RESEARCH

Comparative Effect of Human Platelet Derivativeson Proliferation and Osteogenic Differentiationof Menstrual Blood-Derived Stem Cells

Somaieh Kazemnejad • Roghaieh Najafi •

Amir Hassan Zarnani • Saman Eghtesad

Published online: 14 September 2013

� Springer Science+Business Media New York 2013

Abstract Menstrual blood has been recognized as an easily

accessible and inexpensive source of stem cells, in recent

years. To establish a safe and efficient protocol for develop-

ment of menstrual blood-derived stem cells (MenSCs) into

osteoblasts, the effect of substitution of fetal bovine serum

(FBS) with human platelet derivatives (HPDs) was evaluated

during proliferation and osteogenic differentiation of MenS-

Cs. To this aim, parallel experiments were carried out on

cultured MenSCs in the presence of platelet-rich plasma,

platelet-poor plasma, platelet gel supernatant, or human

platelet releasate (HPR), and compared with cells cultured in

conventional growth medium containing FBS. There was no

significant difference between growth curves of cultured

MenSCs in presence of different fortified media. However, the

MenSCs demonstrated variant differentiation patterns in

response to FBS replacement with HPDs. Mineralization, as

judged by Alizarin red staining, was significantly higher in

cells differentiated in the presence of HPR compared to cells

that were fortified with other medium supplements. A greater

osteocalcin production level, alkaline phosphatase activity,

and mRNA expression of osteogenic-specific genes in dif-

ferentiated MenSCs under HPR condition further confirmed

our previous findings. Based on our data, FBS substitution by

HPDs not only allows for successful MenSCs proliferation,

but also promotes MenSCs development into osteoblasts. The

effectiveness of HPR on osteogenic differentiation of MenSCs

represents an important novel step toward safe and applied

stem cell therapy of bone diseases.

Keywords Menstrual blood stem cells � Osteoblast �Differentiation � Human platelet releasates

Introduction

Bone and periodontal diseases are commonly treated by

autologous bone grafts. However, some potential compli-

cations such as invasive method of operation, chronic pain

and risk of infection limit the applicability of this technique

for clinical use. To remove the disadvantages of bone auto-

graft, stem cell therapy has been sought as a novel thera-

peutic strategy [1]. The numerous ethical dilemmas sur-

rounding the use of embryonic stem cells (ESCs) have

triggered the interest in application of adult stem cells in

therapy of different diseases. However, problems such as

less availability, invasive methods for sample collection

and lower proliferation capacity in comparison with ESCs

limit applicability of adult stem cells for clinical therapy of

liver diseases [2–4]. Pertaining to other sources of stem

cells, regardless of great achievements in generating ter-

minally differentiated cells from human-induced pluripo-

tent stem (iPS) cells, limitations such as the risk of tumor

formation are yet to be addressed [5, 6].

S. Kazemnejad (&)

Department of Embryology and Stem Cells, Reproductive

Biotechnology Research Center, Avicenna Research Institute,

ACECR, POB 19615-1177, Tehran, Iran

e-mail: s.kazemnejad@avicenna.ac.ir;

kazemnejad_s@yahoo.com

S. Kazemnejad � R. Najafi

Department of Biochemistry, Paramedical Faculty of Guilan

University of Medical Sciences, Langroud, Guilan, Iran

A. H. Zarnani

Nanobiotechnology Research Center, Avicenna Research

Institute, ACECR, Tehran, Iran

e-mail: zarnania@gmail.com

S. Eghtesad

Department of Biochemistry and Molecular Biology, University

of Maryland School of Medicine, Baltimore, MD, USA

123

Mol Biotechnol (2014) 56:223–231

DOI 10.1007/s12033-013-9698-9

Several studies have reported that menstrual blood (MB)

