Leukocyte-reduced platelet-rich plasma stimulates the in vitro proliferation of adipose-tissue derived mesenchymal stem cells depending on PDGF signaling

Abstract

BACKGROUND:

Platelet-rich Plasma (PRP) is suggested as xenoprotein-free cell-culture medium replacement for animal-derived supplements.

OBJECTIVE:

The aim of this study was to investigate PRP-triggered signaling in adipose derived mesenchymal stem cells (ASCs).

METHODS:

PRP was obtained from 4 male patients. We incubated ASCs in α-MEM with different Platelet derived growth factor (PDGF) subtypes or 10% or 20% pooled PRP or 20% fetal calf serum (FCS) prior to determination of the S-phase fraction (SPF). To investigate the influence of PDGF signaling on ASCs, PDGF receptor β inhibitor was added, and protein expression of ASCs was measured.

RESULTS:

ASCs exposed to 20% PRP, PDGF-AB and – BB demonstrated significant higher SPF in comparison to PDGF-AA and 20% FCS after 48 hours (all P < 0.05). PDGF receptor β inhibition diminished the PRP-induced SPF increase of ASCs significantly after 48 hours (P < 0.01). ASCs with PDGF receptor β inhibition showed significant higher PDGF receptor β and significant lower c-MYC expression compared to untreated cells in presence of 20% PRP after 48 hours (both P < 0.05).

CONCLUSIONS:

The proliferation promoting effect of PRP on ASCs is mediated by PDGF signaling and is associated with c-MYC overexpression.

Keywords

Platelet-rich Plasma mesenchymal stem cells PDGF receptor β inhibition proliferation cell culture

1 Introduction

Mesenchymal stem cells (MSCs) are multipotent cells that can be easily harvested from adipose tissue (ASCs). They turned into a fundamental component in regenerative therapies due to their multilineage potential and their paracrine secretory ability [1 –5]. For most clinical application, MSCs have to be isolated and expanded in vitro to purify the population and to gain higher cell numbers [6]. In many laboratory standard operating protocols (SOP) fetal calf serum (FCS) represents a medium supplement during cell expansion. Nevertheless, the immunological risks of using animal derived products for human application have to be considered [6 –8]. Therefore, a growing number of studies suggests Platelet-rich Plasma (PRP) as a reasonable, xenoprotein-free medium additive [9 –12]. PRP has been described as an autologous cocktail of growth factors and biomolecules, including platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF). This cocktail is able to induce chemotactic, proliferative, angiogenic and anabolic cellular responses [12, 13]. Amongst others, PDGF is known to play a decisive role in the stimulation of MSC proliferation in vitro [14, 15]. The PDGF family consists of four homodimers that include A-, B-, C-, or D-polypeptides and one heterodimeric AB-isoform [17]. They are interacting with PDGF receptor α and β expressed on MCSs’ surface [17]. Both receptors belong to the family of receptor protein tyrosine kinases [18], and can activate the same phosphoinositide 3 kinase (PI3K), Phospoinositol-Phospholipase C γ (PLC γ), and mitogen activated protein kinase (MAPK) signaling pathway amongst others [19].

At present, we have limited information about the relationship between growth-factors in PRP and their promoting effect on the proliferation of MSCs, with particular attention to the cellular signaling cascades. As robust MSC expansion and differentiation are basic requirements in preclinical cell expansion, it becomes important to determine PRP induced cellular mechanisms that are determining the fate of MSCs. The identification of these mechanisms will contribute to establish PRP as a reliable medium supplement to grow MSCs for regenerative therapies in accordance with the requirements of good manufacturing practice (GMP) [20].

In previous studies we established a modified PRP preparation protocol for a commercially available PRP preparation kit to reduce the number of white blood cells in PRP [21]. We indicated a positive dose-dependent effect of leukocyte-reduced PRP as medium supplement on the proliferation of ASCs. Moreover, we identified several regulatory proteins in ASCs, whose expression was altered significantly in presence of PRP in comparison to FCS [22]. Ultimately, we identified PDGF receptor β and c-MYC among the most significantly differentially regulated proteins.

The aim of the present study was to assess the role of PDGF signaling in ASC proliferation in PRP supplemented monolayer culture.

2 Methods

2.1 Ethics statement

The study was approved by the ethics committee of the University of Regensburg and written informed consent was obtained from each volunteer in accordance with the declaration of Helsinki prior to blood drawing and elective liposuction.

