Lai et al. Stem Cell Research & Therapy (2018) 9:107
https://doi.org/10.1186/s13287-018-0851-z

RESEARCH

Open Access

Platelet-rich plasma enhances theproliferation of human adipose stem cells
through multiple signaling pathways
Fangyuan Lai1, Natsuko Kakudo1*, Naoki Morimoto1, Shigeru Taketani2, Tomoya Hara1,3, Takeshi Ogawa1
and Kenji Kusumoto1

Abstract
Background: Platelet-rich plasma (PRP) is an autologous blood product that contains a high concentration ofseveral growth factors. Platelet-derived growth factor (PDGF)-BB is a potential mitogen for human adipose-derived
stem cells (hASCs). PRP stimulates proliferation of hASCs; however, the signaling pathways activated by PRP remain
unclear.
Methods: hASCs were cultured with or without PRP or PDGF-BB, and proliferation was assessed. hASCs were also
treated with PRP or PDGF-BB with or without imatinib, which is a PDGF receptor tyrosine kinase inhibitor, or
sorafenib, which is a multikinase inhibitor. Inhibition of cell proliferation was examined using anti-PDGF antibody(Abcam, Cambridge, UK), by cell counting. We assessed the effects of inhibitors of various protein kinases such as
ERK1/2, JNK, p38, and Akt on the proliferation of hASCs.
Results: The proliferation was remarkably promoted in cells treated with either 1% PRP or 10 ng/ml PDGF-BB, andboth imatinib and sorafenib inhibited this proliferation. Anti-PDGF antibody (0.5 and 2 μg/ml) significantly
decreased the proliferation of hASCs compared with control. PRP-mediated hASC proliferation was blocked byinhibitors of ERK1/2, Akt, and JNK, but not by an inhibitor of p38.
Conclusions: PRP promotes hASC proliferation, and PDGF-BB in PRP plays a major role in inducing the proliferationof hASCs. PRP promotes hASC proliferation via ERK1/2, PI3K/Akt, and JNK signaling pathways.

Background
Human adipose-derived stem cells (hASCs) were firstisolated from human adipose tissue and identified by
Zuk et al. in 2001 [1]. These cells can differentiate toward multiple lineages, such as osteogenic [2], chondrogenic [3], adipogenic [4], cardiac [5], epidermal [6], andneurogenic [7] lineages. hASCs are used widely in thefield of regenerative medicine, including to promotebone regeneration [2], tooth and periodontal regeneration [8], cartilage regeneration [9], wound healing [6,10], and nerve regeneration to cure Parkinson’s disease[11], as well as to suppress aging [10]. Due to the advantages of the autologous source of these cells and theirrelative abundance and ease of isolation, hASCs have
* Correspondence: kakudon@hirakata.kmu.ac.jp
1
Department of Plastic and Reconstructive Surgery, Kansai Medical University,
2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan
Full list of author information is available at the end of the article

also been widely used in the fields of plastic surgery andregenerative medicine [12].
However, the proliferation and differentiation capacities of hASCs decrease with age [13, 14], body massindex [14], diabetes mellitus [12, 15], radiation exposure[16], and tamoxifen treatment [17]. hASCs account for
about 16–30% of the stromal vascular fraction [18]. To
obtain a sufficient amount of cells for therapeutic purposes, in-vitro proliferation of the cells is required. Fetal
bovine serum (FBS) is widely used for this purpose inmultiple types of cells in vitro. However, due to the riskof heterologous immunization and zoonosis, FBS has
limited clinical use.
Platelet-rich plasma (PRP) is a blood portion that isenriched with platelets [19]. Upon activation, platelets in
PRP release granules containing molecules including
growth factors and regulatory proteins, such as plateletderived growth factor (PDGF), epidermal growth factor© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Lai et al. Stem Cell Research & Therapy (2018) 9:107(EGF), insulin-like growth factors (IGFs), transforming
growth factor beta (TGF-β), vascular endothelial growth
factor (VEGF), and others [19–21]. These growth factors
play important roles in cell proliferation, migration, anddifferentiation.
Our previous study revealed that activated PRP has apotential effect on the proliferation of hASCs andhuman dermal fibroblasts (hDFs) compared with nonactivated PRP [22]. Furthermore, we also reported that
activated PRP induces hDF proliferation via the activation of ERK1/2 signaling [23]. Recently, other investigators reported that PDGF also enhances proliferation of
hASCs through the JNK pathway [24]. However, the signaling pathways involved in PRP-stimulated proliferationof hASCs have not been clarified.
In the present study, we show that PRP stimulated cellproliferation by ERK1/2, JNK, and Akt activation. We
compared this effect with the proliferative effect of
PDGF-BB, a major growth factor in PRP.

