

about 2.5-fold for OCT4A, 1.6-fold for NANOG, and 1.2-fold for SOX2

(Figure 4b).

After multiple nsPEFs stimulation, cell differentiation potential

was assayed by culturing in various differentiation media for 14 days,

followed by trilineage differentiation staining and qRT-PCR

(Figure 4c). Multiple nsPEFs also enhanced the differentiation ability

of prolong cultured, as evidenced by 1.4-fold increased Oil red O

staining for adipogenic differentiation, 1.2-fold increased Alizarin red

staining for osteogenic differentiation, and 1.5-fold increased Alcian

blue staining for chondrogenic differentiation (Figure 4d,e). Compared

with single nsPEFs stimulation, MSCs treated with multiple nsPEFs

stimulation showed significantly higher intensities of Oil red O and

Alcian blue staining (Figure S5A), with slightly reduced Alizarin red

staining (Figure S5A). Multiple nsPEFs stimulation significantly

enhanced expression of adipogenic genes (3.4-fold for PPAR-γ,

2.5-fold for LPL, and 1.4-fold for AP2; Figure 4f), osteogenic genes

(sevenfold for RUNX2, 2.1-fold for OC, and 2.4-fold for ALP;

Figure 4g), and chondrogenic genes (3.4-fold for SOX9, 10.0-fold for

COL II, and 1.9-fold for ACAN; Figure 4h).

Multiple nsPEFs at 10 ns at 20 kV/cm yielded similar enhance-

ment on the expression levels of pluripotency genes (Figure S4G)

F IGURE 3 Single nanosecond pulsed electric fields (nsPEFs) stimulation enhanced gene expression levels of OCT4A, NANOG, and SOX2, aswell as differentiation potential of mesenchymal stem cells cultured on poly(L-lactic acid)/graphitized-carboxylated functionalized carbonnanotubes (PLLA/CNT) film. (a) Gene expression levels of OCT4A, NANOG, and SOX2 after single nsPEFs stimulation (100 ns at 10 kV/cm) onPLLA/CNT films (n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). (b) After single nsPEFs stimulation, mesenchymal stem cells (MSCs)

were cultured on differentiation media for 14 days before collection and analysis of trilineage differentiation staining and gene expression. (c andd) Trilineage differentiation potential after single nsPEFs stimulation evaluated by Oil red O, Alizarin red and Alcian blue staining. (c) Microscopicimages of the staining, scale bar, 50 μm. (d) Spectrophotometric quantification of Oil red O, Alizarin red and Alcian blue staining of MSCs aftersingle nsPEFs stimulation (fold of control, n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). Control: without nsPEFs stimulation. (e–g)Trilineage differentiation potential after single nsPEFs stimulation evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).(e) Adipogenic differentiation gene marker expression of MSCs after single nsPEFs stimulation (PPAR-γ, LPL, and AP2). (f) Osteogenicdifferentiation gene marker expression of MSCs after single nsPEFs stimulation (RUNX2, OC, and ALP). (g) Chondrogenic differentiation genemarker expression of MSCs after single nsPEFs stimulation (SOX9, COLII A1, and ACAN; n = 6, mean values ± SD, *P < 0.05, **P < 0.01,***P < 0.001) [Colour figure can be viewed at wileyonlinelibrary.com]

1142 CHEN ET AL.

and trilineage differentiation capacity of MSCs, compared with

100 ns 10 kV/cm, as evidenced by expression of adipogenic genes

(3.6-fold for PPAR-γ, 2.3-fold for LPL, and 1.1-fold for AP2;

Figure S4J), osteogenic genes (7.9-fold for RUNX2, 2.1-fold for OC,

and 2.2-fold for ALP; Figure S4K), and chondrogenic genes (3.8-fold

for SOX9, 12.6-fold for COL II, and 2.3-fold for ACAN; Figure S4L).

Additionally, Oil red O staining was enhanced 1.5-fold, mineral

deposition in the accumulated ECM enhanced by 1.3-fold, and pro-

teoglycan deposition (Alcian blue staining) enhanced by 1.5-fold

(Figure S4H,I).

