RESEARCH

Donor age negatively imm

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and an inability of the body to maintain tissue turnoverand homeostasis. As a result the number of elderly med-

MSCs possess a multitude of potential applications inregenerative medicine, being able to proliferate and dif-

Choudhery et al. Journal of Translational Medicine 2014, 12:8http://www.translational-medicine.com/content/12/1/8inflammatory factors. To date, bone marrow is the bestArizona, PO Box 245221, 85724 Tucson, AZ, USAFull list of author information is available at the end of the articleical patients have also significantly increased, making thema major target population that could potentially benefitfrom cell based therapies. As autologous cell sources arepreferred for economical and logistical reasons (along withfewer potential side-effects), the effect of donor age on re-generative potential should be determined before clinical

ferentiate in vitro into multiple lineages [3-5]. Low im-munoreactivity and high immunosuppressive propertiesmake MSCs a suitable stem cell source for therapy [6,7].In various animal models it has been shown that MSCscan be used to successfully treat cardiovascular [2,8],neurological [9] and musculoskeletal disorders [10] eitherby differentiation into competent cardiomyocytes, neuron-like cells and chondrocytes, respectively; or by a paracrineeffect via the secretion of growth, anti-apoptotic and anti-* Correspondence: davidh@email.arizona.edu2Department of Immunobiology, College of Medicine, The University oforganisms reparative and regenerative potential, but little and conflicting information is available about the effectsof age on the quality of human adipose tissue derived MSCs (hAT-MSCs).

Methods: To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs fromyoung (60 years) individuals were investigated. MSCs were characterizedfor expression of the genes p16INK4a and p21 along with measurements of population doublings (PD), superoxidedismutase (SOD) activity, cellular senescence and differentiation potential.

Results: Aged MSCs displayed senescent features when compared with cells isolated from young donors,concomitant with reduced viability and proliferation. These features were also associated with significantly reduceddifferentiation potential in aged MSCs compared to young MSCs.

Conclusions: In conclusion, advancing age negatively impacts stem cell function and such age related alterationsmay be detrimental for successful stem cell therapies.

Keywords: Adipose tissue, Mesenchymal stem cells, Donor age, Regenerative potential, Growth kinetics, In vitrodifferentiation potential

BackgroundThe average human life expectancy has significantly in-creased due to advances in medical research and im-provements in general life style. Unfortunately however,human aging is associated with many clinical disorders

use. In recent years, many studies have demonstrated theclinical potential of mesenchymal stem cells (MSCs), bothin vivo and in vitro [1,2]. However, using MSC collectedfrom the elderly who are most likely to benefit from thistechnology raises some practical concerns.tissue-derived mesenchyand differentiationMahmood S Choudhery1,2, Michael Badowski2, Angela M

Abstract

Background: Human adipose tissue is an ideal autologouregenerative medicine and tissue engineering strategies.for many promising applications. It has long been known 2014 Choudhery et al.; licensee BioMed CenCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Access

pacts adiposeal stem cell expansion

se2, John Pierce3 and David T Harris2*

source of mesenchymal stem cells (MSCs) for varioused patients are one of the primary target populationsat advanced age is negatively correlated with antral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/2.0), which permits unrestricted use,, provided the original work is properly cited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in this

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Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 2 of 14http://www.translational-medicine.com/content/12/1/8characterized source of MSCs and most clinical datais based on bone marrow studies. However, there arelimitations to the use of bone marrow-derived MSCs(BM-MSCs), e.g. painful acquisition process, use of ex-tensive anesthesia, and low cell yield. Murine BM-MSCshave been shown to exhibit a decline in MSC numbers,proliferation, angiogenic and wound healing properties,and differentiation, along with enhanced apoptotic andsenescent traits [2,11,12] with advancing donor age. Re-cently, other MSC sources have gained clinical interestfor use in regenerative medicine; and adipose tissue rep-resents one of these sources with a broad spectrum ofbenefits. hAT-MSCs possess morphological, phenotypicand functional characteristics similar to BM-MSC [13],are stable over long term culture, expand efficiently in vitroand possess multi-lineage differentiation potential [5,14].Human adipose tissue represents a more practical au-tologous source of MSCs for various tissue engineeringstrategies. However, the effectiveness of these cells whenobtained from and utilized in elderly patients must beconsidered when contemplating cell-based therapies.Cell-based therapies will be significantly influenced by

the expansion and differentiation potential of any cellsto be used which in turn may be influenced by donorage. In the present report, we thus sought to study thegrowth characteristics and in vitro regenerative potentialof hAT-MSCs obtained from donors of various age groups(young, adult and aged) in combination with gene expres-sion profiles, superoxide dismutase (SOD) activity and sen-escence levels. Morphologically and immunophenotypicallycells obtained from all donors were similar regardless ofdonor age. However, we observed significant decreases inMSC number, frequency and population doublings con-comitant with an increase in senescence levels withincreasing donor age. Our qualitative and quantitativeobservations indicated that although adipogenic (andpossibly neurogenic) potential was maintained duringadvancing age, the osteogenic and chondrogenic abil-ities were negatively affected by donor age.