Methods
Preparation of activated PRP

Activated PRP was obtained using the double-spin
method as described previously [23]. Briefly, after
obtaining informed consent from healthy adult volunteers (n = 3), blood was collected in tubes containing an
acid-citrate-dextrose solution formula A anticoagulant,
and spun in a standard laboratory centrifuge for 7 min
at 450×g. The yellow plasma with buffy coat, containing
platelets, leukocytes, and some erythrocytes from two
tubes, was collected in a monovette via a long cannulaand centrifuged for 5 min at 1600×g. Platelets that accumulated in the thrombocyte pellet in 1.0 ml plasma wereused as PRP. A separate sample of 8 ml blood wasallowed to stand for 30 min at room temperature in a tube
without anticoagulant and then spun for 8 min at 2015×g.
The supernatant was collected as an autologous thrombin.
A 1:1 (v/v) mixture of 0.5 M CaCl2 and autologous thrombin was prepared in advance as an activator. A 10:1 (v/v)
mixture of PRP and activator was incubated for 10 min atroom temperature. Activated PRP was centrifuged at 90×g
and then 9000×g for 10 min each; the supernatant was filtered through a 0.22-μm membrane (Millex GP; Merck
Millipore, Tullagreen, Carrigtwohill, Co. Cork, Ireland)and stored at −80 °C until use.
Measurement of platelet concentrations and growth
factor levels

The number of platelets in whole plasma and PRP wascounted using an XE-2100 automated hematology system (Sysmex Corp., Tokyo, Japan). PDGF-BB, IGF, and
EGF levels in whole plasma and activated PRP were determined using commercially available ELISA kits (R&D

Page 2 of 10

Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
Isolation of hASCs

Unnecessary adipose tissue was obtained from a 61year-old male patient who had previously provided
informed consent and underwent plastic surgery.
hASCs were isolated using a method described previously [25]. After washing extensively with phosphatebuffered saline (PBS), the adipose tissues were cut
into small pieces and incubated with 3 volumes of 0.
1% collagenase (Sigma-Aldrich, St. Louis, MO, USA)
solution with constant shaking at 40 °C for 40 min.
After adding DMEM containing 10% FBS (Hyclone,
Logan, UT, USA) and antibiotics (complete medium),the tissue was centrifuged at 400×g for 3 min. Afterremoving cellular debris through a 100-μm nylon
mesh (BD Falcon, Bedford, MA, USA), the cells wereincubated in complete medium in a dish. The primary hASCs were cultured for 4–5 days until theyreached confluence. These cells were defined as passage “0”. For all experiments, cells from passages 7–
9 were used.
Cell proliferation assay

For the cell proliferation assays, hASCs were seeded at adensity of 1.0 × 104 cells/well in 24-well culture plates
and incubated in complete medium overnight. The cell
medium was then replaced with serum-free DMEM.
After 6 h of incubation, hASCs were treated with PRP or
human recombinant PDGF-BB (PeproTech EC Ltd, London,UK) at the stated concentrations in serum-free DMEM for
48 h. Inhibitors included the PDGF receptor tyrosine kinase
inhibitor imatinib (Wako Co., Ltd, Tokyo, Japan), themultikinase inhibitor sorafenib (AdooQ, Irvine, CA, USA),
the MEK inhibitor PD98059, the phosphatidylinositol-3kinase-Akt inhibitor LY294002, the p38 inhibitor SB203580(Calbiochem-Novabiochem, San Diego, CA, USA), and the
JNK inhibitor SP600125 (Sigma). Inhibitors were added 1 h
before the incubation with PRP or PDGF-BB. Cell proliferation was determined using Cell Counting Kit-8 (Dojindo
Molecular Technologies, Kumamoto, Japan), according to
the manufacturer’s instructions. Absorbance was read at
450 nm on a multiwell plate reader (EnSpire 2300 Multilabel
Reader; PerkinElmer, Inc., Waltham, MA, USA).
To estimate the cell number from the absorbance, astandard curve was established. hASCs were seeded atdensities of 0, 6250, 12,500, 25,000, 50,000, and 100,000
cells/well in 24-well plates for 3 h with 10% FBS in
DMEM. The cells were then incubated with Cell Counting Kit-8 solution for 1 h, and the absorbance was read.
The standard curve was established by plotting the number of cells versus the absorbance.

Lai et al. Stem Cell Research & Therapy (2018) 9:107

BrdU incorporation

hASCs were seeded at a density of 2 × 103 cells/well in
96-well culture plates containing complete medium.
After overnight incubation, the hASCs were first starved
in serum-free DMEM for 6 h and then treated with PRP,
PDGF-BB, human recombinant IGF, or human recombinant EGF (PeproTech EC Ltd) in serum-free DMEM
for 48 h. Inhibitors were added 1 h before incubation
with PRP or PDGF-BB. Quantification of cell proliferation was determined using the Cell Proliferation ELISABrdU kit (Roche, Mannheim, Germany).
Cell cycle assay

hASCs (1 × 106 cells) were seeded in a 10-cm culture
dish containing complete medium and cultured overnight. The medium was then replaced with serum-free
DMEM for 6 h, and the cells were treated with reagentsat the stated concentrations for 48 h. Treated cells werecollected by trypsinization. After washing with ice-cold
PBS twice, cells were fixed in 70% ethanol at −20 °C for
3 h. The fixed cells were then stained with Muse™ Cell
Cycle reagent (Millipore) in the dark at room
temperature for 30 min. Cell cycle phases were analyzed
by flow cytometric quantification of DNA with the
Muse™ Cell Analyzer (Millipore).