F IGURE 4 Multiple nanosecond pulsed electric fields (nsPEFs) stimulation further enhanced and maintained gene expression levels ofOCT4A, NANOG, and SOX2, as well as differentiation potential of mesenchymal stem cells (MSCs) cultured on poly(L-lactic acid)/graphitized-carboxylated functionalized carbon nanotubes (PLLA/CNT) films. (a) MSCs were cultured on PLLA/CNT films (gray) with different timings ofnsPEFs stimulation of 100 ns at 10 kV/cm, before collection and analysis (blue). s1 3d refers to MSCs that were stimulated with nsPEFs once(s1) and then collected and analyzed after 3 days (3d). (b) Gene expression levels of OCT4A, NANOG, and SOX2 after multiple nsPEFs stimulationon PLLA/CNT films. Control: without nsPEFs stimulation. (c) After multiple nsPEFs stimulation, MSCs were cultured on differentiation media for14 days before collection and analysis. (d and e) Trilineage differentiation potential after multiple nsPEFs stimulation evaluated by Oil red O,Alizarin red, and Alcian blue staining. (d) Microscopic images of the staining, scale bar, 50 μm. (e) Spectrophotometric quantification of Oil red O,Alizarin red, and Alcian blue staining of MSCs after multiple nsPEFs stimulation (fold of control, n = 6, mean values ± SD, *P < 0.05, **P < 0.01,***P < 0.001). (f–h) Trilineage differentiation potential after multiple nsPEFs stimulation evaluated by quantitative real-time polymerase chainreaction (qRT-PCR). (f) Adipogenic differentiation gene marker expression of MSCs after multiple nsPEFs stimulation (PPAR-γ, LPL, and AP2).(g) Osteogenic differentiation gene marker expression of MSCs after multiple nsPEFs stimulation (RUNX2, OC, and ALP). (h) Chondrogenicdifferentiation gene marker expression of MSCs after multiple nsPEFs stimulation (SOX9, COLII A1, and ACAN; n = 6, mean values ± SD, *P < 0.05,**P < 0.01, ***P < 0.001) [Colour figure can be viewed at wileyonlinelibrary.com]

CHEN ET AL. 1143

3.5 | Multiple nsPEFs stimulation recovered thepluripotency genes of prolong cultured MSCs and donot cause cell senescence

During routine in vitro culture of MSCs, multipotency gradually

declines with time, decreased gene expression levels of OCT4A,

NANOG, and SOX2 can be seen (Figure 1b). Single nsPEFs enhanced

expression of pluripotency genes rapidly, peaking at Day 3 after

nsPEFs stimulation, and soon gradually declined back to the baseline

by Day 7 (Figure 5a–c). Notably, four times of nsPEFs stimulation

enhanced expression of pluripotency genes and maintained high

expression levels for 16 days after stimulation (threefold and fourfold

for OCT4A and NANOG, around twofold for SOX2, Figure 5a–c).

Although a mild decrease could happen afterwards, the expression of

pluripotency markers could also be maintained at relatively higher

levels than routine culture until 23 days after stimulation (2.5-fold for

OCT4A, 1.6-fold for NANOG, and 1.2-fold for SOX2, Figure 5a–c).

Compared with single nsPEFs stimulation, multiple nsPEFs stimulation

prolonged high expression levels of pluripotency genes and extended

the trilineage differentiation window. To further study if the nsPEFs

have side effects on stem cell senescence, we evaluated the cell telo-

mere length before and after single or multiple nsPEFs stimulation,

and there was no significant difference in telomere length between

the control (without nsPEFs stimulation) and experimental groups

(Figure 5e).

4 | DISCUSSION

nsPEFs have been demonstrated to be an effective strategy for

maintaining multipotency of MSCs in early (Ning, Guo, et al., 2019)

and current study. As a physical cell stimulus, nsPEFs are able to regu-

late cell behaviors and induce varied biological effects on organelles,

partially because it can change the membrane permeability, and there

are many unknowns need further study (Ning, Zhang, et al., 2019).