2.2 PRP samples

Venous blood (15 ml) was drawn directly into the Arthrex Double Syringe (Arthrex, Inc., Naples, FL, USA) for the production of autologous conditioned plasma (ACP) using a winged infusion set (Sarstedt AG & Co., Nümbrecht, Germany). The ACP double syringe was processed using a Hettich Rotofix 32a centrifuge at 1500 rpm for 4 minutes with brake disabled as characterized previously [21]. With this settings an average platelet count of 548.5±104.9×10³/μl in the plasma product can be achieved, while reducing the white blood cell count to 0.05±0.11×10³/μl, as shown before [21]. Blood was separated into two distinct layers by centrifugation whereas a plasma layer appeared on the top and the red/white blood cell layer was apparent on the bottom. The plasma containing the platelets (platelet rich plasma, PRP) was isolated by drawing the inner syringe according to the manufacturer‘s instructions. PRP samples were stored at –20°C until they were pooled for further experiments (n = 4). As PRP was deep freezed right after centrifugation, no anticoagulant has been used. For the usage in the cell culture experiments PRP was activated by thawing.

2.3 Adipose-tissue derived stem cell (ASC) isolation

Human ASCs were isolated from solid subcutaneous adipose tissue, which was obtained from 4 patients undergoing elective body-contouring procedures, as described previously [21]. The samples were processed within 24 hours after harvesting. Briefly, subcutaneous fat tissue was washed in phosphate-buffered saline, and minced into pieces of <2 mm³. Serum-free α-MEM (1 ml/g tissue) and Liberase Blendzyme 3 (Roche Diagnostics, Rotkreuz, Switzerland) (2 U/g tissue) were added and incubated under continuous shaking at 37°C for 45 minutes. The digested tissue was sequentially filtered through 100-μm and 40-μm filters (Fisher Scientific GmbH, Schwerte, Germany) and centrifuged at 450 g for 10 min. The supernatant was discarded and remaining cell pellet was washed twice with Hanks’ balanced salt solution (Cellgro, Mediatech Inc. Manassas, VA, USA) and finally resuspended in α-MEM growth medium containing 20% FCS (Pan Biotech Gmbh, Aidenbach, Germany), 2 mM L-glutamine (Life technologies, Carlsbad, CA, USA), 100 U/ml penicillin, 100 g/ml streptomycin (both Sigma-Aldrich, Saint Louis, MO, USA). Cells were plated at a density of 3×10⁴ cells/cm² in 175 cm² cell culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. All non-adherent cells were removed after 18 hours of incubation by washing culture dishes with Dulbecco’s phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) and ASCs received fresh growth media every other day. For further experiments and analysis, 80% confluent cell layers of passage 0 were frozen in 90% FCS containing 10% dimethyl sulfoxide (DMSO, Invitrogen, Life Technologies GmbH, Darmstadt, Germany).

2.4 Cell culture experiment with PRP

2.5×10⁵ human ASCs at passage 1–2 (n = 4) were seeded in a 100 mm cell culture dish (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and cultured in 7 ml of α-MEM supplemented with 20% FCS, 5 mM glutamine and 100 U/ml penicillin with 100 g/ml streptomycin for 48 hours. Afterwards, the medium was changed to serum-free α-MEM and cells were incubated for 24 hours for cell cycle synchronization. The medium was replaced by 5 ml media for all 6 experimental groups: Group 1: α-MEM containing 20% FCS, group 2: α-MEM containing 20% FCS with 10 ng/ml recombinant PDGF-AA, group 3: α-MEM containing 20% FCS with 10 ng/ml recombinant PDGF-AB, group 4: α-MEM containing 20% FCS with 10 ng/ml recombinant PDGF-BB, group 5: α-MEM containing 10% PRP, group 6: α-MEM containing 20% PRP. PRP from 4 volunteers was pooled and distributed according to the mentioned PRP concentrations. Control samples were taken right after synchronization (T0) and prepared for cell cycle analysis and Western Blot analysis (WB) as described below. Cells were incubated in corresponding medium for 6, 12, 24, 48 and 72 hours. Thereafter, the cells were harvested by trypsination for cell cycle analysis and Western Blot analysis.