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the presence of ATP. The reaction mixture was separated with SDS-PAGE and transferred to a PDVF membrane. The membrane was incubated with rabbit antiphospho-c-Jun (1:1000) at 4 °C overnight, followed byincubation with peroxidase-linked anti-rabbit IgG atroom temperature for 30 min. The labeled proteins werealso detected with enhanced chemiluminescence using
the Prime Western blotting detection system. All of theexperiments were replicated three times.
Statistical analysis

Data are presented as the mean ± standard deviation(SD). The Mann–Whitney U test was used to evaluate
differences among groups. P < 0.05 and P < 0.01 wereconsidered statistically significant.

Results
Concentrations of platelet and growth factors in PRP andblood

Compared with whole plasma, PRP showed a 10.1-foldenrichment in platelets and a 25.9-fold enrichment in
PDGF-BB. In contrast, the concentrations of EGF werecomparable between PRP and whole plasma. IGF in PRP
was present at a concentration of only 60% that in wholeplasma (Table 1).

Western blot analysis

PRP stimulated proliferation of hASCs

The cells were treated with the indicated compounds
and lysed. Extracted cellular proteins (20 μg) wereseparated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and then transferred
to a polyvinylidene difluoride (PVDF) membrane. The membrane was first blocked with Blocking One-P reagent (Nacalai Tesque, Kyoto, Japan) for 30 min at room temperature,and then incubated with the following primary antibodies:
rabbit anti-phospho-ERK1/2 (1:1000; Epitomics Inc.,
Burlingame, CA, USA), rabbit anti-phospho-Akt, rabbit
anti-Akt (1:5000; Abcam, Cambridge, UK), rabbit antiERK1/2 (1:1000; Cell Signaling Technology, Beverly, MA,
USA), or rabbit anti-β-actin (1:1000; Cell Signaling
Technology) at 4 °C overnight. This was followed by incubation with peroxidase-linked secondary antibody (1:20000; GE Healthcare, Little Chalfont, UK) at room
temperature for 30 min. The labeled proteins were detected with enhanced chemiluminescence using the Prime
Western blotting detection system (GE Healthcare).

Cell proliferation was increased by treatment with 0.2%
PRP (P < 0.01 vs control), and 1% PRP stimulated cellproliferation to a greater extent (P < 0.01 vs control andP < 0.01 vs 0.2% PRP). Thus, PRP stimulated proliferation of hASCs in a dose-dependent manner between 0
and 1% PRP (Fig. 1a). The proliferation was decreased
when cells were treated with 3% and 5% PRP, comparedwith 1% (data not shown). The cell growth stimulated by
PRP was confirmed by observation with phase-contrast
microscopy (Fig. 1b).

In-vitro JNK activity assay

Activity of the JNK pathway was analyzed with the
SAPK/JNK Kinase Assay Kit (Cell Signaling Technology). Briefly, cell lysates were immunoprecipitated withthe anti-phospho-JNK antibody coupled to JNK sepharose beads. The concentrated active JNK protein wasthen reacted with the substrate, c-Jun fusion protein, in

PRP containing PDGF-BB promoted hASC proliferation

Cell proliferation of hASCs was also enhanced by treatment with 2 ng/ml PDGF-BB (P < 0.05 vs control).
PDGF-BB at a concentration of 10 ng/ml markedly stimulated cell proliferation (P < 0.01 vs control and P < 0.01
vs 0.2% PRP). PDGF-BB displayed a dose-dependent
stimulation of hASC proliferation between 0 and 10 ng/
ml (Fig. 2a). Treatment with imatinib (5 μM) or sorafenib (5 μM) reduced the PRP-stimulated hASC proliferation (Fig. 2b). Similarly, both imatinib and sorafenib
significantly inhibited the proliferation of hASCs induced by PDGF-BB (10 ng/ml). Inhibition with sorafenib
was more potent than that with imatinib (Fig. 2c).
Furthermore, treatment with anti-PDGF antibody inhibited
PRP-stimulated growth of hASCs in a dose-dependent
manner (Fig. 2d).

Lai et al. Stem Cell Research & Therapy (2018) 9:107

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Table 1 Platelet concentrations and growth factor levels in serum and PRP (n = 3)
Platelets (×1010/L)

PDGF-BB (ng/ml)

EGF (pg/ml)

IGF-I (ng/ml)

Plasma15.5 ± 2.3

1.5 ± 0.4534 ± 8226 ± 6

PRP

157.5 ± 10.6

38.8 ± 0.7

639 ± 17145 ± 4

Ratio (PRP/plasma)

10.1

25.9

1.2

0.6

PDGF platelet-derived growth factor, EGF epidermal growth factor, IGF insulin-like growth factor, PRP platelet-rich plasma

PDGF-BB stimulated DNA synthesis

Cell proliferation of hASCs was also evaluated using
BrdU incorporation assays. Compared with control, PRP
induced a 5.42-fold increase in BrdU incorporation, andimatinib decreased the incorporation 2.97-fold. Thus,
PRP significantly stimulated DNA synthesis in hASCs,
and imatinib inhibited DNA synthesis (Fig. 3a).
We also tested the stimulating effect of growth factors.
PDGF-BB markedly enhanced DNA synthesis (10.08-fold vscontrol). In contrast, treatment with either IGF-I (10 ng/ml)(3.33-fold vs control) or EGF (10 ng/ml) (2.02-fold vs control) showed a minimal effect on DNA synthesis in hASCs.
PDGF-BB plus either IGF or EGF stimulated DNA synthesis
to a greater extent than IGF or EGF alone (Fig. 3b).
Promotion of cell cycle transition from G0/G1 to S phaseby PRP and PDGF-BB