MethodsCollection and processing of adipose tissue-derivedmesenchymal stem cellsConsent was obtained from all donors before the liposuc-tion procedure. All protocols were approved by the localInstitutional Review Board (IRB). Human adipose tissuewas obtained from liposuction procedures under localanesthesia. All adipose tissue samples were processed underthe same conditions. Initial experiments (see Figure 1 andAdditional file 1: Figure S1) were performed using subjectsdivided into 50 years of age (N = 6; 1 male and 5 females)

to examine if there were indeed any effects of donor ageon AT-MSC. In subsequent experiments additionaldonors were recruited and divided into 3 age groups:group 1 ( 60 years; N = 11; mean age 66.0 1.4; 2 males and 9females).The raw lipoaspirate was processed using a previously

described method [5]. Cells from adipose tissue sampleswere isolated by enzymatic digestion. Briefly, 2 mL of tis-sue slurry was placed in a 50-mL Falcon tube and washedvigorously five times with 5 mL of phosphate-bufferedsaline (PBS). Cells in the wash fraction were retained. Thefatty tissue was treated with an equal volume of 0.2%collagenase type IV (Sigma) at 37C for 15 min. Completemedium (Minimal Essential Medium; Thermo Scientific,USA), 20 mL, supplemented with 10% fetal bovine serum(FBS; Hyclone) and 1% each of non-essential aminoacids, sodium pyruvate, glutamine and streptomycin/penicillin solution was added in the digested tissue toneutralize collagenase, passed through a 40-m filterand centrifuged at 150 g for 10 min. The cells fromboth the wash fraction and the digested fraction weresuspended in complete medium and counted usingtrypan blue and Turks stains. Cells were plated in25 cm2 culture flasks and maintained at 37C/5% CO2in expansion medium with humidity. MSCs adheredto the culture flasks whereas other cells were depletedby replacing the spent medium with fresh medium.The medium was changed twice a week thereafter. Toprevent spontaneous differentiation, cells were main-tained at sub-confluent levels (70-80%) and were har-vested with 0.05% trypsin-EDTA for use in subsequentexperiments.

Phenotypic characterization by flow cytometryCultured cells (passage 1) were trypsinized and stainedwith a panel of antibodies for fluorescence-activated cellsorting (FACS) analysis. Approximately, 1 105 cells werere-suspended in phosphate buffered saline (PBS) and incu-bated with IgG block for 5 minutes to block non-specificbinding. The following antibodies were used: AF-700conjugated CD3 (BD BioSciences, USA), PE conju-gated CD14 (BD, Immunocytometry, USA), APC con-jugated CD19 (BD BioSciences, USA), PE conjugatedCD34 (BD, BioSciences, USA), APC conjugated CD44(BD, Pharmingen, USA), FITC conjugated CD45 (BDPharmingen, USA), PE conjugated CD73 (BD Pharmingen,USA), AF-700 conjugated CD90 (Biolegend, USA) andAPC conjugated CD105 (Biolegend, USA). Cells werestained for 30 minutes at 4C with the antibodies. Afterwashing, samples were analyzed on a LSR II flow cytometer(BD, USA) and at least 10,000 events were acquired for

each population. Data acquisition and analysis were per-formed using FACS DIVA software (BD Biosciences, USA).

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 3 of 14http://www.translational-medicine.com/content/12/1/8Unstained cells were used to establish flow cytometer set-tings. Debris and cells/particles with auto-fluorescence wereremoved by using a threshold on the forward scatter.

Cell proliferation assaysAssay for colony forming unit (CFU-assay)CFU-assays were performed to determine MSC frequency.The processed lipoaspirates after collagenase digestion wereplated in 25 cm2 culture flasks in limiting dilutions (105, 5 104, 104, etc.) to verify the ability to form colonies. Cultureswere maintained for 14 days at 37C/5% CO2 in expansionmedium. At day 14, medium was removed and resultantcolonies were washed twice with PBS, fixed with absolutemethanol and stained with 0.1% crystal violet for 60 minutesat room temperature [5]. The flasks were washed with waterand colonies with more than 30 cells were counted undera microscope by two independent observers.

Figure 1 Comparison of age related parameters in AT-MSCs isolatedindicate that gene expression of p16 and p21 is higher is AT-MSCs isolated froas compared to untreated control cells. (C, D) Similarly, aged cultures show seyoung cultures (data shown as percent positive cells). Concomitant witshown as absorbance values) and viability after stress (F; data shown adeviation. *P < 0.01 for young AT-MSCs versus aged AT-MSCs. AT-MSCs:senescence associated beta galactosidase.Cumulative growth indexMSCs were serially passaged for cumulative populationdoubling analysis as described [2]. The first confluentcultures were designated as passage 0 (P0) and were dis-sociated with trypsin/EDTA, counted by hemacytometerand re-plated at a 1:10 dilution. The cell number was re-corded for each passage until the cells stop dividing. Theaverage cell number was expressed with respect to timein culture to obtain a growth curve. The populationdoublings (PDs) and doubling time (DT) were calculatedusing the following equations [2],

PDs Log N=N0 3:31DT CT=PDs

Where, PDs represent population doublings, N isthe final number of cells, No is the initial number of

from young and aged donors. (A, B) Gene profiling of AT-MSCsm aged as compared to young donors. Data is shown as fold-inductionnescent features as determined by SA--gal staining compared toh these features young AT-MSCs have higher level of SOD (E; datas percent viable cells). Results are expressed as Mean Standardadipose tissue derived mesenchymal stem cells, SA--gal:

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 4 of 14http://www.translational-medicine.com/content/12/1/8cells seeded DT is doubling time and CT is the time inculture.