2.5 Cell cycle analysis

Cells were washed twice with ice-cold PBS containing 2% FCS and incubated on ice overnight in 70% methanol. Afterwards, cells were washed twice with PBS and incubated in the presence of 10 μg/ml RNAse for 30 minutes at 37°C. The DNA intercalating 4^′,6-Diamidin-2-phenylindol (DAPI) fluorochrome was added at final concentration of 1 μg/ml 15 minutes prior to analysis to ensure quantitative DNA staining.

Flow cytometric analyses were performed on each sample with 3×10⁵ DAPI stained cells using a FACSCanto-II flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a blue (488 nm), a red (633 nm), and a violet (405 nm) laser and standard optical configuration. The instrument was operated with the FACSDiva software Version 7.0 (BD Biosciences). The DNA dye DAPI was excited with the violet excitation line and fluorescence emission was detected by the optical trigon unit equipped with a 450/50 bp filter. DNA histograms were plotted on a linear scale upon cell doublet, aggregate, and debris discrimination via pulse processing. Cell cycle fractions (i.e. percentages of cells in G0/G1-, S- and G2/M-phase) were quantified using the ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA). Treatment effects are expressed by the S-phase fraction (SPF) compared to untreated cells.

2.6 Western blot analysis

Cell culture media was removed and ASCs were washed twice in ice-cold PBS, and lysed in RIPA lysis buffer (Upstate, EMD Millipore Corporation, Temecula, CA, USA), including protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The proteins were separated on SDS/PAGE gel and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk, and subsequently incubated with rabbit anti PDGF receptor beta antibody (ab32570, 1 : 1000), rabbit anti MEK-1 antibody (ab96379, 1 : 2500), rabbit anti-c-MYC (ab32072, 1 : 5000), and mouse anti-GAPDH (ab8245, 1 : 5000) (all from Abcam, Cambridge, UK) for 1 hour at room temperature. Following incubation with goat anti-mouse IRDye 680 (LI-COR 926-68070, 1 : 15000) and goat anti-rabbit IRDye 800 (LI-COR 926-32211, 1 : 15000) secondary antibody, labeled proteins were detected using the LI-COR Odyssey detection system and the LI-COR Image Studio (LI-COR Biosciences, Lincoln, NV, USA).

2.7 PDGF receptor β Inhibition

PDGF signaling was inhibited with CP-673451 (Selleck Chemicals, Houston, TX, USA), a selective PDGF receptor β inhibitor. 2.5×10⁵ human ASCs at passage 1–2 (n = 3) were seeded in a 100 mm cell culture dish and expanded for 48 hours as described above before starving for 24 hours in serum-free medium as described above. Afterwards, the medium was changed to α-MEM with 3 μM inhibitor and cells were incubated for 30 minutes at 37°C and 5% CO₂ prior to exchanging medium and incubation with α-MEM containing: 20% FCS or 20% PRP. After 48 hours of incubation cells were harvested and prepared for cell cycle analysis and western blot as described above.

2.8 Statistics

Statistical analysis was performed using SPSS software package (version 19, IBM SPSS, Chicago, IL, USA) whereas all graphs were prepared by using GraphPad Prism (version 5, Statcon, La Jolla, CA, USA). All data were tested for normal distribution applying the Shapiro-Wilk test. Descriptive data are expressed in terms of mean±standard deviation (SD) as indicated. Differences between the SPF and protein expression of ASCs at different timepoints and in different cell culture conditions obtained by cell cycle analysis and by WB were investigated by One-Way Analysis of Variance (ANOVA) with Bonferroni correction. The paired t-test with Bonferroni correction was applied to identify significant differences between SPF and protein expression in ASCs treated with PDGF receptor β inhibitor and without receptor inhibition. The level of significance was set at P = 0.05 for all statistical tests.