When treated with PRP compared to control, the flow
cytometry showed a trend in which cells in the S and
G2/M phases increased (Fig. 4a). A histogram of theabFig. 1 PRP stimulated proliferation of hASCs. Cells were incubated with PRP in serum-free DMEM for 48 h. Growth examined with Cell Counting
Kit-8 by reading absorbance at 450 nm. a PRP stimulated hASC proliferation in a dose-dependent manner (n = 7). *P < 0.01. b Phase-contrast micrographs show increase in hASCs after treatment with PRP. PRP platelet-rich plasma

Lai et al. Stem Cell Research & Therapy (2018) 9:107

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a

b

c

d

Fig. 2 PDGF-BB mediated the stimulatory effect of PRP. Cells were incubated with reagents at indicated concentrations in serum-free DMEM for
48 h. Cell proliferation assessed with Cell Counting Kit-8. a PDGF-BB enhanced hASC proliferation in a dose-dependent manner (n = 7). b Effect ofimatinib (5 μM) and sorafenib (5 μM) on PRP-dependent proliferation of hASCs (n = 7). c Effect of imatinib (5 μM) and sorafenib (5 μM) on PDGFBB-dependent proliferation of hASCs (n = 7). d Anti-PDGF antibody inhibited PRP-stimulated proliferation of hASCs (n = 4). *P < 0.05. PDGF
platelet-derived growth factor, PRP platelet-rich plasma

flow cytometry results is shown in Fig. 4b. The percent
of cells treated with PRP that were in the S phase (15.44± 7.31%) was significantly higher compared with control(3.67 ± 0.91%). Similarly, the percent of cells treated with
PRP that were in the G2/M phase (32.11 ± 5.5%) was
also significantly higher compared with control (13.61 ±
6.63%). The percent of cells treated with PRP plus imatinib in the S and G2/M phases (8.81 ± 3.27% and 17.28± 3.15%, respectively) and the percent treated with
PDGF-BB (9.01 ± 4.54% and 19.75 ± 2.97%, respectively)
were not significantly different compared to control (3.
67 ± 0.91% and 13.61 ± 6.63%, respectively). PRP stimulated cell progression to the S and G2/M phases, andimatinib inhibited this effect. PDGF-BB showed a small
effect on the cell cycle (Fig. 4a, b).
Activation of ERK1/2, Akt, and JNK signaling pathways
with PRP and PDGF-BB

To examine the signaling pathways involved in stimulation of hASCs by PRP, cells were treated with an

ERK1/2 inhibitor (PD98059, 20 μM), PI3K/Akt inhibitor (LY294002, 10 μM), JNK inhibitor (SP600125,
20 μM), or p38 inhibitor (SB203580, 10 μM). PRPinduced cell proliferation was suppressed by PD98059,
LY294002, and SP600125, but not SB203580 (Fig. 5).
The signaling pathways activated by these treatments
were further analyzed in hASCs with western blotting anda JNK activity assay. Phosphorylation of ERK1/2 and Akt
increased following treatment with PRP or PDGF-BB.
Imatinib inhibited the phosphorylation of these enzymes
in the presence of PRP (Fig. 6a, b). Phosphorylation of
JNK was not detected under these conditions. Next, wemeasured the activity of JNK using the substrate c-Jun. As
shown in Fig. 6b, PRP markedly activated JNK. Thus,
stimulation of cell growth by PRP was mediated throughmultiple signaling pathways.

Discussion
We demonstrated that PRP enhanced the proliferationof hASCs through multiple signaling pathways by

Lai et al. Stem Cell Research & Therapy (2018) 9:107

abFig. 3 DNA synthesis in PRP-treated hASCs. Cells were incubated with PRP(1%) or PDGF-BB (10 ng/ml) in serum-free DMEM for 48 h. DNA synthesis
measured by incorporation of BrdU. a Effect of PRP on DNA synthesis in
hASCs (n = 7). b Effects of growth factors on stimulation of DNA synthesis
in hASCs (n = 6). *P < 0.01. BrdU 5-bromo-2′-deoxyuridine, PRP platelet-richplasma, PDGF platelet-derived growth factor, IGF insulin-like growth factor,
EGF epidermal growth factor

activating ERK1/2, JNK, and Akt. The proliferative effectof PRP, which was similar to the proliferative effect of
PDGF-BB alone, was inhibited by the tyrosine kinase inhibitor imatinib and by the multikinase inhibitor sorafenib. The proliferative effect of PRP was also lowered byadding anti-PDGF antibody to the medium, indicatingthat PDGF-BB, which was abundant in activated PRP,
played a major role in the proliferative effect of PRP. In
addition, PRP induced the proliferation of cells in the Sphase of the cell cycle, concomitant with an increase in
BrdU uptake. Addition of PRP activated ERK, JNK, andAkt, and PRP-mediated proliferation was blocked by inhibitors of these signal transduction enzymes. On theother hand, PDGF-BB alone only slightly activated ERK,JNK, and Akt (Fig. 6a, b). These results indicated that