contains a unique population of cells with properties similar

to adult stem cells [7–9]. Compared to other sources of adult

stem cells, such as bone marrow and umbilical cord blood,

the MB offers much relative ease and wider window of

harvesting the cells (i.e., cord blood only available during

birth). Menstrual blood-derived stem cells (MenSCs) exhibit

long-term self-renewal ability, greater proliferation capacity

compared to umbilical cord- and bone marrow-derived

mesenchymal stem cells (BMSCs) and have minimal risk of

karyotypic abnormalities [10–13]. In addition, recent studies

have showed that reprogramming efficiency for generation

of iPS cells could be increased using MenSCs as a cell

source even in the absence of ectopic expression of c-Myc

[14, 15]. Such ready availability, ease of access and the

possibility of cyclic sample collection should allow autol-

ogous transplantation of MenSCs for large-scale clinical

application. Finding the male counterpart of these stem cells

(e.g., testis-derived cells) [16, 17] will further advance the

MenSCs potential for autologous transplantation. Mean-

while, along the line of investigations on therapeutic indi-

cations rendered by umbilical cord- and bone marrow-

derived mesenchymal stem cells, allogeneic transplantation

of MenSCs could be a potential approach to cell therapy. Of

note, MenSCs, referred to as endometrial regenerative cells,

have been shown to be safe when transplanted in male

patients with multiple sclerosis, characterized by absence of

immunological reactions or treatment-associated adverse

effects [18].

Recently, we showed that MenSCs could be committed

to osteogenic lineage, although with relatively lower

capacity compared to BMSCs [19]. As the MenSCs have

the potential to become an important future stem cell

source for basic research as well as clinical application,

protocols for an improved osteo-induction of MenSCs are

of particular interest.

Human platelet derivatives (HPDs) have been regarded

as a promoting factor on process of osteogenesis in vitro

and in vivo through releasing growth factors inside platelet

a-granules [19–25]. In some applications, platelets have

been used directly as platelet-rich plasma (PRP) [26, 27].

In other applications, platelet gels and releasates (or lysate)

have been used that were prepared from PRP. Moreover,

promoting effect of plasma and cryoprecipitate on regen-

eration and remodeling has been reported in some studies

[20, 21, 24, 28–30].

We recently demonstrated that fetal bovine serum (FBS)

substitution by human platelet releasate (HPR; as one human

platelet derivative) in culture medium of MenSCs, sub-

stantially improves the differentiation ability of MenSCs

into osteogenic lineage [19]. This replacement not only

promotes MenSCs development into osteoblasts, overcomes

potential hurdles associated with FBS-based culture such as

high cost and risks of infectious disease transmission and

immunological reaction due to the xenogenic origin of FBS

[31, 32]. However, based on available evidence, the quali-

tative and/or quantitative alterations in platelets potentially

affect the regenerative capacity of HPDs [27, 33, 34]. In the

current study, to establish a safe and efficient protocol for

induction of differentiation of MenSCs into osteoblasts, the

effects of different products of human platelet, such as PRP,

platelet-poor plasma (PPP), platelet gel supernatant (PGS),

or human platelet releasate (HPR) are evaluated. Parallel

experiments were carried out to determine mineralization

and expression levels of alkaline phosphatase (ALP) and

osteocalcin (OCN) in cultured cells in each condition.

Materials and Methods

Preparation of Human Platelet Derivatives

Seven units of PRPs were provided from Iranian Blood

Transfusion Organization. The PRPs were pooled and cell

content was subsequently determined using Sysmex K-1000

(Kobe, Japan). Approximately, 1.5 9 109 platelets/ml was

obtained that were divided into three equal portions. One

portion of PRP samples was frozen at -20 �C until use and

two other portions were processed for preparation of PGS

and HPR. Preparation of PGS was performed according to

established protocols with a modification [35, 36]. In brief,

1 ml of prepared thrombin was added to 4 ml of PRP and

incubated for 1 h at room temperature to nucleate platelet

gel. When clotting was complete, the supernatant was sep-

arated by centrifugation (4,0009g for 5 min at 4� C) and

frozen at -20� C. HPR was prepared according to our

protocol described previously [19]. Briefly, PRPs were

centrifuged at 3,0009g, 20 �C for 10 min to obtain platelet

pellet and PPP. The platelet concentrates were incubated at

room temperature for 30 min on a rotating platform to

eliminate platelet aggregates. After cell counting using

Sysmex K-1000, platelet density was adjusted to final con-

centration equivalent to PRP sample using phosphate buffer

saline (PBS). Next, the samples were activated by adding

thrombin in ratio of 1:4 to facilitate growth factor release.

The platelet releasate was centrifuged at 4,0009g for 5 min

to eliminate platelet membrane fragments. The supernatant

was filtered through 0.22 lm filter, divided into aliquots and

frozen at -20 �C for future use.