3 Results

3.1 Time dependent effect of PRP on ASC proliferation

Flow cytometric analyses of ASCs revealed an increasing SPF in ASCs from 4.6±1.6% (T0), 4.4±2.8% at 6 hours and 3.6±1.3% at 12 hours, to the highest value at 24 hours (21.8±12.0%) in the presence of 20% FCS (Fig. 1A). After 48 hours SPF decreased to 10.2±4.1% and to 8.1% ±3.2% after 72 hours. A similar course could be observed in the presence of 10% PRP (Fig. 1B): 3.2±0.7% to 4.0±1.9%, to 33.9±12.4%, to 7.0±2.7%, to 5.8±1.3%. In the presence of 20% PRP, the increase of the SPF after 24 hours was even more pronounced with 25.6±10.8%. The highest SPF of 32.1±3.8% was detected after 48 hours, which dropped significantly to 5.8±0.7% after 72 hours (P < 0.01) (Fig. 1C). The highest SPF of ASCs was significantly increased after 48 hours in the presence of 20% PRP compared to 20% FCS and 10% PRP (both P < 0.01) (Fig. 3).

Fig.1

Flow cytometric analyses of time-dependent proliferation of human ASCs under different media conditions. Flow cytometric analyses of S-Phase fraction (SPF) of human ASCs in the presence of 20% FCS (A), 10% PRP (B) and 20% PRP (C) as medium supplement over a period of 72 hours in cell culture. Values represent the mean±SD of 4 experiments (^*P < 0.05 as indicated).

Fig.2

Influence of PDGF receptor β inhibition on the proliferation of human ASCs in flow cytometric analysis. Flow cytometric analyses of S-Phase fraction (SPF) of human ASCs in the presence of 20% FCS and 20% PRP, and additional PDGF receptor β inhibition (CP-673451) at 48 hours. Values represent the mean±SD of 4 experiments (^*P < 0.05 as indicated).

Fig.3

Proliferation of human ASCs after 48 hours under different media conditions. Flow cytometric analyses of S-Phase fraction (SPF) of human ASCs in the presence of 20% FCS and 10 ng recombinant PDGF subtypes -AA, -AB and -BB, 10% PRP and 20% PRP as medium supplement at 48 hours. Values represent the mean±SD of 4 experiments (^*P < 0.05 as indicated).

3.2 PRP stimulates ASC proliferation depending on signaling via PDGF receptor β

Cell cycle analysis of the SPF of ASCs revealed a significant higher percentage of cells in the S-Phase in the presence of 20% PRP in comparison to 20% FCS (P < 0.01) after 48 hours, with an SPF of 32.1±3.8% and 10.2±4.1%, respectively (Fig. 2).

Additional PDGF receptor β inhibition resulted in similar SPFs of 16.1±3.1% in the presence of 20% PRP in comparison to 10.4±4.2% in the presence of 20% FCS after 48 hours. Moreover, PDGF receptor β inhibition revealed a significant reduction in the SPF of ASCs in 20% PRP supplemented medium (P < 0.01) (Fig. 2). In contrast, similar SPFs of ASCs were found in 20% FCS supplemented medium with and without PDGF receptor β inhibitor.

3.3 PDGF subtypes -AB and -BB stimulate ASC proliferation

Flow cytometric analyses of ASCs revealed similar SPF in ASCs in the presence of 20% FCS and further supplementation of 10 ng PDGF -AA after 48 hours (9.0±1.3%). SPF of ASCs supplemented with 10 ng PDGF -AB (17.0±3.5%) was significantly higher than the SPF of cells with 10 ng PDGF -AA supplement (P < 0.05) (Fig. 3). Addition of 10 ng PDGF -BB resulted in a significantly higher SPF of 22.0±5.1% in comparison to 10 ng PDGF -AA after 48 hours (P < 0.01) (Fig. 3). No difference between the SPF of ASC cultured with 10 ng PDGF – BB and 10 ng PDGF – AB supplement was detected after 48 hours (Fig. 3). There was no significant difference between the SPF of ASCs in the presence of 10 ng PDGF-BB (22.0±5.1%) and 20 ng PDGF-BB (22.0±1.9%) (P = 0.21) (data not shown). The highest SPF was found in ASCs cultured with 20% PRP supplemented medium in comparison to the SPF of cells in presence of all other supplements respectively (32.1±3.8%, all P < 0.05) (Fig. 3).

3.4 Time dependent expression of PDGF receptor β, c-MYC, and MEK-1

Western blot analysis of PDGF receptor β expression in ASCs revealed a peak after 12 hours when cultured in α-MEM containing 20% FCS (P < 0.05) (Fig. 4A). In cells cultured in the presence of 10% or 20% PRP, the PDGF receptor β expression decreased significantly after 6 hours (both P < 0.01) and remained at a low level at all time-points in comparison to baseline T0 (all P > 0.05) (Fig. 4B and C).

