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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
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]
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]
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
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]
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]
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]