Isolation and Culture of MenSCs

MB was obtained from five healthy adult women of ages

ranged between 22 and 30 years without vaginal discharge

or infection using sterile Diva cup (Diva International,

lunette, Finland). The women enrolled to the study were

224 Mol Biotechnol (2014) 56:223–231

123

screened serologically for such sexually transmitted dis-

eases as HIV, Chlamydia, and herpes simplex virus. They

were also negative for HBV and HCV infections. All

participants had signed informed consent form approved by

the medical ethics committee of Guilan University of

Medical Sciences guidelines prior to providing samples for

these studies. Isolation of stem cells from MB was per-

formed as described earlier [19]. Briefly, mononuclear cells

were separated using Ficoll-Hypaque (GE-Healthcare,

Uppsala, Sweden) and washed. The cell pellet was sus-

pended in Dulbecco’s modified Eagle’s medium-F12

(DMEM-F12) (Sigma-Aldrich, MO, USA) containing

2.5 lg/ml fungizone (Gibco, Scotland, UK), 100 lg/ml

streptomycin and 100 U/ml penicillin and cultured in

polystyrene 75-cm2 tissue culture flasks. The flasks were

maintained at 37 �C in a humidified 5 % CO2 incubator.

Following 1–2 days of incubation, non-adherent cells were

discarded and only the adherent cell population remained

for our studies. Culture medium was refreshed every

3–4 days. When the cells reached 70 % confluency, they

were passaged using Trypsin/EDTA (Gibco). All experi-

ments were performed with cells at passage 2–4 from 3 to 6

donors.

Cell Proliferation Assay

MTT reduction test was employed to assess proliferative

capacity of MenSCs in presence of media supplemeted

with different platelet derivatives as described previously

[37]. Briefly, cells were seeded at a concentration of

5 9 103 cells/well in 12-well plates and cultured in

DMEM-F12 fortified with 10 % FBS, PGS, PPP, PRP, or

HPR for 9 days. Optical densities (OD) were measured at

570 nm using ELISA reader (Labsystem Multiskan, Fin-

land), with background subtraction at 670 nm.

Osteogenic Differentiation Protocol

In order to find out most efficient platelet derivative for

osteogenic differentiation of MenSCs, cells at passage 2–3

were plated in 6-well plates at a density of 10 9 103 cells/

well and cultured in DMEM-F12 fortified with distinct

platelet derivatives (PGS, PRP, PPP, HPR) or FBS plus

osteogenic agents: 0.1 lM dexamethasone, 10 lM b-glyc-

erophosphate, and 50 lM ascorbic acid (Sigma-Aldrich)

[38]. The cells were cultured in media supplemented with

15 % FBS or platelet derivatives without osteogenic

inducers served as control cells. Cells were in culture for

14 days and the media were changed twice a week. After

2 weeks, osteogenic properties were assessed in differen-

tiated cells by calcium accumulation staining, ALP activity

assay, OCN level and expression of osteoblast-specific

genes, and were compared to those in control cells.

Evaluation of Differentiated Cells

To evaluate calcium accumulation in mineralized cells,

cultured cells were washed with PBS and fixed in 10 %

neutral-buffered formalin for 20 min. Cells were then

stained with alizarin red (Sigma-Aldrich) for 20 min.

During this time, cells were checked microscopically for

orange-red color development. As soon as the color

appeared, cells were washed in deionized water to

remove excess dye and then checked with invert

microscope (Olympus CKX41, USA). Measurement of

ALP and OCN serves as sensitive means of identification

of osteogenic development [39]. Detection of ALP

activity and OCN production was used for more in-depth

characterization of differentiated MenSCs into osteoblast-

like cells at various culture conditions. For detection of

ALP activity and OCN production, culture media was

collected at day 1, 5, 10, and 14 of differentiation and

ALP activity and OCN production were, respectively,

measured by a colorimetric assay kit using p-nitrophenyl

phosphate as substrate (Pars Azmun, Iran) and human

OCN ELISA quantitative Kit (Invitrogen) according to

the manufacturer’s recommendations.

