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Research study

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Clauser L, Tieghi R, Palmieri A, Carinci F. Adipose-derived stem cells secrete neurotrophic factors. Annals of Oral & Maxillofacial Surgery 2013 Mar 01;1(2):12.

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Adipose-derived stem cells secrete neurotrophic factorsL Clauser1, R Tieghi1, A Palmieri2, F Carinci1*

AbstractIntroductionStructural fat grafting is a widely approved technique in craniomaxil-lofacial reconstructive surgery used to treat several types of diseases including congenital and post-trau-matic deformities, and to fill out tis-sue depression due to orbital and periorbital surgery, scars or cancer resections.

Adipose tissue is considered a secreting organ that produces spe-cial proteins such as adipokine and neurotrophic factors such as nerve growth factor and brain-derived neu-rotrophic factor.

Given their role in stimulating re-pair of peripheral nerves, their ex-pression could influence the good outcome of a structural fat grafting. This study discusses the ability of adipose-derived stem cells to secrete neurotrophic factors.Materials and methodsIn this study, we compared the abil-ity of adipose-derived stem cells and adipocytes, obtained from the same patient, to secrete these two neuro-trophic factors. The expression levels of nerve growth factor and brain-derived neurotrophic factor were measured using quantitative real-time polymerase chain reaction.

ResultsStem cells showed up-regulation of brain-derived neurotrophic factor

and nerve growth factor with respect to adipocytes.DiscussionThese result demonstrated that adi-pose tissue is an abundant and eas-ily accessible wealth of mesenchymal stem cells and that these cells are re-sponsible for the production of nerve growth factor and brain-derived neu-rotrophic factor, which are involved in tissue and nerve repair.ConclusionAdipose tissue is easily obtainable by lipoaspiration with minimal discom-fort to the patient, and its MSC content is adequate for clinical-grade cell ma-nipulation in regenerative medicine.

IntroductionStructural fat grafting is a widely ap-proved technique in craniomaxillo-facial reconstructive surgery. It can be used to treat several types of dis-eases including congenital and post-traumatic deformities, and to fill out tissue depression due to orbital and periorbital surgery, scars or cancer resections1–4.

To restore the normal shape and vol-ume of soft tissue, regenerative medi-cine exploits a natural resource that is human adipose tissue. For years, sev-eral methods followed one another, but the one introduced by Coleman in 19915 is still successfully employed. Briefly, it provides the aspiration, pu-rification and subsequent reinjection of autologous fat. Each step must be performed carefully to prevent fatty structure damage. The importance and usefulness of this approach are due to its high yield in terms of sur-vival rate of fat grafting (of up to 90%).

Adipose tissue is an abundant and easily accessible wealth of mesen-chymal stem cells (MSC), which are able to differentiate into different types of cell lines, thus restoring soft

tissues. It is considered a secreting organ which produces special pro-teins such as adipokine6 and neuro-trophic factors such as nerve growth factor (NGF)7 and brain-derived neu-rotrophic factor (BDNF)8.

NGF is a target-derived neurotro-phin that plays a key role in growth and maintenance of sympathetic in-nervation of tissues such as the adi-pose tissue. NGF is also involved in immune and inflammatory respons-es9. In many studies, it was shown that different tissues including adi-pose tissue secreted NGF7,10.

BDNF is a potent anti-diabetic and anorexigenic factor11, with an impor-tant role in differentiation and plas-ticity of the central nervous system, body weight control and energetic homeostasis12.

Given their role in stimulating re-pair of peripheral nerves, their ex-pression could influence the good outcome of a structural fat grafting.

In this study we compared the ability of adipose-derived stem cells (ADSCs) and adipocytes, derived from the same patient, to secrete these two neurotrophic factors. The expression levels of NGF and BDNF were measured using the quantita-tive real-time polymerase chain reac-tion (qRT-PCR).

Materials and methodsThis study conforms to the values laid down in the Declaration of Helsinki (1964). The protocol of this study has been approved by the relevant ethical committee related to our in-stitution in which it was performed. All subjects gave full informed con-sent to participate in this study.

Stem isolationHuman adipose tissue was obtained by liposuction of adult volunteer

* Corresponding author Email: crc@unife.it; francesco.carinci@ unife.it1 Department of Morphology, Surgery and

Experimental Medicine, University of Ferrara, Ferrara, Italy

2 Department of Experimental, Diagnostic and Specialized Medicine, University of Bologna, Bologna, Italy

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Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Clauser L, Tieghi R, Palmieri A, Carinci F. Adipose-derived stem cells secrete neurotrophic factors. Annals of Oral & Maxillofacial Surgery 2013 Mar 01;1(2):12

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patients. Fat was finely minced with sterile scissors and transferred to a tube containing digestive solution (DMEM containing 1 mg/ml of colla-genase type II). The tube was placed in 37°C water bath for 60 min, swirl-ing occasionally.