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other factors in PRP function in the additive effect oncell growth.
PRP is enriched in platelets, which were collected bycentrifugation of autologous blood. Cytokines including
PDGF, TGF-β, VEGF, IGF, EGF, and basic fibroblast
growth factor (bFGF) are contained in α-granules ofplatelets. PRP was collected without coagulation, andwas then activated by adding autologous thrombin andcalcium chloride. Growth factors in activated PRP are
indispensable for the proliferation of various types ofcells [26]. We now demonstrated that PRP was a potent
inducer of proliferation of hASCs.
Competence activity is known to be stimulated by factors that can make cells become “competent” to replicate
their DNA and divide. Competence growth factors include PDGF [27] and FGF-2 [28]. PDGF and FGF-2
alone act on cells that are in either the G0 or early G1phase of the cell cycle, rendering them competent to initiate DNA replication [28]. In contrast, progression activity refers to activity mediated by factors that can
dictate the ultimate fraction of competent cells that enters the S phase [27]. These typical progression growth
factors are EGF [28] and IGF-I [28]. The progression
growth factors allow cells to progress through the prereplicative phase of the cycle, inducing cells to enter theS, G2, and M phases. PRP comprises a large amount ofcompetence growth factors, such as PDGF and FGF-2,
and the progression growth factors, EGF and IGF-I. This
study strongly suggests that these competence and progression growth factors act on hASCs in a concerted
and compounding manner, and progress the cell cyclefrom the G0 phase to G1 and S phases.
Cell cycle progression is regulated by the expression of
cyclins. Cyclins are factors that bind to and activate thecyclin-dependent kinases (CDKs). There are approximately 20 kinds of cyclins, such as cyclin A2, B1, andD1, and several types of CDKs such as CDK1, CDK2,
and CDK4. These factors and kinases are known to control cell cycle progression by binding with each other indifferent combinations. Cyclin D is expressed in response to mitogens, and then binds with CDK4 or
CDK6. The formed cyclin D complex phosphorylates thetarget protein, progressing the cell cycle from the G1 tothe S phase. It was reported that the expression of cyclin
D1 in hASCs increased with the transition of the cell
cycle from the G1 to the S phase [24]. In addition, thecdc2/cyclin B complex was reported to regulate the G2/
M phase transition [29]. This study found that theaddition of PRP led to an increase in the proliferation of
hASCs in the S and M phases, implying a possible involvement of cyclin D1 and B1.
The proliferative effect of PRP on preadipocytes [30], osteoblasts [31], and bone marrow mesenchymal stem cells[32] has been reported. Furthermore, PRP can enhanceLai et al. Stem Cell Research & Therapy (2018) 9:107

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abFig. 4 Analysis of the cell cycle in PRP-treated and PDGF-BB-treated hASCs. Cells were incubated with PRP (1%) or PDGF-BB (10 ng/ml) in serumfree DMEM for 48 h. Cell cycle stages determined by flow cytometry. a Representative data from four independent experiments. b Cell cycle
distributions in hASCs after treatment with PRP and PDGF-BB (n = 4). *P < 0.01 compared with the control. PRP platelet-rich plasma, PDGF
platelet-derived growth factor

the proliferative effect of mesenchymal progenitor cells by
activating the ERK signaling pathway, and PDGF-BB is a
key factor in this stimulation [33]. Also, the proliferative
effect on chondrocytes can be significantly stimulated by
PRP via the ERK signaling pathway, and platelet-derived
adenosine diphosphate in PRP is a key mediator of proliferation [34]. We found that PRP induces the proliferationof hDFs by the activation of ERK1/2 signaling [23].
Proliferation of hASCs induced by PRP was reported by
Kakudo et al. [22] and Gersch et al. [35], but the signaling
pathways remain unclear. PDGF-BB stimulates DNA synthesis in hASCs and cell proliferation, and these effects
are mediated by JNK activation [24] or Akt activation [36].
Also, FGF-2 [37], EGF [38], or VEGF [39] induces theproliferation of hASCs through ERK1/2 activation. Ourpresent study showed that the addition of PRP to hASCs
activated JNK, ERK1/2, and Akt, and the addition of inhibitors of these kinases reduced the proliferative activity.
Because PRP contains abundant PDGF-BB, FGF-2, EGF,
and VEGF, the interaction among these growth factors
may stimulate cell proliferation through multiple signaling
pathways. The addition of PD98059, SP600125, or LY294002
to PRP-treated hASCs partially inhibited cell proliferation,
which supports this conclusion. Hye Kim et al. [40] reported
that an isoform of PDGF, PDGF-D, showed a strong proliferation effect on hASCs, and thus PDGF-D present in PRP
may also induce proliferation of hASCs through the ERK1/2
and Akt pathways.