Reverse Transcription-Polymerase Chain Reaction

(RT-PCR)

RT-PCR was performed to assess expression of osteocyte-

related genes ALP, OCN, and parathormone receptor

(PTHR). Briefly, total RNA was isolated from 1 9 106

cells by a standard RNA extraction protocol using RNA-

bee (Biosite, Uppsala, Sweden). Reverse transcription was

performed using 2 lg purified RNA, 200 U/ll M-MuLV

Reverse Transcriptase (Fermentase, Vilnius, Lithuania),

20 pM N6 Random-Hexamer (Cybergene, Stockholm,

Sweden), 59 RT buffer, and 20 pM dNTP Mix (Roche,

Mannheim, Germany) in a thermocycler (Eppendorf, Ger-

many) at 42 �C for 60 min. Then 1 ll of cDNA was

admixed with 12.5 ll reaction master mix (Amplicon,

Copenhagen, Denmark) and 1 ll of each primer (Table 1).

As indicated in Table 1, after initial denaturation at 94 �C

for 3 min, PCR amplification was continued at 94 �C for

30 s, annealing temperature for 30 s, and 72 �C for 30 s for

total cycles of 35 and final extension was performed at

72 �C for 7 min. Each PCR was performed under linear

conditions with b-actin used as an internal standard. The

amplified DNA fragments were electrophoresed on 1.5 %

agarose gel and visualized by ultra-violet transilluminator

(Uvitec-USA). For semi-quantitative determination, gel

images were analyzed using the AlphaEase software

(Genetic Technologies, Inc, USA). Values for all genes

were normalized to that of the corresponding b-actin.

Mol Biotechnol (2014) 56:223–231 225

123

Statistical Assay

All measurements were performed in triplicate. The results

are reported as the mean ± SD. Statistical analysis was

performed using non-parametric Mann–Whitney U test.

For all statistical analysis, the SPSS 13 software was used

and p value \0.05 was considered significant.

Results

Effect of Different Culture Media Supplements

on MenSCs Expansion

The MenSCs exhibited spindle-shaped, fibroblastic mor-

phology and a homogeneous cell population with a char-

acteristic, non-haematopoietic phenotype. FBS replacement

with each type of HPDs caused no gross difference in cell

morphology and the cells preserved their fibroblastic

appearance (Fig. 1a). Moreover, the growth-stimulating

activity of HPDs (HPR, PRP, PPP, PGS) in MenSCs cultures

was evaluated by measuring cell proliferation on days 1, 3,

6, and 9 after exposure, and comparing it to the conventional

medium (supplemented by FBS). As shown in Fig. 1b, the

MenSCs revealed an ascensional time-dependent growth

pattern in presence of all culture conditions. On the other

hand, there was no significant difference between the growth

curves of neither cultured MenSCs in presence of different

HPDs, nor those in FBS.

Evaluation of MenSCs Osteogenesis

Mineralization is a hallmark of end-stage osteoblastic dif-

ferentiation that is judged by formation of nodule-like

structures with calcium deposits [38]. Mineralization was

assessed in cultured cells for evaluation of osteoblast dif-

ferentiation. As shown in Fig. 2, MenSCs in the presence

of all of the HPD-supplemented media and osteogenic

inducers successfully reached the mineralization step.

However, the degree of mineralization was dependent on

the contents of culture media. Whenas, substitution of FBS

with PPP in the presence of osteogenic factors had no

appreciable effect on mineralization potential of MenSCs,

the level of calcium deposits increased with replacement of

FBS by PGS, PRP and HPR in media containing osteo-

genic factors. The promoting efficiency of this replacement

on formation of calcium deposits was especially significant

in differentiated MenSCs in the presence of HPR. Omis-

sion of osteogenic inducers resulted in no mineralization in

all groups.

OCN production was examined in cultures derived from

differentiated MenSCs at various post-differentiation time

points (Fig. 3b). Different from the cultured cells in media

without osteogenic factors, the differentiated cells in the

presence of HPDs or FBS supplemented media with oste-

ogenic factors showed enhancing levels of OCN during

differentiation. In parallel to the mineralization level, OCN

levels of differentiated MenSCs in the presence of PRP,

HPR, and PGS were more pronounced in comparison to

those of FBS- and PPP-containing media. In addition, OCN

level of differentiated cells in the presence of HPR was

approximately 2.7 folds higher than the detected level of

OCN in FBS group (p \ 0.001). Moreover, ALP activity in

culture media retrived from differentiated MenSCs was

evaluated at different time points. As shown in Fig. 3a, in

all differentiation groups ALP level of differentiated

MenSCs indicated a gradual increasing pattern during

differentiation. However, only replacement of FBS with

HPR and PGS had a significant effect on ALP level in

differentiated cells (p \ 0.001 and p \ 0.01, respectively).