Fig.4

Western blot analysis of PDGF receptor β expression in ASCs. Western blot analysis of PDGF receptor β expression in ASCs right after starving (T0), at 6, 12, 24 and 48 hours of incubation: ASCs at passage 3 or below were cultured in α-MEM containing 20% FCS (A), 10% PRP (B) and 20% PRP (C). Values represent the mean±SD of 4 experiments (^*P < 0.05 as indicated).

C-MYC expression in ASCs cultured in α-MEM containing 20% FCS also demonstrated a significant peak after 12 hours compared to baseline T0 (P < 0.05) (Fig. 5A). Whereas 10% and 20% PRP media supplementation induced a significant peak of c-MYC expression after 48 hours compared to all previous time points (all P < 0.05) (Fig. 5B and C). Furthermore, c-MYC expression of ASCs treated with 20% PRP substituted medium was significantly higher in comparison to 10% PRP after 48 hours (P < 0.01) (data not shown).

Fig.5

Western blot analysis of c-MYC expression in ASCs. Western blot analysis of c-MYC expression in ASCs right after starving (T0), at 6, 12, 24 and 48 hours of incubation: ASCs at passage 3 or below were cultured in α-MEM containing 20% FCS (A), 10% PRP (B) and 20% PRP (C). Values represent the mean±SD of 4 experiments (^*P < 0.05 as indicated).

MEK1 expression of ASCs cultured in α-MEM containing 20% FCS peaked after 24 hours of incubation (P < 0.01) (supplementary material 1A), whereas MEK1 expression peaked after 6 hours in presence of 10% PRP (P < 0.01) (supplementary material 1B). However, similar MEK1 expression was observed in ASCs cultured in the presence of 20% PRP at every time point respectively (all P > 0.05) (supplementary material 1C).

3.5 Influence of PDGF receptor β inhibition on protein expression in ASCs after 48 hours

PDGF receptor β expression was significantly lower in ASCs cultured for 48 hours in the presence of 20% PRP (0.10±0.02) in comparison to 20% FCS (0.46±0.01) (P < 0.01) (Fig. 6A). Inhibition of PDGF receptor β induced a significant decrease in the expression of PDGF receptor β in the presence of 20% FCS (to 0.09±0.02, P < 0.01) (Fig. 6B). On the contrary, inhibition of PDGF receptor β induced a significant increase in the expression of PDGF receptor β in the presence of 20% PRP (0.28±0.02, P < 0.05) (Fig. 6C).

Fig.6

PDGF Receptor β expression in ASCs after PDGF Receptor β inhibition. Western blot analysis of PDGF Receptor β (PDGFR β) expression in ASCs after 48 h: ASCs below passage 3 were cultured in α-MEM containing 20% FCS and 20% PRP with or without PDGF receptor β inhibitor (CP-673451) for 48 hours. GAPDH was used as a housekeeping gene. Values represent the mean±SD of 3 experiments (^*P < 0.05 as indicated).

C-MYC expression was significantly higher in ASCs cultured for 48 hours in the presence of 20% PRP (0.16±0.04) in comparison to 20% FCS (0.03±0.01, P < 0.05) (Fig. 7A). Inhibition of PDGF receptor β induced a significant reduction of c-MYC expression of ASCs in the presence of 20% FCS (0.0003±0.0006) and 20% PRP (0.002±0.001) (both P < 0.05) (Fig. 7B and C).

Fig.7

C-MYC expression in ASCs after PDGF Receptor β inhibition. Western blot analysis of C-MYC expression in ASCs after 48 h: ASCs below passage 3 were cultured in α-MEM containing 20% FCS and 20% PRP with or without PDGF receptor β inhibitor (CP-673451) for 48 hours. Values represent the mean±SD of 3 experiments (^*P < 0.05 as indicated).

MEK1 expression was similar in ASCs in the presence of 20% FCS (0.45±0.05) and 20% PRP (0.60±0.05) after 48 hours (P = 0.05) (supplementary material 2A). Inhibition of PDGF receptor β did not induce changes of expression levels of MEK1 in the presence of 20% FCS (0.31±0.02) or 20% PRP (0.53±0.13) (both P > 0.05) (supplementary material 2B and C).

