Journal of Biomaterials Applications

http://jba.sagepub.com/Applications

Journal of Biomaterials

http://jba.sagepub.com/content/25/7/743The online version of this article can be found at:

DOI: 10.1177/0885328209360425

2011 25: 743 originally published online 17 March 2010J Biomater ApplR. Nieto-Aguilar, D. Serrato, I. Garzón, A. Campos and M. Alaminos

Cells in a Novel Fibrin-agarose ScaffoldPluripotential Differentiation Capability of Human Adipose-derived Stem

Published by:

http://www.sagepublications.com

can be found at:Journal of Biomaterials ApplicationsAdditional services and information for

http://jba.sagepub.com/cgi/alertsEmail Alerts:

http://jba.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

http://jba.sagepub.com/content/25/7/743.refs.htmlCitations:

What is This?

- Mar 17, 2010 OnlineFirst Version of Record

- Apr 1, 2011Version of Record >>

at Biblioteca Universitaria de Granada on November 20, 2012jba.sagepub.comDownloaded from

http://jba.sagepub.com/

http://jba.sagepub.com/content/25/7/743

http://www.sagepublications.com

http://jba.sagepub.com/cgi/alerts

http://jba.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://jba.sagepub.com/content/25/7/743.refs.html

http://jba.sagepub.com/content/25/7/743.full.pdf

http://jba.sagepub.com/content/early/2010/03/17/0885328209360425.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml


Pluripotential DifferentiationCapability of Human

Adipose-derived Stem Cells in aNovel Fibrin-agarose Scaffold

R. NIETO-AGUILAR, D. SERRATO, I. GARZON, A. CAMPOS

AND M. ALAMINOS*Faculty of Medicine, Department of Histology (Tissue Engineering Group)

University of Granada, Avenida de Madrid 11, E18012 Granada, Spain

ABSTRACT: The potentiality of adipose-derived stem cells (ASCs) cultured on2D systems has been previously established. Nevertheless, very little is known sofar about the differentiation potentiality of ASCs in 3D culture systems usingbiomaterials. In this work, we have evaluated the transdifferentiation capabilitiesof ASCs cultured within a novel fibrin-agarose biomaterial by histologicalanalysis, histochemistry and immunofluorescence. Our results showed that 3Dfibrin-agarose biomaterial is highly biocompatible and supports the transdiffer-entiation capabilities of ASCs to the osteogenic, chondrogenic, adipogenic, andneurogenic lineages.

KEY WORDS: fibrin-agarose, biomaterials, scaffolds, adipose-derived stromalcells, transdifferentiation.

INTRODUCTION

Management of the different conditions that affect the skeletaland neural tissues usually has several anatomic, physiological,

and aesthetic implications. The demand of corrective procedures withthese tissues increases every year due to congenital or acquired diseases,

*Author to whom correspondence should be addressed. E-mail: [email protected] 1–5 appear in color online: http://jba.sagepub.com

JOURNAL OF BIOMATERIALS APPLICATIONS Vol. 25 — March 2011 743

0885-3282/11/07 0743–26 $10.00/0 DOI: 10.1177/0885328209360425� The Author(s), 2010. Reprints and permissions:http://www.sagepub.co.uk/journalsPermissions.nav



including trauma, tumors, infections, and cosmetic corrections [1,2].In most of the cases, surgical treatment of the above mentionedpathologies requires the use of autologous or heterologous tissue grafts[3]. In general, these procedures are associated to different degrees oforganic, metabolic and immune complications such as graft rejection,infection, dehiscence, etc. [4], which can impair the results of thesurgical procedure.

In this context, novel techniques have focused on the efficientgeneration of autologous artificial tissues and organs developed bytissue engineering. Due to its autologous origin, these bioengineeredhuman tissues would not be submitted to the possibility of immunerejection once implanted in the host patient [5]. The construction ofartificial organs by tissue engineering is one of the research fields thathave experienced major progress during recent years [6]. By using tissueengineering techniques and biomaterials, different researchers havedeveloped well-organized artificial substitutes of different organs andtissues for therapeutic use, including, among others, human skin [7],cornea [8,9], bone [10], blood vessels [11], and oral mucosa [12,13].However, autologous generation of these tissues in the laboratory ishighly dependent on the availability of a proper source of adult cells thatare usually obtained from tissue biopsies from the same origin [12–15].

Ideally, for the generation of complex structures consisting on severaltissue types, it should be possible to obtain all these tissue types from asingle and accessible cell source. In this regard, one of the mostpromising sources of human adult stem cells is the adipose tissue, whichis very rich in stromal adipose-derived stem cells (ASCs) with highproliferative and differentiation capabilities [16,17]. So far, ASCs havebeen used for several ex vivo applications, including some of potentialusefulness in regenerative medicine such as the generation ofbioengineered bone [18,19], cartilage [20], neurons [21], and adiposetissue [22]. The clinical need of new sources of human tissues suitablefor skeletal, connective and neuronal reconstruction and repair supportsfurther research of novel methods and techniques based on tissueengineering and cell therapy.