There was no significant difference between ALP activity

of differentiated cells in the presence of PPP and PRP with

that in the FBS-containing media (p \ 0.1 and p \ 0.07,

respectively).

Based on the obtained results of cytochemical and

molecular assays, expression pattern of osteogenic genes

including OCN, PTHR, and ALP in MenSCs differentiated

in the presence of HPR and osteogenic inducers was inves-

tigated in reference to differentiated cells by FBS and oste-

ogenic agents using semi-quantitative RT-PCR (Fig. 3c).

Table 1 Sequence of the

primers used in this study

ALP alkaline phosphatase, OCN

osteocalcin, PTHR

parathormone receptor

Gene Sequence Annealing

temperature

(�C)

NCBI

accession

number

ALP F: 50-CTCTCCAAGACGTACAACACC-30

R: 50-AATGCCCACAGATTTCCCAGC-3052.3 NM_0004784

OSTEO F: 50-GCTGGCCAGGCAGGTGCGA-30

R: 50-CTCCTGAAAGCCGATGTGGTCA-3061.5 NM_199173

PTHR F: 50-CTCCGGGAACAAAAAGTGGAT-30

R: 50-CTGAGACCTCGGTGTATGGTG-3052.7 NM_0003162

b-Actin F: 50-GTGGGGGCGCCCCAGG-30

R: 50-CTCCTTAATGTCACGCACGAT TTC-3060 NM_001101

226 Mol Biotechnol (2014) 56:223–231

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Although there was a significant inter-individual variation in

the expression pattern of the evaluated genes, median level of

OCN and ALP was significantly more upregulated in dif-

ferentiated MenSCs by HPR compared to that of FBS-driven

cells (OCN: p \ 0.05, ALP: p \ 0.019) (Fig. 3d). The

upregulation level of PTHR was not significantly different

between the two groups (p \ 0.8).

Discussion

Bone diseases are common worldwide and have a signifi-

cant impact on overall health status of human population.

Currently, due to potential complications following autol-

ogous bone grafting, including chronic pain, risk of

infection and limited bone availability, stem cell therapy

has been considered as an alternative therapeutic approach

for treating bone defects. However, problems with well-

known stem cell sources, such as low availability, painful

access, or limited proliferative ability [9], has propelled

scientists to take advantage of MenSCs in cell therapy of

bone disorders as well as in other diseases.

In our previous study, considerable differences were

beheld between MenSCs and BMSCs regarding

proliferative capacity, immunophenotype and osteogenic

differentiation potential. We showed that MenSCs possess

some markers of mesenchymal stem cells (MSC) such as

CD29, CD44, CD73, CD105, and CD146. However, these

cells cannot be simply classified as MSC due to their much

higher growth capacity compared to BMSCs, and expression

of ESC marker, OCT-4, which is not commonly expressed in

MSC [19]. We showed that MenSCs could be committed to

osteogenic lineage, although with relatively lower capacity

compared to BMSCs. It seems that immunophenotypic

differences of MenSCs and BMSCs are the main cause of

discrepancy between osteogenic differentiation potential of

these cells. Therefore, finding a proper stimulus to trigger

osteogenic differentiation of MenSCs is needed prior to

using MenSCs for clinical therapy of bone diseases.

Recently, the use of HPDs for expansion and differen-

tiation of stem cells has been suggested as a promising FBS

substitute [19–25, 30, 40]. The regenerative potential of

platelet derivatives depends on two key factors, including

(1) platelet concentration, and (2) the processing technique,

which influences on concentration of platelet-derived

growth factors [33, 34, 36].

In as much as concentration of released secretory pro-

teins would be proportional to the platelet concentration

Fig. 1 Morphology and

proliferation potential of

MenSCs in presence of different

human platelet derivatives.

a The MenSCs showed a

fibroblastic-like shape

regardless of supplement media

type; Scale bar: 100 lm.

b MTT results of expanded

MenSCs in presence of different

human platelet derivatives at

different times after cell

seeding; FBS fetal bovine

serum, HPR human platelet

releasate, PGS platelet gel

supernatant, PRP platelet-rich

plasma, PPP platelet-poor

plasma

Mol Biotechnol (2014) 56:223–231 227

123

Fig. 2 Cytochemical

evaluation of MenSCs

differentiated into osteoblasts.