4 Discussion

As suggested by our previous work [20, 21], we found a significant increase in proliferation of ASCs when incubated in presence of 20% PRP supplementation in comparison to 20% FCS. Here we prove for the first time that this effect is mediated by PDGF signaling, supported by the findings of PDGF receptor β inhibition in PRP cultures, and conversely the supplementation of FCS cultures with recombinant PDGF isoforms. Furthermore, we show a correlation between c-MYC-expression and ASCs proliferation in presence of 20% PRP.

Animal derived cell culture supplements bear the risk of contamination, immunogenicity or change in MSC gene expression level [19 , 22–24]. PRP has been proposed as a potential autologous alternative to xenogenic media supplements, or human allogenic supplements, that often reveal the limitation of suboptimal rate of cell proliferation [25]. The stimulatory effect of PRP on the proliferation of MSCs has been reported before [25 –27]. Taking various PRP preparations into account, it becomes crucial to describe the biological characteristics prior to development of reproducible in vivo or in vitro applications [28]. In a previous study, we characterized the biological composition of the applied leukocyte-poor PRP in detail [21]. We also proofed an unique change in the proteomic profile of ASCs in presence of PRP [22].

Van Pham et al. showed an elevated proliferation curve of ASCs when treated with PRP supplemented medium in comparison to FCS over a period of 105 hours. After 24 hours, ASCs began to proliferate significantly faster in presence of PRP [29]. In the present study, we illustrated a promoting effect of 20% PRP cell culture medium supplement on ASC proliferation after 24 to 48 hours of incubation. This effect peaked after 48 hours, and exceeded standard cell culture medium containing 20% FCS by far, even if recombinant PDGF subtypes – AA or -AB were added. In contrast to Van Pham et al., 20% PRP did not show a proliferation promoting effect on ASCs beyond 48 hours of incubation. Consequently, this implies the need for medium replacement every 3 days in our autologous cell culture setting utilizing 20% PRP as medium supplement. However, considerable cell confluency in our setting might have contributed to contact inhibition of ASCs at this time.

It has been suggested, that PRP promotes the proliferation of ASCs mainly via activation of PDGFR/AKT pathway [10], which is in line with the findings of this manuscript. Our proliferation analysis results further implicate, that PDGF subtypes -AB and -BB are effective in increasing ASCs proliferation comparable to the PPR effect and exceeding the effect of the subtype AA. Relevant in vivo PDGF-PDGF receptor interactions are those of PDGF -AA and PDGF -CC via PDGFR α, and PDGF -BB via PDGF receptor β [19]. Additional to the results of FCS culture supplementation with recombinant PDGF isoforms we highlighted the importance of PDGF receptor β signaling for the proliferation promoting effect of PRP on ASCs by inhibition of PDGF receptor β. Gharibi et al. described the regulation of proliferation and differentiation of MSCs under influence of PDGF -BB in detail. Their results implicate that PDGF receptor β signaling in MSCs simultaneously activates PI3K/AKT/mTOR and ERK signaling as mediator of differentiation [30]. ERK-signaling was described as an inhibitor of MSC differentiation by blocking PPARγ and CEBPα expression, whereas PI3K/AKT signaling was found to be the main contributor in promoting MSC cell cycle progression [30]. Moreover, a negative feedback mechanism between PI3K/AKT and PDGF receptor β expression has been demonstrated [30]. This is consistent with our results describing the inverse relationship between cell proliferation and PDGF receptor β expression in PRP cultured ASCs. Adding PDGF receptor β inhibitor significantly diminished cell proliferation in presence of PRP after 48 hours. In addition, we provide information about protein expression kinetics: The influence of 20% PRP lead to a significantly, ongoing decline of PDGF receptor β expression for at least 72 hours, which apparently has been initiated during the first 6 hours of incubation. It can be assumed, that the subsequent down regulation of PDGF receptor β in PRP-stimulated ASCs is based on the well described effect of receptor complex internalization into endosomes induced by ligand binding [31]. Growth factor triggered PDGF receptor β downregulation is a possible explanation for high PRP concentrations (e.g. 60%) having a minor impact, or even an inhibitory effect on the proliferation of different cell types, than lower concentrations (e.g. 20%) [10 , 31–33]. Nevertheless, in a previous study we found 20% PRP medium supplementation results in a higher proliferation promoting effect on ASCs than 10% PRP [22]. In summary, these results illustrate the importance of PDGF receptor β activation and PI3K/AKT signaling with a negative feedback regulation on PDGF receptor β expression for the proliferation of MSCs cultured in PRP. Thus in line we demonstrated that the PDGF subtypes – AB and – BB in PRP are the main mediators of the proliferation stimulating effect on ASCs.