In vitro differentiation assaysMSCs from young, adult and aged groups were analyzedfor the potential to differentiate into adipose, bone, car-tilage and neuron-like cells. MSCs were induced to dif-ferentiate between passages 2-3, as described below.

Adipogenic differentiationAT-MSCs were seeded in triplicate in 12 well platesat a final cell density of 5,000 cells per cm2 in completeexpansion medium. 24-48 hours later, which was desig-nated as day 0, differentiation was initiated using adipo-genic induction medium (ThermoScientific, USA), asper manufacturers instructions. The medium was chan-ged every 3-4 days thereafter and experiments were ter-minated after 3 weeks. AT-MSCs at the same celldensity were maintained in expansion medium to serve ascontrols.

Oil red O stainingAdipogenesis was confirmed three weeks after inductionby oil Red O staining to visualize accumulated cytoplasmiclipid rich vacuoles [5,14] according to the manufacturersinstructions (IHC World, USA). Briefly, the differentiatedMSCs were fixed with 4% paraformaldehyde (PFA), washedwith pre-stain solution (99% isopropanol) and incubatedwith oil red O solution for 30 minutes at 60C. Oil red Ostaining was followed by washing with 60% isopropanoland then several changes of distilled water. Cells werecounterstained with haematoxylin solution for 1 minuteand visualized under phase contrast microscopy.

In vitro osteogenic differentiationFor osteogenic differentiation 50,000 MSCs per wellwere seeded in 6 well plates in triplicate in expansionmedium. After 24 hours (at 90% confluency) osteogenicdifferentiation was promoted by treating MSC cultureswith osteogenic induction medium (ThermoScientific,USA) for 3 weeks while MSCs maintained in expansionmedium for 3 weeks were used as controls.

Von Kossa stainingOsteogenic potential was confirmed by the von Kossasmethod [15] of extracellular matrix calcification detection.Von Kossas staining was performed by silver nitrate treat-ment using a commercially available kit (IHC World,USA). Briefly, PFA (4%) fixed cultures were treated withsilver nitrate for 60 minutes at room temperature underultraviolet (UV) light, followed by treatment with sodiumthiosulphate for 5 minutes. The cells were counter-stained

with nuclear fast red and then photographed using phasecontrast microscopy. Extracellular matrix calcification wascarried out by detection of the presence of black extracel-lular deposits.

Chondrogenic differentiationChondrogenesis was induced in micromass pellet culturesas described [5,16]. Micromass-pellet cultures were pre-pared from 2.5 105 MSCs in 15 ml conical tubes thatwere centrifuged at 750 rpm for 10 minutes in completeexpansion medium. Cell pellets were incubated with thechondrogenic induction medium (ThermoScientific, USA)after 24-48 hours for 3 weeks. Half of the medium was re-placed with fresh medium twice a week.

Alcian blue stainingEach micromass pellet was parafinized after dehydrationand cut into thin sections (4-5 um). The sections wereanalyzed for chondrogenic differentiation assay with acommercially available Alcian blue kit (IHC World, USA).Sections were fixed with 4% PFA and washed with distilledwater followed by treatment with Alcian blue for 20 mi-nutes at room temperature. The stained sections were vi-sualized under phase contrast microscopy and imageswere captured.

Differentiation of MSCs into neuron-like cells2.5 103 MSCs at passage 2 were propagated in six wellplates in complete growth medium, followed by treatmentwith neuronal induction medium (ThermoScientific, USA)as per manufacturers instructions. After 24-48 hours, thedifferentiated MSCs were stained with H&E (Hematoxylineand Eosin). Briefly, the medium was discarded and the cellswere washed with PBS, fixed with methanol and stainedwith H&E solution for 30-60 seconds, and photographedusing phase contrast microscopy. Cells having a neuron-like morphology were counted in each culture.

Quantification of differentiationThe total number of oil red O positive MSCs werecounted in triplicate in at least 10 non-overlapping highdensity fields. The mean differentiation level (%) wasexpressed as total number of oil red O positive cellsdivided by total number of cells, and then multipliedby 100. Additionally, oil red O uptake was quantifiedcolorimetrically using a previously published method[17]. Briefly, oil red O was extracted with isopropanolcontaining 4% nonidet P-40 detergent overnight atroom temperature and optical density was then mea-sured at 520 nm [5,17]. All analyses were carried outin triplicate.For the quantification of mineralized matrix deposition,

imageJ software (http://rsbweb.nih.gov/ij/) was used whichmeasures the amount of cellular staining (black) in a given

field of view. Percentage positive area was calculated bydividing the positively stained area divided by the total

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 5 of 14http://www.translational-medicine.com/content/12/1/8area, multiplied by 100. All analyses were carried out intriplicate.Alcian blue uptake was analyzed using a colorimetric

assay as described [18]. Briefly, after 21 days, the micro-mass cultures were fixed with methanol and the wholemount stained with Alcian blue. Alcian blue was ex-tracted with 6 M guanidine HCl and absorbance wasread at 620 nm.