The sample was centrifuged at 1300 rpm for 5 min. Then, it was removed from centrifuge, shaken vigorously (to complete separation of stromal cells from primary adi-pocytes) and centrifuged again for 5 min. The oil on the top of the tube (which includes primary adipocytes) was aspirated and discarded, while the stromal fraction at the bottom was washed three times with 10 ml of PBSA 1× and centrifuged again for 5 min. After the last wash, the pellet was resuspended in 10 ml of Alphamem medium (Sigma Aldrich, Inc., St. Louis, MO), supplemented with 10% foetal calf serum, antibiot-ics (penicillin 100 U/ml and strepto-mycin 100 µg/ml—Sigma, Chemical Co., St. Louis, MO) and amino acids (l-glutamine—Sigma, Chemical Co., St. Louis, MO). The medium was changed after 2–3 days. Cells were characterized for staminality by flow cytometric analyses.

Flow cytometric analysesThe purity of cell cultures was deter-mined by analysis of different anti-gens after staining with fluorochrome (FITC- or PE-) conjugated mAbs anti-human CD14-FITC, CD14-PE, CD34-FITC, CD45-FITC, CD90-PE, CD105-PE (Immunotech, Marseille, France), and analysed by FACScan. The non-spe-cific mouse IgG was used as isotype control (Immunotech). To avoid non-specific fluorescence from dead cells, live cells were gated tightly using for-ward and side scatter (Figure 1).

Primary human dermal fibroblast (HFb) cells cultureFragments of dermal tissue of healthy volunteers were collected during op-eration. The pieces were transferred to 75 cm2 culture flasks containing

11,0000g for 1 min, to permit RNA to bind at the membrane. Contaminat-ing DNA was removed using rDNase solution, which was directly applied onto the silica membrane.

Samples were washed with two dif-ferent buffers to remove salts and me-tabolites. Pure RNA was finally eluted with RNase-free water and quantified at NanoDrop (Thermo Scientific).

cDNA synthesisTotal RNA (500 ng) was mixed with 50 ng of random nonamers primer, dNTP mix (10 mM each) and RNase-free water, in a final volume of 12 µl.

The reaction was heated to 65°C for 5 min and quick-chilled on ice. Then the First-Strand Buffer (Invitro-gen) and 0.1 M DTT were added 5×. After incubation at 25°C for 2 min, 1 µl (200 units) of SuperScript™ II en-zyme (Invitrogen) was added.

DMEM medium (Sigma Aldrich, Inc., St. Louis, MO) supplemented with 20% foetal calf serum and antibiotics (penicillin 100 U/ml and streptomy-cin 100 µg/ml—Sigma Aldrich, Inc., St Louis, MO).

Cells were incubated in a humidi-fied atmosphere of 5% CO2 at 37°C. Medium was changed the next day and twice a week. After 15 days, the pieces of dermal tissue were removed from the culture flask. Cells were har-vested after 24 days of incubation.

RNA processingTotal RNA was isolated from ADSCs, HFb and adipose tissue using Nucle-ospin RNA tissue (Machery Nagel).

Cells and tissues were lysed by incubation in the lysis buffer (RA1). After addition of 70% ethanol, the lysate was loaded onto the Nucle-ospin column and centrifuged at

Figure 1: Surface antigene profile of ADSCs. Phenotypic characterization by flow cytometry of cell cultures derived from adipose tissue by staining with the indicated mAb. Representative dot plots documenting the purity of cell preparations and the homogenously CD105+, CD90+, CD34−, CD45, CD14− surface antigen profile, are shown. Irr., irrelevant, isotype control Ab.

Page 3 of 5

Research study

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Clauser L, Tieghi R, Palmieri A, Carinci F. Adipose-derived stem cells secrete neurotrophic factors. Annals of Oral & Maxillofacial Surgery 2013 Mar 01;1(2):12

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ResultsADSCs were phenotypically char-acterized by flow cytometric analy-ses. Cell preparations derived from adipose tissue were homogenously CD105+, CD90+, CD34−, CD45−, CD14−, which is a typical MSC surface anti-gen profile (Figure 1).

The expression level of two growth factors, NGF and BDFN, was com-pared in three cell types: adipocytes, HFb and ADSCs.

Gene expression was quantified using qRT-PCR and normalized to the expression of the housekeeping gene RPL13.

Comparing expression levels of ADSCs and HFb, we found that NGF gene was over-expressed in stem cells. Conversely BDFN was down-regulated (Figure 2).

These two genes were down-regu-lated in adipocyte when compared with fibroblast (Figure 3) and stem cells.

Stem cells show up-regulation of BDFN and NGF with respect to adi-pocyte (Figure 4).

DiscussionRecent studies have shown that adi-pose tissue is a paracrine and endo-crine organ, able to produce signal proteins termed adipokines, proteins involved in the regulation of energy balance, insulin sensitivity and glu-cose tolerance, vascular homeostasis, lipid metabolism, angiogenesis and inflammation6,14.

Adipokines are responsible for the complex processes of adipose tissue remodelling with repair of peripher-al nerves and changes in the density of blood vessels.