The nuclear transcriptional factor c-MYC is a key mediator of PDGF-induced mitogenesis [34]. C-MYC transmits proliferative signals, regulates cellular growth by transversing into S phase of the cell cycle and is involved in differentiation [35 –37]. Stimulated proliferation of ASCs cultured in 20% PRP substituted medium was attributed to c-MYC over-expression in our study. The results of the PDGF receptor β inhibition experiments emphasize the dependency of ASC proliferation from c-MYC overexpression in presence of PRP. Jones et al. suggested that there is a common signaling cascade, by which mitogens drive arrested cells into the cell cycle involving the input of c-MYC, PI3K and MEK [38]. However, we were not able to detect a significant influence of PRP medium supplementation or PDGF receptor β inhibition on MEK1 expression in ASCs.

Noteworthy, overexpression of the oncoprotein c-MYC is also correlated with tumorigenesis and could lead to spontaneous transformation in MSCs [38 –40]. Assessing this major risk for the clinical application, a first study indicated a good resistance of ASCs to spontaneous transformation during in vitro expansion in the presence of pooled allogeneic human serum, despite high c-MYC protein expression associated with enhanced proliferation rates [41]. For clinical preparation of ASCs, extensive proliferation on the one hand and differentiation on the other hand is necessary. It has been shown that enforced c-MYC overexpression blocks terminal cell differentiation in different cell types [41 –43]. Interestingly, a reduced adipogenic differentiation capacity of ASCs cultured with PRP associated with PPARγ downregulation has recently been described by Amable et al. [44]. However, the influence of c-MYC overexpression on ASC differentiation in PRP supplemented medium remains to be described in detail.

These results taken together show clearly that c-MYC is an important mediator for ASC proliferation and differentiation.

The knowledge about PRP-triggered mechanisms that direct ASCs’ fate is a key to tissue engineering approaches, and crucial to meet GMP requirements for autologous cell culture settings. Adjusted growth factor concentrations in PRP are necessary for gaining optimal proliferation rates of ASCs. Considering paradoxical inhibitory effects of high growth factor concentrations, the “more is better” attitude of many PPR preparations [13 , 45] has to be questioned. This study highlights that PRP as culture medium supplement in ASC expansion is depending on PDGF receptor β signaling and provides robust ASC proliferation in a time dependent manner. Our findings identified c-MYC as an important PDGF receptor β downstream effector protein, mediating cell cycle progress and proliferation of ASCs in presence of PRP for the first time. The results of the present study advocated that PDGF plays the leading role in triggering the mitogenic signal-transduction leading to ASC proliferation.

5 Conclusion

PRP-induced PDGF signaling plays a decisive role in the stimulation of proliferation of ASCs, and regulates the protein expression over time. PRP can be considered a highly effective substitute for allogenic cell culture medium supplement. 20% PRP culture needs medium replacement every 3 days. C-MYC is a key-meditator of ASC proliferation in the presence of PRP, however its’ distinct role has to be further evaluated with special regard to the influence on ASC differentiation.

6 Limitations of this study

Several limitations of this study need to be noted: First, PRP samples were stored at –20 °C prior to cell culture experiments. Damage to platelets during storing could lead to activation of the platelets with subsequent release of growth factors [46, 47]. Therefore, the obtained data may not correctly mimic in vivo environment. Second, cell culture experiments were not performed with autologous PRP. However, all data originated from the same samples. Third, the low sample size of this study has to be considered, when interpreting the results. Last, the biological characteristics of the leukocyte-reduced PRP used in the present study have been described in detail previously [21], however it represents only one out of many PRP preparations available.

Competing Interests

Peter Angele is an expert advisor for Arthrex Inc. (Naples, FL, USA). All other authors declare that there is no conflict of interests regarding the publication of this paper.

Footnotes

Acknowledgments

The authors would like to thank Elke Gerstl for the support with all experiments. We would like to acknowledge Arthrex Inc. (Naples, FL, USA) for contributing to the sampling material.

The supplementary material is available in the electronic version of this article: .

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