Senescence-associated -galactosidase Staining (SA--gal)To detect cellular senescence the SA--gal staining kitwas used (Cell Signalling, USA). Briefly, 5 103 cellswere seeded in 12 well plates incubated with freshly pre-pared -gal-staining solution for 60 minutes at 37C inthe absence of CO2. MSCs were washed with water andthe blue color (i.e., senescent cells) was observed undermicroscopy. Phase contrast images were taken and thepercentage SA--gal positive cells were calculated bydividing blue stained cells by the total number of cells,multiplied by 100.

Superoxide dismutase (SOD) activityAge-related differences in SOD activity were determinedusing a commercially available colorimetric assay kit(Abcam, USA) according to the protocol provided by themanufacturer. Briefly, protein was isolated using a lysisbuffer and SOD activity was measured using 10 ug of thetotal protein extract. Absorbance values were measuredby using a Spectra max PLUS 384 (Molecular Devices,USA) at 450 nm.

ImmunocytochemistryAfter culture in neuronal differentiation medium, differ-entiated cells were washed with PBS and incubated withthe rabbit-anti-human nestin (US Biological, USA) over-night at 4C. The cells were then washed with PBS andincubated with PE-conjugated goat-anti-rabbit (SantaCruz, USA) antibody for 60 minutes at 37C. Cells weremounted using vecta-sheild mounting medium (VectorLaboratories, USA) containing DAPI (4,6-diamidino-2-phenylindole) to stain nuclei and observed using fluores-cence microscopy (Zeiss, USA).

Total RNA extractionGene expression was assayed at the mRNA level. Total cel-lular RNA was extracted using TRIzol reagent (Invitrogen,USA) and an Rneasy Mini Kit (Qiagen, USA). All proce-dures were carried out according to the protocol rec-ommended by manufacturers. RNA concentration wasdetermined using a ND-1000 spectrophotometer (NanoDropTechnologies). cDNA synthesis was performed by using 1ug of total RNA in a 20 ul reaction mixture containing 1

ul of 10 uM oligodt primer and 1 ul of reverse transcript-ase enzyme (RT-enzyme) with the SuperScript III FirstStrand synthesis system (Invitrogen, USA). The manufac-turers instructions were followed.

Quantitative RT-PCRReal time PCR was performed using iTaq SYBR Greensupermix with ROX (Bio-Rad, USA) in an ABI PRISM7300 sequence detection system. The final reaction con-tained template cDNA, iTaq SYBR Green and gene specificprimers (see Additional file 2: Table S1). The followingPCR conditions were used: 50C for 2 minutes and 95Cfor 10 minutes, followed by 40 cycles for 30 sec at 95C,45 sec at 60C and 72C for 30 sec. Beta actin was used asan internal control. The CT (cycle threshold) values ofbeta actin and other specific genes were acquired afterpolymerase chain reaction. The normalized fold expres-sion was obtained using the 2-CT method. The resultswere expressed as the normalized fold expression for eachgene as compared to untreated (i.e., un-induced) controlcells. In order to minimize PCR reaction variations, allsamples were transcribed simultaneously.

Cell viabilityTo determine the level of cell viability in response to in-cubation in H2O2, the trypan blue exclusion assay wasused. Sub-confluent cultures of MSCs were incubatedwith 200 um H2O2 for 90 minutes. Cells were trypsinize-dand counted in a hemacytometer. The number of viablecells was calculated by dividing the number of trypanblue-negative cells by the total number of cells exam-ined, multiplied by 100.

Statistical analysisExperimental data was analyzed using Graphpad Prism 5Software. One-way ANOVA was used when three or moregroups within one variable were compared. To analyzetwo groups the unpaired-t-test was used. The data areexpressed as mean standard deviation. Values of P

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 6 of 14http://www.translational-medicine.com/content/12/1/8Figure S1B shows the relative percentage expression foreach marker used in the experiment.

Cellular senescence increases in elderly AT-MSCSenescence was evaluated in two ways: by measuringmRNA expression of the p16 and p21 genes and, by de-tection of SA--gal expression [2,12]. Initially, twogroups of donors (50 years old do-nors) were examined. The mRNA levels of the p16 andp21 genes (Figure 1A), which are associated with senes-cence, were analyzed by PCR [21]. The expression ofboth genes was significantly higher in the aged group(>50 years) than in the young group (

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 7 of 14http://www.translational-medicine.com/content/12/1/8than in the other groups (Figure 3B-C), but this differencewas not significant (Figure 3D) (67.4% 7.1% in young vs.70.8% 5.6% and 62.2% 13.0% positive, respectivelyin adult and aged). Quantification of oil red O uptakeindicated a statistically non-significant difference amongMSCs obtained from young, adult and aged individuals(Figure 3E). Histochemistry findings were confirmed by realtime RT-PCR analysis. The expression of the adipogenesis-specific genes, peroxisome proliferator-activated-receptor-gamma (PPAR-) and lipoprotein lipase (LPL), wasanalyzed [17,20]. The expression of both adipogenic spe-cific genes (LPL: 22.0 8.1 (young), 26.0 8.3 (adult),35.3 16.2 (aged) fold-expression, and PPAR-: 129.3 9.5 (young), 101.7 15.3 (adult), 116.0 32.6 (aged) fold-expression) was significantly higher in induced MSCs ascompared to control MSCs. However, there was no signifi-cant difference among the various age groups (Figure 3F).