Lai et al. Stem Cell Research & Therapy (2018) 9:107

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abFig. 5 Pharmacological inhibition of PRP-induced proliferation
through JNK and p38 pathways. After incubation in serum-free
DMEM for 6 h, cells were treated with inhibitors at thedesignated concentrations for 48 h. Cell proliferation assessedwith Cell Counting Kit-8. Cells treated with ERK1/2 inhibitor(PD98059, 20 μM), PI3K/Akt inhibitor (LY294002, 10 μM), JNK
inhibitor (SP600125, 20 μM), or p38 inhibitor (SB203580, 10 μM)(n = 4). *P < 0.01 compared with no inhibitor. ERK extracellularsignal-regulated kinase, JNK c-Jun N-terminal kinase, Akt protein
kinase B, PRP platelet-rich plasma

It is noted that the use of stem cells for therapeutic
applications is influenced by their proliferative and differentiation potential, which is affected by the age of thedonors. It was previously reported that compared withyoung cells, aged hASCs exhibited increased cellularsenescence features [41–43], a decline in both stromal
vascular fraction (SVF) cell yield [43] and hASC proliferation rate [41–44], a decreased differentiation potential[42] toward adipogenic [41, 43, 44], osteogenic [41, 43],and chondrogenic [41] lineages, negative effects on
hASC frequency [43], fewer progenitor cell numbers[41], and impaired migration ability [43]. Based on thesereports, we believe that hASCs obtained from older patients may have limitations in clinical application. Our
study had limited sources of adipose acquisition due tothe lack of younger patients, and thus it was impossible
to compare the differences between young hASCs andaged hASCs. However, based on our experimental results, as proliferation of aged hASCs can still be stimulated by PRP, we speculate that young hASCs will havehigher proliferative ability by PRP stimulation.
In future studies, we will use young hASCs to examine
the effects of PRP on proliferation promotion.

Fig. 6 Phosphorylation of PRP-treated hASCs. After incubation inserum-free DMEM for 6 h, cells were stimulated with PRP (1%) or
PDGF-BB (10 ng/ml) for 30 min. Imatinib added for 1 h before stimulation with PRP. a Immunoblots of p-Akt and p-ERK1/2. b JNK activity
examined in cell lysates, c-Jun as the substrate. Amounts of c-Jun
quantitated in comparison with Ponceau S staining (lower panel).
ERK extracellular signal-regulated kinase, Akt protein kinase B, PRP
platelet-rich plasma, PDGF platelet-derived growth factor

Conclusion
We found that both PRP and PDGF-BB can induce theproliferation of hASCs by activating ERK1/2, Akt, and JNK
signaling pathways. This study clarified that PDGF-BB
present in PRP plays an essential role in the proliferation
via multiple signaling pathways, and is not limited to stimulation by PDGF-BB. The reason for the potent effect of
PRP may be due to the presence of various factors involvedin a variety of proliferative activities.
Thus, PRP is a powerful promoter to proliferate
hASCs in vitro. Future studies are required to clarify theinteraction of these factors that are present in PRP.
Acknowledgements
The authors thank the Central Research of Laboratory of the Kansai Medical
University for their technical assistance.
Funding
This work was supported by the research grant D2 from Kansai Medical University.
Availability of data and materials
All data generated and/or analyzed during this study are included in thispublished article.
Authors’ contributions
FL conceived the work, acquired data, drafted the manuscript, and approved
the final version. NK and ST conceived the work, revised the manuscript, andapproved the final version. TH and TO acquired data, and approved the final
version. NM and KK revised the manuscript, and approved the final version.
All authors read and approved the final manuscript.

