Platelet-rich plasma enhances the proliferation of human adipose stem cells through multiple signaling pathways
Lai et al. Stem Cell Research & Therapy
Platelet-rich plasma enhances the proliferation of human adipose stem cells through multiple signaling pathways
Fangyuan Lai 0
Natsuko Kakudo 0
Naoki Morimoto 0
Shigeru Taketani 2
Tomoya Hara 0 1
Takeshi Ogawa 0
Kenji Kusumoto 0
0 Department of Plastic and Reconstructive Surgery, Kansai Medical University , 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 , Japan
1 Department of Oral Implantology, Osaka Dental University , Osaka 573-1121 , Japan
2 Department of Microbiology, Kansai Medical University , Osaka 573-1010 , Japan
Background: Platelet-rich plasma (PRP) is an autologous blood product that contains a high concentration of several 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, and both 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 by inhibitors 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 proliferation of hASCs. PRP promotes hASC proliferation via ERK1/2, PI3K/Akt, and JNK signaling pathways.
Background
Human adipose-derived stem cells (hASCs) were first
isolated 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
], and
neurogenic [
7
] lineages. hASCs are used widely in the
field of regenerative medicine, including to promote
bone 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 their
relative abundance and ease of isolation, hASCs have
also been widely used in the fields of plastic surgery and
regenerative medicine [
12
].
However, the proliferation and differentiation
capacities of hASCs decrease with age [
13, 14
], body mass
index [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 in
multiple types of cells in vitro. However, due to the risk
of heterologous immunization and zoonosis, FBS has
limited clinical use.
Platelet-rich plasma (PRP) is a blood portion that is
enriched 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
(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, and
differentiation.
Our previous study revealed that activated PRP has a
potential effect on the proliferation of hASCs and
human 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 proliferation
of hASCs have not been clarified.
In the present study, we show that PRP stimulated cell
proliferation 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-c (...truncated)