Figure 2 Yield and growth characteristics of MSCs. Cells per gram of adwas performed to enumerate number of cells in SVF that can form colonies. Npotential is associated with donor age as indicated by number and time for pdecreases (C) while time per population doubling increases (D) with age of tmesenchymal stem cells, CFU, colony forming unit assay. *P < 0.01 for youngOverall our findings indicated that the adipogenicpotential of AT-MSCs was independent of donor ageand thus it appeared that AT-MSCs could potentiallybe used for tissue engineering applications that re-quire adipocyte production without concern for ageof the donor.

AT-MSC osteogenic potential declines with donor ageAT-MSCs isolated from each age group were induced todifferentiate into osteoblasts. The cells proliferated rap-idly in a tightly packed monolayer culture to form aggre-gates with calcium deposition. After 3 weeks, Von Kossastaining [15] revealed a positive extracellular matrix forma-tion in the induced AT-MSCs (Figure 4A-C). AT-MSCs ob-tained from younger (Figure 4A) donors produced morematrix than AT-MSCs obtained from both adult (Figure 4B)and aged (Figure 4C) donors. Cells in the control group

ipose tissue decreases with increased age of the donors (A). CFU assayumber of CFUs deceases with age of the donor (B). Proliferativeopulation doublings. Number of population doublings of MSCshe donor. Results are expressed as mean standard deviation. MSCs:AT-MSCs versus aged AT-MSCs.

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Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 8 of 14http://www.translational-medicine.com/content/12/1/8did not show such changes (Figure 4D-F). Comparativequantification of Von Kossa staining by ImageJ soft-ware is shown for all age groups (Figure 4G). Signifi-cant age-related differences among young (20.0% 1.7% positive cells), adult (15.2% 1.3% positive cells) andaged (8.9% 2.2% positive cells) were observed in this re-gard. Osteogenic induction was further evaluated by realtime RT-PCR analysis of lineage-specific expression of two

Figure 3 Adipogenic potential of AT-MSCs is independent of donor afrom young, adult and aged individuals. Insets show MSCs cultured in norm21 days and oil red O was used to stain for lipid rich vacuoles, as shown (Fwas determined, followed by quantification of oil red O uptake. Adipogeni(C) donors stained positive for oil red O. Differentiation levels varied betwepositive cells (D) and colorimetrically by evaluating oil red O uptake (E). Adipoand a non-significant difference was found when different age groups were costeogenic genes, alkaline phosphatase and osteocalcin[23]. When comparing the effect of donor age onosteogenic potential, we observed a higher expressionof the osteogenic specific genes in young AT-MSC com-pared to the other age groups (Figure 4H), (Osteocalcin:28.3 5.9 (young), 22.0 6.1 (adult), 11.7 1.4 (aged) fold-expression, and Alkaline phosphatase: 129.0 14.6 (young),63.7 3.2 (adult), 36.3 5.2 (aged) fold-expression).

ge. Adipogenic differentiation was carried out for AT-MSCs isolatedal expansion medium. Adipogenic experiments were terminated afterigure 3A-C). The percentage of cells that stained positive for oil red Oc differentiated MSCs isolated from young (A), adult (B) and ageden groups but non-significantly as indicated by counting oil red Ogenic differentiation was further confirmed through real time RT-PCRompared (F). Results are expressed as mean standard deviation.

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 9 of 14http://www.translational-medicine.com/content/12/1/8Chondrogenic potential of AT-MSCs declines with ageChondrogenic differentiation of AT-MSCs was performedin micromass pellet cultures. Thin sections of the pelletswere stained with Alcian blue (Figure 5A-C) to detect sul-fated proteoglycans in the cartilage matrix [24]. Uptakeof Alcian blue staining was quantified colorimetrically(Figure 5D-E) and quantitatively (Figure 5F). Differenceswere observed when comparing AT-MSCs obtained from

Figure 4 Effect of donor age on osteogenic potential of AT-MSCs.(A-C) Representative figures showing matrix mineralization in induced culturstain positive with von Kossa staining. Possible age related differences in ostefrom young individuals revealed more matrix mineralization than adult and aOST and ALP was analyzed through quantitative RT-PCR (H). Results are expreyoung AT-MSCs versus aged AT-MSCs, #P for adult versus aged AT-MSCs. OSTthe various age groups (0.51 0.06 vs. 0.26 0.04 vs. 0.17 0.02 OD units). At higher microscopic magnification(insets) the cartilage-like tissue appeared to be composedof rounded cells, surrounded by lacunae and lying in aproteoglycan rich extracellular matrix [24,25]. Significantage-related differences were observed in the chondrogenicdifferentiation potential of AT-MSCs isolated from variousage groups when real time RT-PCR analysis (Figure 5F)

Osteogenic induction was assessed by von Kossa staining.es of young, adult and aged, respectively. (D-F) Control AT-MSCs did notogenic potential were measured using ImageJ software. AT-MSCs isolatedged groups (G). Similar, results were obtained when gene expression ofssed as mean standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 for: osteocalcin, ALP: Alkaline phosphatase.