Many strategies have been studied to prevent stem cells from los-

ing of differentiation ability after prolonged culture (Ball et al., 2012;

Kim et al., 2018b). We here found that nsPEFs enhanced the expres-

sion of stem cell potency genes with comparable magnitude as other

methods. For example, the levels of gene expression of pluripotency

genes OCT4, NANOG, and SOX2 elevated by nsPEFs (Figures 3a and

4b) at the same levels of cultured in low oxygen tension (1.5-fold for

NANOG; Fehrer et al., 2007), calcium ions (threefold for OCT4A and

fourfold for NANOG; Kim et al., 2018a), small molecule Pluripotin SC1

(fourfold for OCT4A and NANOG, twofold for SOX2; Al-Habib

et al., 2013), inhibition of PDGF receptor (sixfold for OCT4A, fourfold

for NANOG; Ball et al., 2012; Ball et al., 2014), inhibition of Akt and

mTOR (1.5- and 1.2-folds for OCT4A, threefold for NANOG of both;

B. Gharibi et al., 2014) or formation sphere morphology of MSCs (1.2-

to 1.4-folds for OCT4A, 2- to 2.2-folds for NANOG, 1- to 1.2-folds for

SOX2; Huang et al., 2011). When stimulated bone marrow derived

MSCs were cultured in differentiation medium, their trilineage differ-

entiation capacity was shown to be enhanced (Figures 3c–g and 4d–

h).

Previous studies used traditional conductive gap cuvettes to

deliver nsPEFs, which is limited to the suspended cells with one shot

and cannot continually give the stimulation during cell culture. In

order to apply nsPEFs to growing cells without interfering the culture

condition, we developed a conductive cell culture platform with bio-

compatible materials that support the cell attachment and further pro-

liferation and differentiation. Biomaterials made from PLA-based

containing CNTs have been proven as good substrate with ability in

F IGURE 5 Compared to the effects of single nanosecond pulsed electric fields (nsPEFs) stimulation, the effects of multiple nsPEFsstimulation maintained longer, even up to 23 days of high expression levels of OCT4A, NANOG, and SOX2. (a–c) Comparison of gene expressionlevels of OCT4A, NANOG, and SOX2 between single and multiple nsPEFs stimulation on poly(L-lactic acid)/graphitized-carboxylated functionalizedcarbon nanotubes (PLLA/CNT) films. (d) Schematic diagram of comparison of pluripotency gene expression within in vitro culture, after single ormultiple nsPEFs stimulation. (f) RelativeT/S ratio of telomere length after single (s1 3d) and multiple (s4 3d) nsPEFs stimulation. Compared withPassage 4 MSCs cultured on tissue culture plates (TCPs) for 3 days (n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001) [Colour figurecan be viewed at wileyonlinelibrary.com]

1144 CHEN ET AL.

promoting adhesion and growth of MSCs (Li et al., 2016; Shao

et al., 2011). We here adopted the PLLA/CNT as the conductive cell

culture surface and successfully applied multiple nsPEFs during the

long-term cell culture. Our results indicated that nsPEFs stimulation

could be applied to adherent cells directly with the PLLA/CNT film

repeatedly. In addition to PLLA/CNT film, there are several types of

conductive scaffold or hydrogel with suitable conductivity have been

reported (Bei et al., 2019; Gelmi et al., 2016; Saravanan et al., 2018)

and have the potential to be used as cell culture platform to perform

living nsPEFs stimulation. Our finding here suggests a new way to

expand the application of nsPEFs to adherent 2D and 3D cell cultures

in the near future.

nsPEFs have also been shown to be effective over an extended

period of time without the need for complicated pre-treatment steps.