Adipose tissue is innervated by sympathetic nerves and secretes two neurotransmitters, NGF and BDFN, involved in the stimulation of lipolysis15, control of cell number and growth and maintenance of sympa-thetic neurons within tissues16.

Their expression could influence the good outcome of structural fat grafting, a procedure larg- ely employed in craniomaxillofacial

The reaction was incubated at 42°C for 50 min and inactivated by heating at 70°C for 15 min.

Real-time PCRcDNA was amplified by real-time PCR by using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) and the specific assay designed for the investigated genes.

SYBR® Green assays reactions were performed in a 20 µl volume using the ABI PRISM 7500 (Applied Biosystems, Foster City, CA). Each reaction contained 10 µl 2× Power SYBR® Green PCR Master Mix (Ap-plied Biosystems, Foster City, CA), 400 nM concentration of each primer and cDNA.

Forward and reverse primers for the selected genes were designed

using primer express software (Ap-plied Biosystems, Foster City, CA), and are listed in Table 1.

All experiments were performed including non-template controls to exclude reagent contamination. PCRs were performed with two biological replicates.

Expression was quantified using qRT-PCR. The gene expression levels were normalized to the expression of the housekeeping gene RPL13. Quan-tification was done with the delta/delta calculation method13.

Statistical analysesComparison of gene expression be-tween three types of cells was per-formed with two-tailed ANOVA using Excel spreadsheets (Microsoft Office 2003).

Figure 2: Relative gene expression in ADSCs compared with HFb.

Table 1 Primer sequences for SYBR® green assay

Gene name Primer sequences (5´–3´)

NGF F-CGTCCGGACCCAATAACAGTR-AGTGTGGTTCCGCCTGTATG

BDFN F-CGTGAGGTTTGTGTGGACCCR-CCACCTTGTCCTCGGATGTT

RPL13 F-CTGGAGGAGAAGAGGAAAGAGAR-TTGAGGACCTCTGTGTATTTGTCAA

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Research study

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Clauser L, Tieghi R, Palmieri A, Carinci F. Adipose-derived stem cells secrete neurotrophic factors. Annals of Oral & Maxillofacial Surgery 2013 Mar 01;1(2):12

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In this study, we have compared the ability of ADSCs and adipocytes, derived from the same patient, to se-crete these two neurotrophic factors. The expression levels of NGF and BDNF were measured using qRT-PCR.

The expression level of NGF and BDFN was compared in three cell types: adipocytes, stem cells isolated from adipose tissue and dermal fi-broblasts.

Obtained results showed that AD-SCs over-expressed NGF and BDFN genes, compared with adipocyte, in-dicating that stem cells are the main producers of neurotrophic factor in adipose tissue.

NGF expression is also up-regulat-ed in stem cells in respect to dermal fibroblast.

Adipose tissue is typically used in structural grafting to restore the dermal tissue of origin. Evidence that stem cells from adipose tissue over-expressing NGF in respect to fibroblasts make these cells a useful source for tissue regeneration.

ConclusionThese results demonstrate that the production of NGF and BDFN is due to the stem cells present in the adi-pose tissue rather than adipocytes.

The frequency of stem cells within adipose tissue ranges from 1:100 to 1:1500 cells, which far exceeds the frequency of marrow stromal cells (MSCs) in the bone marrow.

Adipose tissue is an ideal source of autologous stem cells, particularly in comparison with the traditional bone marrow cells procurement procedure, as it is easily obtainable by lipoaspiration under local anaes-thesia with minimal discomfort to the patient, and its MSC content is adequate for clinical-grade cell ma-nipulation in regenerative medicine.

References1. Clauser L, Polito J, Mandrioli S, TieghiR, Denes SA, Galiè M. Structural fat graft-ing in complex reconstructive surgery. J Craniofac Surg. 2008 Jan;19(1):187–91.

into different types of cell lines, thus restoring soft tissues17.

Many studies have demonstrated that differentiated ADSCs produce neurotrophic factors, such as BDNF and NGF,18–20 which are responsible for their neurotrophic effects. The ease of harvesting and the rapid expansion capability make the ADSC cultures ide-al candidates in regenerative medicine and peripheral nerve regeneration.

reconstructive surgery, to restore congenital and post-traumatic de-formities and to fill out tissue depres-sion due to orbital and periorbital surgery, scars or cancer resections1–4.

Adipose tissue represents a com-plex organ of preadipocytes, adipo-cytes, endothelial cells, stem cells and nerve fibres. MSCs (ADSCs) are very abundant and easily isolable in this tissue. They are able to differentiate

Figure 3: Relative gene expression in ADSCs compared with adipocytes.

Figure 4: Relative gene expression in adipocytes compared with HFb.

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Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Clauser L, Tieghi R, Palmieri A, Carinci F. Adipose-derived stem cells secrete neurotrophic factors. Annals of Oral & Maxillofacial Surgery 2013 Mar 01;1(2):12

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