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 10 of 14http://www.translational-medicine.com/content/12/1/8was performed using the lineage specific genes, aggre-can and collagen type 2 [26,27] (Aggrecan: 10.0 1.5(young), 4.3 0.2 (adult), 1.8 0.4 (aged) fold-expression,and Collagen type 2: 1.9 0.1 (young), 1.3 0.1 (adult),1.3 0.2 (aged) fold-expression). These latter two findingsindicated that donor age negatively regulated AT-MSCdifferentiation into cartilage.

Figure 5 Chondrogenic potential of AT-MSCs is compromised by donmedium for 21 days in micromass pellet culture. Alcian blue staining was per(C) aged individuals. At higher microscopic magnification (shown in insets) thsurrounded by lacunae lying in a proteoglycan rich extracellular matrix. Chonin which Alcian blue uptake (blue color) was extracted with 6 M guanmicromass 30 minutes after incubation and (D2) after 120 minutes. (E) abscompared to aged. (F) Quantitative RT-PCR was performed for mRNA expresswas observed for both genes. Results are expressed as mean standard deviaAT-MSCs, #P for adult versus aged AT-MSCs.AT-MSCs undergo a neuronal-like differentiation in vitroindependent of donor ageAT-MSC were cultured in neurogenic differentiationmedium and stained with H&E (Hematoxyline and Eosin).The AT-MSCs displayed a neuronal-like differentiationwith prominent and elongated neuronal structures [5] re-gardless of donor age (Figure 6A-C). More than 95% of

or age. Differentiation of AT-MSCs was induced chondrogenic inductionformed for induced AT-MSCs isolated from (A) young, (B) adult ande cartilage like tissue appeared to be composed of rounded cells,drogenic in vitro potential was quantified by a colorimetric assayidine HCl and absorbance was read at 620 nm. (D1) showingorbance values were significantly higher for young and adult AT-MSCsion of aggrecan and collagen type 2. Age related decline in mRNA leveltion. *P < 0.05, **P < 0.01, ***P < 0.001 for young AT-MSCs versus aged

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 11 of 14http://www.translational-medicine.com/content/12/1/8the cells displayed a neuron-like morphology in each cul-ture as indicated in Figure 6D (99.7 0.3 in young vs.95.7 4.3 in adult vs. 98.0 1.5 (aged) positive cells).Induction into neuron-like cells was also confirmedby the assessment of nestin expression by immuno-cytochemistry (Figure 6E). Again we did not observe anyvariation in the percentage of neuron-like positive cellsbetween the different age groups (Figure 6F) (58.2 14.4in young vs. 53.6 17.9 in adult vs. 52.4 19.2 (aged)percent positive cells). A quantitative increase in mRNAlevels for the neurofilament (NFM) and neuron-specific-enolase (NSE) genes [17,28] after neurogenic inductionsupported the observation of neuronal differentiation.However, when AT-MSCs isolated from young, adultand aged donors were compared the expression of theNFM and NSE genes were similar (Figure 6G) (NFM:46.3 16.2 N = 3 (young), 39.7 6.7 (adult), 45.3 17.3(aged) fold-expression, and NSE: 24.3 6.4 (young),25.7 3.5 (adult), 22.4 15.2 (aged) fold-expression).Although we did not find differences between agegroups when AT-MSC were induced at initial pas-sages, other experiments indicated that younger AT-MSC cells were better than aged cells when expandedAT-MSC cultures were neurally induced at latter pas-sages (data not shown).

Figure 6 The neuron-cell-like morphology of AT-MSCs isolated from yobserved in MSCs isolated from young, adult and aged individuals, respectRepresentative slide showing nestin expression as determined by immunoftime RT-PCR analysis showed equivalent up-regulation of neurogenic speciage (G). NSE: Neuron-Specific-Enolase, NFM: Neurofilament.DiscussionIn view of conflicting reports, we have undertaken acomprehensive analysis of age-related AT-MSC charac-teristics in a single study using donors of broad agerange. Our study focused on the parameters of MSCyield, frequency, replication and differentiation into adipo-genic, osteogenic, chondrogenic and neurogenic lineages,along with other age-related parameters (senescence, SODactivity and viability under cell stress). The present studydemonstrated the effect of donor age on the cell expan-sion and differentiation potential of AT-MSCs, and repre-sents one of the most comprehensive studies providinginsight into whether cell-based therapies will be negativelyaffected by donor age. It is assumed that organismal agingis linked to diminished organ repair due to reduced func-tional capacity of tissue resident stem cells. It is believedthat such cells residing in the elderly are subjected to age-related changes and thus contribute less to tissue rejuven-ation. Similarly, age-related diseases such as diabetesand heart failure also negatively impact the function ofendogenous progenitor cells [29]. As stem cells are thebasis of tissue regeneration therapies, a diminished func-tionality of these cells in the elderly may result in reducedefficacy of autologous cell therapies. With an increasein the aging population, cellular therapies are becoming

oung, adult and aged donors. (A-C) Neuron-like-morphology wasively. (D) Percentage of AT-MSCs showing neuron-like morphology.luorescence staining (E) with similar expression in all groups (F). Realfic genes (NFM and NSE) in AT-MSCs isolated from donors of different