The effects of single stimulation of nsPEFs usually lasts for around

3 days, and the differentiation potency of MSCs could only be par-

tially and transiently recovered (Figures S1B and 3a). When the cells

were cultured on the PLLA/CNT film, multiple nsPEFs stimulation can

be continuously delivered to the organelles, thus keep the gene

expression of OCT4A, NANOG, and SOX2 and trilineage differentiation

stay high (Figure 4b–h). With our conductive cell culture platform,

multiple nsPEFs stimulation successfully enhanced and maintained

the multipotency of MSCs for over 23 days, which is far beyond the

previous methods. For instance, enhanced expression of pluripotency

genes can last for around 1 week with the following methods, such as

repression of Zeb1 and Hypoxia (Liu et al., 2013), depletion of histone

demethylase KDM2A (Dong, Yao, Du, Wang, & Fan, 2013), inhibition

of PDFG receptor (Ball et al., 2012), inhibition of Akt and mTOR

(B. Gharibi et al., 2014), knockdown of p21Cip1/Waf1 (Yew et al., 2011),

or formation of sphere morphology of MSCs (after 7 or 10 days, ret-

urned to normal level; Huang et al., 2011). Moreover, these various

aforementioned techniques need preconditioning for a long duration

compared to nsPEFs stimulation, such as PDGFR inhibitor (24 h),

rapamycin to inhibit Akt and mTOR (35 days), lentiviral transduction

technology for knockdown of p21 (7 and 8 weeks), or KDM2A (at least

7 days) expression within cells. nsPEF technique is thus an easy and

effective method for the long-term culture duration without the need

for time-consuming preconditioning steps of handling MSCs, thereby

avoiding potential damage or contamination.

As a biophysical stimulus, nsPEFs could effectively reverse

decreased differentiation potentials of MSCs during prolonged in vitro

culture. Furthermore, this technology does not depend on molecules

or permanent genome modifications, but only mildly and reversely

modify signaling pathways and epigenetic expression. These relatively

mild effects of nsPEFs facilitate clinical translation. The advanced utili-

zation of nsPEFs in this study shed a light on rescuing the functions of

MSCs after prolonged culture, which might have much potential for

clinical applications in cell therapy. On the other hand, by utilizing a

PLLA/CNT film, multiple nsPEFs stimulation could be easily and

repeatedly perform without complex pretreatment. This can greatly

expand the application of nsPEFs, such as combination with electro-

poration to direct DNA transfection to nucleus in vitro or combination

with patch clamp to inhibit specific voltage-gated ionic current.

5 | CONCLUSIONS

In summary, through PLLA/CNT films, we successfully applied multi-

ple nsPEFs to attach MSCs, which could significantly increase differ-

entiation potential of MSCs, as proved by gene expression and

trilineage differentiation. In conclusion, multiple nsPEFs stimulation

could effectively mitigate decreasing multipotency of MSCs during

prolonged in vitro culture.

ACKNOWLEDGEMENTS

The authors would like to thank Mr. Kaile Wang for operating the

nsPEFs equipment. This work was supported by the National Natural

Science Foundation of China grant (81772334) and Peking University

Medicine Seed Fund for Interdisciplinary Research

(BMU2018ME001).

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS

Jiaqing Chen: conception and design, collection and assembly data,

data analysis and interpretation, manuscript writing, and final approval

of manuscript; Yiqian Huang: materials fabrication, manuscript writing,

and final approval of manuscript; Jiabei Yang: data interpretation,

manuscript writing, and final approval of manuscript; Kejia Li: data

interpretation and manuscript writing; Yangzi Jiang: data interpreta-

tion and manuscript writing; Boon Chin Heng: manuscript writing;

Qing Cai: collection and assembly data, data analysis and interpreta-

tion, and final approval of manuscript; Jue Zhang: conception and

design and final approval of manuscript; Zigang Ge: conception and

design, manuscript writing, and final approval of manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All animal experiments were approved by the Institutional Animal

Care and Use Committee of Peking University.

ORCID

Qing Cai https://orcid.org/0000-0001-6618-0321

Jue Zhang https://orcid.org/0000-0003-0440-1357

Zigang Ge https://orcid.org/0000-0002-5202-0973

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SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section at the end of this article.

Figure S1. Effects of nsPEFs stimulation. (A) The schematic diagram

of nsPEFs stimulation with conductive gap cuvette. (B) Gene expres-

sion levels of OCT4A, NANOG and SOX2 with single nsPEFs stimula-

tion with conductive gap cuvette, as quantified by qRT-PCR (n = 6,

mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure S2. Effects of electrical direction of nsPEFs stimulation and

mitomycin c treatment on gene expression. (A) Schematic diagram of

electrical current direction optimization. Group A: electric current

along the film; Group B: electric current through the film. (B) Effects

of different electrical current direction of nsPEFs stimulation of the

gene expression levels of OCT4 and NANOG on day 3. (n = 3, mean

values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). (C) After nsPEFs

stimulation, MSCs were treated with mitomycin c for 2 h and cultured

for 3d before collection and analysis. (D) Gene expression levels of

OCT4A, NANOG and SOX2 in MSCs treated with mitomycin c, C:

mitomycin c treatment; NC: no mitomycin c treatment. (n = 6, mean

values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure S3. Selection of parameters of nsPEFs stimulation on

PLLA/CNT films. (A) MSCs were cultured on PLLA/CNT film (gray) in

growth media (red) for 3 days after nsPEFs stimulation with different

field strengths and duration before collection and analysis (blue).