The accumulation of calcium

deposits in differentiated cells in

the presence of conventional

osteogenic medium fortified

with FBS (a), or osteogenic

medium supplemented with

PGS (b), PRP (c), HPR (d), and

PPP (e) is shown by Alizarin

red staining. Cultured MenSCs

in the presence of FBS (f), PGS

(g), PRP (h), HPR (i), and PPP

(j) without osteogenic inducers

served as control. Scale bar:

100 lm

228 Mol Biotechnol (2014) 56:223–231

123

ratio, the concentration of platelets in different HPDs was

assimilated. However, as anticipated, differences in pro-

cessing techniques result in variation in platelet deriva-

tives, leading to different growth factor concentrations and

consequent impacts on osteogenic differentiation ability of

cells.

Therefore, it is crucial to optimize conditions, in which

MenSCs proliferate and differentiate in response to specific

growth factors or platelet derivatives. Data demonstrated in

this study suggest that FBS replacement with each of the

platelet derivatives had no significant effect on MenSC

growth, while the cells revealed different behavior in

response to variant supplemented differentiation media. On

the other hand, intensity of mineralization in parallel to

functionality markers, such as ALP activity and OCN pro-

duction, was most pronounced in differentiated MenSCs in

the presence of HPR compared to other groups. The cyto-

chemical and functional observations were corroborated

with more extent of osteogenic-specific markers including

OCN and PTHR in differentiated cells cultured in the pre-

sence of HPR compared to differentiated cells in conven-

tional differentiation media (FBS plus osteogenic inducers).

Although molecular mechanisms involved in improve-

ment of MenSC differentiation in presence of HPR are

unclear, it seems that the specific differentiation promoting

growth factors that are present in higher concentrations in

HPR in comparison with other HPDs interfere in this

process. This precept is specially supported with observa-

tion that PPP had the weakest effect on osteogenic differ-

entiation of cultured MenSCs. Therefore, the determination

of key growth factors concentrations such as TGF-b and

PDGF [41–43] in HPR compared to other HPDs is a major

issue which helps to gain broader insights about the

obtained results. Moreover, reason of different pattern of

HPR influence on MenSCs proliferation and differentiation

is remained to be determined. It sounds that cocktail of

competence and progressive growth factors and cytokines

in HPR activates signaling pathways of MenSCs in line of

differentiation promotion more than proliferation. In fact,

platelet derivatives affect on mitogenic and differentiation

potential depend on cell type and growth factors/cytokines

concentration [44–47].

Based on accumulative data presented here, all tested

human supplements support MenSCs proliferation and

osteogenic differentiation. HPR, however, seems to be the

optimal component, assuring efficient osteogenic devel-

opment of MenSCs. Further in vivo studies focusing on

genomic stability or lack of transformation are currently

underway to assure that the osteocyte-like cells driven from

MenSCs have biological application for clinical therapy.

Fig. 3 Functional and Molecular characterization of differentiated

MenSCs into osteoblasts. Alkaline phosphatase (ALP) activity and

osteocalcin (OCN) levels in cell supernatants through differentiation

are shown in a and b; � Significant difference between specified day

and the previous time period of the same group (p \ 0.05),� Significant difference (p \ 0.05) between groups at the same day.

c Expression pattern of osteogenic-specific transcripts; osteocalcin

(OCN), parathormone receptor (PTHR), and ALP in differentiated

cells in the presence of conventional osteogenic medium supple-

mented with HPR and FBS. d Densitometric analysis of RT-PCR

results; data of differentiated cells were normalized to corresponding

b-actin and calculated in reference to contol cells. � Significant

difference between FBS and HPR groups (p \ 0.05)

Mol Biotechnol (2014) 56:223–231 229

123

Acknowledgments This work was supported by a research grant

from Research Vice-chancellor of Guilan University of Medical

Science. We thank Mojtaba Hosseinpoor for technical assistance.

Conflict of interest The authors indicate no potential conflicts of

interest.

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