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 12 of 14http://www.translational-medicine.com/content/12/1/8more relevant for aged patients who are the main targetpopulation for such therapies. It is therefore important toinvestigate donor age as a critical factor in determiningwhether cell therapies can achieve the desired results inthese individuals using autologous stem cells.Analysis indicated that the overall yield of nucleated

cells was significantly and negatively affected by donorage. Similar observations have been reported in literatureby assessing the yield of bone marrow-derived MSCsand circulating endothelial progenitor cells [30,31].These results indicated that age-related changes in MSCnumber should be taken into account whenever thesecells are considered for clinical applications in the eld-erly. Although AT-MSCs from all age groups had theability to form colonies (an indication of cell function),AT-MSC from younger donors produced more coloniescontaining larger numbers of cells. Other investigatorshave reported that the number of cells forming coloniesdecreased significantly with increasing donor age and is inaccordance with the results of our current study [20].These findings are important as it indicates that harvest-ing AT-MSCs from elderly donors may require either thecollection of more adipose tissue or require pretreatmentstrategies to enhance cell proliferation and expansion.AT-MSCs isolated from each age group (young, adult

and aged) exhibited a fibroblastic morphology and pheno-type common to MSC that was independent of donor age[19]. That is, regardless of donor age, AT-MSC expressedCD44, CD73, CD90 and CD105 (mesenchymal markers)while lacking expression of CD3, CD14, CD19, CD34 andCD45 (hematopoietic markers). These results were inagreement with previous reports [19,20], although Stolzinget al. [24] observed age-related changes in expression ofsome cell surface markers such as CD44, CD90, CD105and Stro-1 when bone marrow-derived MSCs wereanalyzed.AT-MSCs obtained from aged donors displayed in-

creased senescent features as indicated by higher expres-sion of the p16 and p21 genes. Recent evidence suggeststhat p16 and p21 are markers of senescence [24]. Inaddition, SA--gal expression was also measured and wasfound at higher levels in aged AT-MSC cultures, whileSOD activity was decreased [24]. The ability to expandcells without loss of function is one of the most importantconsiderations when culturing MSC for therapeutic pur-poses. We evaluated whether donor age had an effect onproliferation. There was an age-related difference in thenumber of maximum population doublings, being statisti-cally higher for AT-MSCs isolated from young donors ver-sus adult or aged donors. AT-MSCs from young donorsalso proliferated at a higher rate than other groups. Thedoubling time of the AT-MSC was significantly increased

with advanced age as well. These findings were not un-expected as the expression of other age-related markers(i.e., SA--gal, P16 and p21) was found to be significantlyhigher in aged donors compared to young donors.Published studies have demonstrated the multi-lineage

differentiation potential of AT-MSCs [5,14]. However,recent studies have raised questions about the usefulnessof AT-MSCs collected from aged donors [15,20]. Wethus have analyzed the differentiation capability of AT-MSCs relative to age of the donor. AT-MSCs obtainedfrom donors of each age group efficiently differentiatedinto adipocytes. Quantification of oil red O content didnot indicate a significant correlation between donor ageand adipogenesis. The number of oil red O positive cellswas also equivalent between age groups although morevariation was observed in the aged group as comparedto the young and adult groups. Similarly, we observedvery little fluctuation in the mRNA levels of the adipo-genic specific genes, PPAR- and LPL, due to donor age.Digirolamo et al. [32] reported similar results, as hasZhu et al. [15]. Overall, the results of our current studyindicated that adipogenic potential of AT-MSCs was wellpreserved in advanced age and AT-MSC of any agecould potentially be used for tissue engineering applica-tions that require fat production, or potentially for cos-metic/plastic surgery applications.Concomitant with aging, the chance of bone fractures

is significantly increased while the ability to heal suchfractures is lost. A significant drop in osteogenic poten-tial of AT-MSCs with increased age was observed, asmeasured qualitatively by matrix calcification and quan-titatively by real time RT-PCR, using osteocalcin and alka-line phosphatase gene expression. We observed a lineardecrease in osteogenic potential with increasing age. Zhuet al. [15] reported a similar decline in osteogenic poten-tial starting in middle age (40-49 years). However, Zhuet al. [15] only studied female patients with a different agerange. A decrease in circulating oestrogen levels has beenshown to be responsible for loss of osteogenic potential ofstem cells in females [33,34]. Other conflicting reportshave been published; e.g. Shi et al. [35] reported no changewith age while Khan et al. [36] found age-related differ-ences in osteogenic potential of AT-MSCs. These incon-sistent results may be due to the different age ranges andthe health status of the donors that were studied. Overall,the majority of reports found results similar to our currentstudy; describing an overall decline in osteogenic potentialwith donor age (regardless of species).Damaged cartilage does not heal well and stem cells

might be potential candidates for cartilage repair. How-ever, the relationship between stem cell aging and thepotential to undergo chondrogenesis has not been wellestablished. We observed that there was a significantage-related decline in the chondrogenic potential of AT-

MSCs. Similarly, mRNA expression of the aggrecan andcollagen type 2 genes was significantly reduced in the

our study. In combination, these findings and our osteo-

Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ,

Choudhery et al. Journal of Translational Medicine 2014, 12:8 Page 13 of 14http://www.translational-medicine.com/content/12/1/8genic results indicate that donor age may negatively im-pact the use of AT-MSC for orthopedic applicationswhich are not uncommon as one grows older.Studies have indicated that MSCs could be a potential

treatment for various neurodegenerative disorders. Wehave previously shown that hAT-MSCs can be differenti-ated into neuron-like cells in vitro [5]. In the currentstudy although cell outgrowths were more prominent inyoung AT-MSC cultures, no significant differences werefound in the total number of neuron-like cells from anyage group. Similarly, we observed no significant differ-ence in the expression of the nestin gene, as the percent-age nestin expression was independent of donor age.Real time RT-PCR also indicated equivalent expressionof the neuronal specific genes NFM and NSE in all agegroups. Therefore, it may still be feasible to consider useof AT-MSC for neurological applications at any point inthe donors life (e.g., for stroke, Parkinsons disease, etc.).