(B) Gene expression levels of OCT4A, NANOG and SOX2 in MSCs after

single nsPEFs stimulation with different field strength and duration on

PLLA/CNT films, as quantified by qRT-PCR. (n = 6, mean values ± SD,

*P < 0.05, **P < 0.01, ***P < 0.001).

Figure S4. Group of 10 ns at 20 kV/cm nsPEFs stimulation showed

similar effects as group of 100 ns at 10 kV/cm. (A) Gene expression

levels of OCT4A, NANOG and SOX2 after single nsPEFs stimulation on

PLLA/CNT films under 10 ns at 20 kV/cm. (B-C) Tri-lineage differenti-

ation potential after single nsPEFs stimulation evaluated by Oil red O,

Alizarin red and Alcian blue staining. B, Microscopic images of the

staining, Scale bar, 50 μm. C, Spectrophotometric quantification of Oil

CHEN ET AL. 1147

red O, Alizarin red and Alcian blue staining of MSCs after single

nsPEFs stimulation. (n = 6, mean values ± SD, *P < 0.05, **P < 0.01,

***P < 0.001). (D-F) Tri-lineage differentiation potential after single

nsPEFs stimulation evaluated by qRT-PCR. D, Adipogenic differentia-

tion gene marker expression of MSCs after single nsPEFs stimulation

(PPAR-γ, LPL and AP2). E, Osteogenic differentiation gene marker

expression of MSCs after single nsPEFs stimulation (RUNX2, OC and

ALP). F, Chondrogenic differentiation gene marker expression of

MSCs after single nsPEFs stimulation (SOX9, COLII A1 and ACAN).

(n = 6, mean values ± SD, *P < 0.05, **P < 0.01, ***P < 0.001).

(G) Gene expression levels of OCT4A, NANOG and SOX2 after multiple

nsPEFs stimulation on PLLA/CNT films under 10 ns 20 kV/cm. (H-I)

Tri-lineage differentiation potential after multiple nsPEFs stimulation

evaluated by Oil red O, Alizarin red and Alcian blue staining. H, Micro-

scopic images of the staining, Scale bar, 50 μm. I, Spectrophotometric

quantification of Oil red O, Alizarin red and Alcian blue staining of

MSCs after multiple nsPEFs stimulation. (n = 6, mean values ± SD,

*P < 0.05, **P < 0.01, ***P < 0.001). (J-L) Tri-lineage differentiation

potential after multiple nsPEFs stimulation evaluated by qRT-PCR. J,

Adipogenic differentiation gene marker expression of MSCs after mul-

tiple nsPEFs stimulation (PPAR-γ, LPL and AP2). K, Osteogenic differ-

entiation gene marker expression of MSCs after multiple nsPEFs

stimulation (RUNX2, OC and ALP). L, Chondrogenic differentiation

gene marker expression of MSCs after multiple nsPEFs stimulation

(SOX9, COLII A1 and ACAN). (n = 6, mean values ± SD, *P < 0.05,

**P < 0.01, ***P < 0.001).

Figure S5. Comparison of tri-lineage differentiation staining with sin-

gle versus multiple nsPEFs stimulation. (A) 100 ns at 10 kV/cm; (B)

10 ns at 20 kV/cm.

How to cite this article: Chen J, Huang Y, Yang J, et al.

Multiple nanosecond pulsed electric fields stimulation with

conductive poly(L-lactic acid)/carbon nanotubes films

maintains the multipotency of mesenchymal stem cells during

prolonged in vitro culture. J Tissue Eng Regen Med. 2020;14:

1136–1148. https://doi.org/10.1002/term.3088

1148 CHEN ET AL.