Adipose-derived stem cells secrete neurotrophic factors

Abstract Introduction Structural fat grafting is a widely approved technique in craniomaxillofacial reconstructive surgery used to treat several types of diseases including congenital and post-traumatic deformities, and to fill out tissue depression due to orbital and periorbital surgery, scars or cancer resections. Adipose tissue is considered a secreting organ that produces special proteins such as adipokine and neurotrophic factors such as nerve growth factor and brain-derived neurotrophic factor. Given their role in stimulating repair of peripheral nerves, their expression 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 methods In this study, we compared the ability of adipose-derived stem cells and adipocytes, obtained from the same patient, to secrete these two neurotrophic factors. The expression levels of nerve growth factor and brainderived neurotrophic factor were measured using quantitative realtime polymerase chain reaction.


Introduction
Structural fat grafting is a widely approved technique in craniomaxillofacial reconstructive surgery.It can be used to treat several types of diseases including congenital and posttraumatic deformities, and to fill out tissue depression due to orbital and periorbital surgery, scars or cancer resections [1][2][3][4] .
To restore the normal shape and volume of soft tissue, regenerative medicine exploits a natural resource that is human adipose tissue.For years, several methods followed one another, but the one introduced by Coleman in 1991 5 is still successfully employed.Briefly, it provides the aspiration, purification 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 survival rate of fat grafting (of up to 90%).
Adipose tissue is an abundant and easily accessible wealth of mesenchymal 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 proteins such as adipokine 6 and neurotrophic factors such as nerve growth factor (NGF) 7 and brain-derived neurotrophic factor (BDNF) 8 .
NGF is a target-derived neurotrophin that plays a key role in growth and maintenance of sympathetic innervation of tissues such as the adipose tissue.NGF is also involved in immune and inflammatory responses 9 .In many studies, it was shown that different tissues including adipose tissue secreted NGF 7,10 .
BDNF is a potent anti-diabetic and anorexigenic factor 11 , with an important role in differentiation and plasticity of the central nervous system, body weight control and energetic homeostasis 12 .
Given their role in stimulating repair of peripheral nerves, their expression 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 quantitative real-time polymerase chain reaction (qRT-PCR).

Materials and methods
This 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 institution in which it was performed.All subjects gave full informed consent to participate in this study.

Stem isolation
Human adipose tissue was obtained by liposuction of adult volunteer patients.Fat was finely minced with sterile scissors and transferred to a tube containing digestive solution (DMEM containing 1 mg/ml of collagenase type II).The tube was placed in 37°C water bath for 60 min, swirling 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 adipocytes) 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, antibiotics (penicillin 100 U/ml and streptomycin 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.

Primary human dermal fibroblast (HFb) cells culture
Fragments of dermal tissue of healthy volunteers were collected during operation.The pieces were transferred to 75 cm 2 culture flasks containing 11,0000g for 1 min, to permit RNA to bind at the membrane.Contaminating DNA was removed using rDNase solution, which was directly applied onto the silica membrane.
Samples were washed with two different buffers to remove salts and metabolites.Pure RNA was finally eluted with RNase-free water and quantified at NanoDrop (Thermo Scientific).

cDNA synthesis
Total RNA (500 ng) was mixed with 50 ng of random nonamers primer, dNTP mix (10 mM each) and RNasefree water, in a final volume of 12 µl.
Cells were incubated in a humidified atmosphere of 5% CO 2 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 harvested after 24 days of incubation.

RNA processing
Total RNA was isolated from ADSCs, HFb and adipose tissue using Nucleospin 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 Nucleospin column and centrifuged at
The expression level of two growth factors, NGF and BDFN, was compared 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 downregulated (Figure 2).
These two genes were down-regulated in adipocyte when compared with fibroblast (Figure 3) and stem cells.
Stem cells show up-regulation of BDFN and NGF with respect to adipocyte (Figure 4).

Discussion
Recent studies have shown that adipose tissue is a paracrine and endocrine organ, able to produce signal proteins termed adipokines, proteins involved in the regulation of energy balance, insulin sensitivity and glucose tolerance, vascular homeostasis, lipid metabolism, angiogenesis and inflammation 6,14 .
Adipokines are responsible for the complex processes of adipose tissue remodelling with repair of peripheral 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 lipolysis 15 , control of cell number and growth and maintenance of sympathetic neurons within tissues 16 .
Their expression could influence the good outcome of structural fat grafting, a procedure largely 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 PCR
cDNA 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 (Applied 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 (Applied 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.Quantification was done with the delta/ delta calculation method 13 .

Statistical analyses
Comparison of gene expression between three types of cells was performed with two-tailed ANOVA using Excel spreadsheets (Microsoft Office 2003).In this study, we have compared the ability of ADSCs and adipocytes, derived from the same patient, to secrete 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 fibroblasts.
Obtained results showed that AD-SCs over-expressed NGF and BDFN genes, compared with adipocyte, indicating that stem cells are the main producers of neurotrophic factor in adipose tissue.
NGF expression is also up-regulated 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.

Conclusion
These results demonstrate that the production of NGF and BDFN is due to the stem cells present in the adipose 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 anaesthesia with minimal discomfort to the patient, and its MSC content is adequate for clinical-grade cell manipulation in regenerative medicine.into different types of cell lines, thus restoring soft tissues 17 .
Many studies have demonstrated that differentiated ADSCs produce neurotrophic factors, such as BDNF and NGF, [18][19][20] which are responsible for their neurotrophic effects.The ease of harvesting and the rapid expansion capability make the ADSC cultures ideal candidates in regenerative medicine and peripheral nerve regeneration.reconstructive surgery, to restore congenital and post-traumatic deformities and to fill out tissue depression due to orbital and periorbital surgery, scars or cancer resections [1][2][3][4] .
Adipose tissue represents a complex organ of preadipocytes, adipocytes, endothelial cells, stem cells and nerve fibres.MSCs (ADSCs) are very abundant and easily isolable in this tissue.They are able to differentiate

Figure 1 :
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.

Figure 2 :
Figure 2: Relative gene expression in ADSCs compared with HFb.
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 Competing interests: none declared.Conflict of interests: none declared.All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.

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

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