Lai et al. Stem Cell Research & Therapy (2018) 9:107

Ethics approval and consent to participate
The study was approved by the Ethics Review Board of Kansai Medical
University in accordance with the ethical guidelines of the Helsinki
Declaration of 1975. All specimens were collected and used with informed
consent from the donors.
Competing interests
The authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
Author details
1
Department of Plastic and Reconstructive Surgery, Kansai Medical University,
2-5-1 Shin-machi, Hirakata, Osaka 573-1010, Japan. 2Department ofMicrobiology, Kansai Medical University, Osaka 573-1010, Japan. 3Departmentof Oral Implantology, Osaka Dental University, Osaka 573-1121, Japan.
Received: 9 January 2018 Revised: 18 March 2018
Accepted: 20 March 2018
References
1. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz
HP, Hedrick MH. Multilineage cells from human adipose tissue: implications
for cell-based therapies. Tissue Eng. 2001;7:211–28.
2. Zhang M, Zhang P, Liu Y, Zhou Y. GSK3 inhibitor AR-A014418 promotes
osteogenic differentiation of human adipose-derived stem cells via ERK andmTORC2/Akt signaling pathway. Biochem Biophys Res Commun. 2017;
490(2):182–8.
3. Galeano-Garces C, Camilleri ET, Riester SM, Dudakovic A, Larson DR, Qu W,
Smith J, Dietz AB, Im HJ, Krych AJ, et al. Molecular Validation ofchondrogenic differentiation and hypoxia responsiveness of platelet-lysate
expanded adipose tissue-derived human mesenchymal stromal cells.
Cartilage. 2017;8:283–99.
4. Paul NE, Denecke B, Kim BS, Dreser A, Bernhagen J, Pallua N. The effectof mechanical stress on the proliferation, adipogenic differentiation andgene expression of human adipose-derived stem cells. J Tissue Eng
Regen Med. 2017;12(1):276–84.
5. Joo HJ, Kim JH, Hong SJ. Adipose tissue-derived stem cells for myocardial
regeneration. Korean Circ J. 2017;47:151–9.
6. Hur W, Lee HY, Min HS, Wufuer M, Lee CW, Hur JA, Kim SH, Kim BK, Choi
TH. Regeneration of full-thickness skin defects by differentiated adiposederived stem cells into fibroblast-like cells by fibroblast-conditioned
medium. Stem Cell Res Ther. 2017;8:92.
7. Jahromi M, Razavi S, Amirpour N, Khosravizadeh Z. Paroxetine can enhanceneurogenesis during neurogenic differentiation of human adipose-derived
stem cells. Avicenna J Med Biotechnol. 2016;8:152–8.
8. Hu L, Liu Y, Wang S. Stem cell-based tooth and periodontal regeneration.
Oral Dis. 2017. https://onlinelibrary.wiley.com/doi/epdf/10.1111/odi.12703.
9. Szychlinska MA, Castrogiovanni P, Nsir H, Di Rosa M, Guglielmino C, Parenti
R, Calabrese G, Pricoco E, Salvatorelli L, Magro G, et al. Engineered cartilage
regeneration from adipose tissue derived-mesenchymal stem cells: amorphomolecular study on osteoblast, chondrocyte and apoptosis
evaluation. Exp Cell Res. 2017;357(2):222–35.
10. Gaur M, Dobke M, Lunyak VV. Mesenchymal stem cells from adipose tissue
in clinical applications for dermatological indications and skin aging. Int J
Mol Sci. 2017;18(1):E208. http://www.mdpi.com/1422-0067/18/1/208/htm.
11. Takahashi H, Ishikawa H, Tanaka A. Regenerative medicine for Parkinson's
disease using differentiated nerve cells derived from human buccal fat padstem cells. Hum Cell. 2017;30:60–71.
12. Tobita M, Tajima S, Mizuno H. Adipose tissue-derived mesenchymal stem
cells and platelet-rich plasma: stem cell transplantation methods thatenhance stemness. Stem Cell Res Ther. 2015;6:215.
13. Geissler PJ, Davis K, Roostaeian J, Unger J, Huang J, Rohrich RJ. Improving
fat transfer viability: the role of aging, body mass index, and harvest site.
Plast Reconstr Surg. 2014;134:227–32.
14. van Harmelen V, Skurk T, Rohrig K, Lee YM, Halbleib M, Aprath-Husmann I,
Hauner H. Effect of BMI and age on adipose tissue cellularity anddifferentiation capacity in women. Int J Obes Relat Metab Disord.
2003;27:889–95.