ConclusionsStem cell research and stem cell therapy is expandingrapidly. However, a number of issues still need to be ad-dressed to make such therapies more useful. AutologousMSC source is widely used cell source in cell based ther-apy for patients. However, certain limitations are appliedsuch as poor functionality of MSCs isolated from elderly.AT-MSCs isolated from younger donors are anticipatedto be a more useful cell source for tissue engineeringand regenerative medicine applications. Cell based thera-peutic approaches for the elderly should focus on alter-native strategies such as banking younger adipose tissuefor later use. Preservation of stem and progenitor cells ata younger age while when biological activity is at itsgreatest potential could provide an ideal situation for fu-ture regenerative medicine applications.Overall, results of the current study indicated that

aged MSCs displayed senescent features when comparedwith cells isolated from young donors. The results dem-onstrated that the growth kinetics and the osteogenicand chondrogenic potentials of AT-MSCs were adverselyaffected by increased donor age. However, the adipo-genic and possibly the neurogenic potential of the AT-MSCs seemed to be maintained during aging.

Additional files

Additional file 1: Figure S1. Phenotypic characterization. Flowcytometric analysis of cells show that AT-MSCs were positive for CD44,aged group as compared to the other groups. Murphyet al. [37] has also reported an age-related decline inchondrogenic potential of MSC similar to the results ofCD73, CD90 and CD105, while being negative for hematopoieticmarkers CD3, CD14, CD19, CD34 and CD45. (A) representative graphics,and (B) analysis of expression. -:99.0%.

Additional file 2: Table S1. The primer sequences (5-3) for the primerpairs used.

AbbreviationsMSCs: Mesenchymal stem cells; hAT-MSCs: Human adipose tissue derivedMSCs; PDs: Population doublings; SOD: Superoxide dismutase; BM-MSCs: Bonemarrow derived MSCs; PBS: Phosphate buffered saline; FBS: Fetal bovine serum;FACS: Fluorescence activated cell sorting; CFU: Colony forming unit;DT: Doubling time; PFA: Paraformaldehyde; H&E: Hematoxylin and eosin; SA--gal: Senescence-Associated -galactosidase Staining; PPAR-: Peroxisomeproliferator activated receptor-gamma; LPL: Lipoprotein lipase;NFM: Neurofilament; NSE: Neuron specific enolase.

Competing interestsDr. Choudhery and Ms Muise do not have financial or non-financial competinginterests. Dr. Harris is the CSO for Adicyte, Inc. Dr. Badowski is a consultant toAdicyte.

Authors contributionsMSC was involved in the design and experimentation of the study as wasMB; AM performed experimentation and data acquisition; JP was alsoinvolved in data acquisition, experimental design and data analysis; DTHperformed the overall experimental design and final data analysis. All authorshave seen and agreed to the submitted version of the manuscript.

AcknowledgementsThis work was supported, in part, by Adicyte, Inc. The authors also wish toacknowledge our undergraduate students for their help in this study.

Author details1Advanced Centre of Research in Biomedical Sciences, King Edward MedicalUniversity, Lahore, Pakistan. 2Department of Immunobiology, College ofMedicine, The University of Arizona, PO Box 245221, 85724 Tucson, AZ, USA.3Aesthetic Surgery of Tucson, Tucson, AZ, USA.

Received: 23 September 2013 Accepted: 3 December 2013Published: 7 January 2014

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AbstractBackgroundMethodsResultsConclusions

BackgroundMethodsCollection and processing of adipose tissue-derived mesenchymal stem cellsPhenotypic characterization by flow cytometryCell proliferation assaysAssay for colony forming unit (CFU-assay)Cumulative growth indexIn vitro differentiation assaysAdipogenic differentiationOil red O stainingIn vitro osteogenic differentiationVon Kossa stainingChondrogenic differentiationAlcian blue stainingDifferentiation of MSCs into neuron-like cellsQuantification of differentiationSenescence-associated -galactosidase Staining (SA--gal)Superoxide dismutase (SOD) activityImmunocytochemistryTotal RNA extractionQuantitative RT-PCRCell viability

Statistical analysis

ResultsAT-MSC morphology and phenotype is independent of donor ageCellular senescence increases in elderly AT-MSCSuperoxide dismutase (SOD) activityEffect of donor age on cell viability after stressEffect of donor age on cell yield, viability and MSC frequencyEffect of donor age on AT-MSC growthAssay for multi-lineage differentiation potentialAdipogenic differentiation potential of AT-MSCs is independent of donor ageAT-MSC osteogenic potential declines with donor ageChondrogenic potential of AT-MSCs declines with ageAT-MSCs undergo a neuronal-like differentiation invitro independent of donor age

DiscussionConclusionsAdditional filesAbbreviationsCompeting interestsAuthors contributionsAcknowledgementsAuthor detailsReferences

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