Page 9 of 10

15. van Tienen FH, van der Kallen CJ, Lindsey PJ, Wanders RJ, van Greevenbroek
MM, Smeets HJ. Preadipocytes of type 2 diabetes subjects display an
intrinsic gene expression profile of decreased differentiation capacity. Int J
Obes. 2011;35:1154–64.
16. Poglio S, Galvani S, Bour S, Andre M, Prunet-Marcassus B, Penicaud L,
Casteilla L, Cousin B. Adipose tissue sensitivity to radiation exposure. Am J
Pathol. 2009;174:44–53.
17. Pike S, Zhang P, Wei Z, Wu N, Klinger A, Chang S, Jones R, Carpenter J,
Brown SA, DiMuzio P, et al. In vitro effects of tamoxifen on adipose-derived
stem cells. Wound Repair Regen. 2015;23:728–36.
18. Zuk P. Adipose-derived stem cells in tissue regeneration: a review. ISRN
Stem Cells. 2013;2013:35.
19. Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor
analysis from platelet-rich plasma: implications for wound healing. Plast
Reconstr Surg. 2004;114:1502–8.
20. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology
and applications in plastic surgery. Plast Reconstr Surg. 2006;118:147e–59e.
21. Lubkowska A, Dolegowska B, Banfi G. Growth factor content in PRP and theirapplicability in medicine. J Biol Regul Homeost Agents. 2012;26:3S–22S.
22. Kakudo N, Minakata T, Mitsui T, Kushida S, Notodihardjo FZ, Kusumoto K.
Proliferation-promoting effect of platelet-rich plasma on human adiposederived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 2008;
122:1352–60.
23. Hara T, Kakudo N, Morimoto N, Ogawa T, Lai F, Kusumoto K. Platelet-richplasma stimulates human dermal fibroblast proliferation via a Rasdependent extracellular signal-regulated kinase 1/2 pathway. J Artif Organs.
2016;19:372–7.
24. Kang YJ, Jeon ES, Song HY, Woo JS, Jung JS, Kim YK, Kim JH. Role of c-Jun
N-terminal kinase in the PDGF-induced proliferation and migration ofhuman adipose tissue-derived mesenchymal stem cells. J Cell Biochem.
2005;95:1135–45.
25. Kakudo N, Shimotsuma A, Miyake S, Kushida S, Kusumoto K. Bone tissue
engineering using human adipose-derived stem cells and honeycomb
collagen scaffold. J Biomed Mater Res A. 2008;84:191–7.
26. Burnouf T, Strunk D, Koh MB, Schallmoser K. Human platelet lysate:
replacing fetal bovine serum as a gold standard for human cell
propagation? Biomaterials. 2016;76:371–87.
27. Stiles CD, Capone GT, Scher CD, Antoniades HN, Van Wyk JJ, Pledger WJ.
Dual control of cell growth by somatomedins and platelet-derived growth
factor. Proc Natl Acad Sci U S A. 1979;76:1279–83.
28. Bramanti V, Grasso S, Tibullo D, Giallongo C, Raciti G, Viola M, Avola R.
Modulation of extracellular signal-related kinase, cyclin D1, glial fibrillary
acidic protein, and vimentin expression in estradiol-pretreated astrocyte
cultures treated with competence and progression growth factors. J
Neurosci Res. 2015;93:1378–87.
29. Perdiguero E, Nebreda AR. Regulation of Cdc25C activity during the meiotic
G2/M transition. Cell Cycle. 2004;3:733–7.
30. Fukaya Y, Kuroda M, Aoyagi Y, Asada S, Kubota Y, Okamoto Y, Nakayama T,
Saito Y, Satoh K, Bujo H. Platelet-rich plasma inhibits the apoptosis of highly
adipogenic homogeneous preadipocytes in an in vitro culture system. Exp
Mol Med. 2012;44:330–9.
31. Slapnicka J, Fassmann A, Strasak L, Augustin P, Vanek J. Effects of activatedand nonactivated platelet-rich plasma on proliferation of human osteoblasts
in vitro. J Oral Maxillofac Surg. 2008;66:297–301.
32. Oliva A, Passaro I, Di Pasquale R, Di Feo A, Criscuolo M, Zappia V, Della
Ragione F, D'Amato S, Annunziata M, Guida L. Ex vivo expansion of bonemarrow stromal cells by platelet-rich plasma: a promising strategy inmaxillo-facial surgery. Int J Immunopathol Pharmacol. 2005;18:47–53.
33. Gruber R, Karreth F, Kandler B, Fuerst G, Rot A, Fischer MB, Watzek G.
Platelet-released supernatants increase migration and proliferation, anddecrease osteogenic differentiation of bone marrow-derived mesenchymal
progenitor cells under in vitro conditions. Platelets. 2004;15:29–35.
34. Zhou Q, Xu C, Cheng X, Liu Y, Yue M, Hu M, Luo D, Niu Y, Ouyang H, Ji J,
Hu H. Platelets promote cartilage repair and chondrocyte proliferation via
ADP in a rodent model of osteoarthritis. Platelets. 2016;27:212–22.
35. Gersch RP, Glahn J, Tecce MG, Wilson AJ, Percec I. Platelet rich plasma
augments adipose-derived stem cell growth and differentiation. Aesthet
Surg J. 2017;37:723–9.
36. Chen QJ, Chen L, Wu SK, Wu YJ, Pang QJ. rhPDGF-BB combined with ADSCs
in the treatment of Achilles tendinitis via miR-363/PI3 K/Akt pathway. Mol
Cell Biochem. 2017;438(1-2):175–82.

Lai et al. Stem Cell Research & Therapy (2018) 9:107

Page 10 of 10

37. Zaragosi LE, Ailhaud G, Dani C. Autocrine fibroblast growth factor 2
signaling is critical for self-renewal of human multipotent adipose-derived
stem cells. Stem Cells. 2006;24:2412–9.
38. Baer PC, Schubert R, Bereiter-Hahn J, Plosser M, Geiger H. Expression of afunctional epidermal growth factor receptor on human adipose-derived
mesenchymal stem cells and its signaling mechanism. Eur J Cell Biol. 2009;88:273–83.
39. Song SY, Chung HM, Sung JH. The pivotal role of VEGF in adipose-derivedstem-cell-mediated regeneration. Expert Opin Biol Ther. 2010;10:1529–37.
40. Hye Kim J, Gyu Park S, Kim WK, Song SU, Sung JH. Functional regulation of
adipose-derived stem cells by PDGF-D. Stem Cells. 2015;33:542–56.
41. Ye X, Liao C, Liu G, Xu Y, Tan J, Song Z. Age-related changes in theregenerative potential of adipose-derived stem cells isolated from theprominent fat pads in human lower eyelids. PLoS One. 2016;11:e0166590.
42. Choudhery MS, Badowski M, Muise A, Pierce J, Harris DT. Donor age
negatively impacts adipose tissue-derived mesenchymal stem cell
expansion and differentiation. J Transl Med. 2014;12:8.
43. Liu M, Lei H, Dong P, Fu X, Yang Z, Yang Y, Ma J, Liu X, Cao Y, Xiao R.
Adipose-derived mesenchymal stem cells from the elderly exhibit
decreased migration and differentiation abilities with senescent properties.
Cell Transplant. 2017;26:1505–19.
44. Yang HJ, Kim KJ, Kim MK, Lee SJ, Ryu YH, Seo BF, Oh DY, Ahn ST, Lee HY,
Rhie JW. The stem cell potential and multipotency of human adipose
tissue-derived stem cells vary by cell donor and are different from those ofother types of stem cells. Cells Tissues Organs. 2014;199:373–83.

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