In general, bioengineered tissues consist of 3D structures and stemcells that are grown on different biomaterials used as scaffolds. In thissense, we recently developed a novel biomaterial consisting in a mixtureof human fibrin and agarose [12–14]. Although several types of epithelialand connective cells isolated from the human cornea and oral mucosahave shown proper attachment and differentiation on this biomaterial,the influence of fibrin-agarose matrices on the multilineage differentia-tion capabilities of human ASCs have not been explored to the date.

744 R. NIETO-AGUILAR ET AL.



In this work, we have efficiently isolated and cultured ASCs in a 2Dculture system as well as immersed in a 3D scaffold of fibrin and agaroseand we have induced them to differentiate to adult cells of bone, cartilage,adipose, and neuron tissue phenotypes. Then, we have carried out ahistological and genetic study of the differentiated cells in order to estimatethe cell changes that take place during the differentiation process.

MATERIALS AND METHODS

Human Tissue Samples

In this work, biopsies of complete thickness of skin were obtained underlocal anesthesia. Average size of the samples used were approximately3� 2.5� 2 cm3. All tissues corresponded to healthy donors who weresubmitted to minor aesthetic surgical procedures at Division of PlasticSurgery of the University Hospital Virgen de las Nieves of Granada. Rightafter the excision, all tissues were submerged in Dulbecco’s modifiedEagle’s medium (DMEM) (Sigma-Aldrich Inc. St. Luis, MO, USA)supplemented with antibiotics and antimycotics (100 U/mL of penicillinG, 100 mg/mL of streptomycin and 0.25 mg/mL of B amphotericin) (Sigma-Aldrich) and stored at 48C until the moment of use.

As controls, normal human tissues corresponding to bone, cartilage,adipose tissue, and brain were used.

All donors provided their written consent for the use of the tissues inthis work, and this research was approved by the local Research andEthics committees at the University of Granada (Spain).

Generation of Primary Cultures of ASCs

On arriving to the laboratory, samples were washed in phosphatebuffered saline (PBS) and the adipose tissue was mechanically isolatedand fragmented into small pieces. Then, the tissue explants wereenzymatically digested in a 3% type I collagenase solution (Gibco BRLLife Technologies, Karlsruhe, Germany) for 8 h at 378C. Finally, isolatedstem cells were harvested by centrifugation and cultured in DMEMsupplemented with 10% fetal bovine serum (Sigma-Aldrich) andantibiotics-antimycotics (100 U/mL of penicillin G, 100 mg/mL of strep-tomycin and 0.25 mg/mL of B amphotericin) to generate 2D ASCs cellcultures (2D-ACC).

The culture medium was renewed every 3 days. Once the cells reachedsubconfluence, the cultures were trypsinized and subcultured on culturechamber slides (NuncTM, Roskilde, Denmark). All cells used in this work

Pluripotential Differentiation Capability of ASCs 745



belonged to the first four passages. As controls human oral mucosafibroblasts were cultured on same culture chamber slides.

Generation of Fibrin-agarose 3D Hydro-gels with ASCsImmersed Within (3D-FAH)

Once cultured ASCs reached subconfluence, the cells were trypsinizedand 175,000 cells per mL were seeded on fibrin-agarose scaffolds usingTranswell� permeable supports (Corning incorporated, NY, USA)following previously published protocols [12–14]. In short, the 3Dstromal substitute was generated by using a mixture of human plasmaand 0.1% of type VII agarose with tranexamic acid and calcium chloride.Once the hydrogel polymerized, the artificial constructs were suppliedwith basal medium.

As controls, fibrin-agarose scaffolds were seeded with 175,000 humanoral mucosa fibroblasts per mL.

Induction of Transdifferentiation to the Osteogenic,Chondrogenic, Adipogenic, and Neurogenic Lineagesin 2D-ACC and 3D-FAH

In order to induce both 2D-ACC and 3D-FAH cell cultures to theosteogenic, chondrogenic, adipogenic, and neurogenic phenotypes,samples were cultured in different inductive media. All these mediaconsisted of a basal DMEM medium 10% of FBS and antibiotic-antimycotics that was supplemented with different growth factors andinductive reagents.

For the induction to the osteoblastic lineage, the basal medium wassupplemented with 100 nM of dexametasone, 10 mM b-glycerol phos-phate and of 50 mM of L-ascorbic acid. For condrogenic induction, weadded 40 mg/mL L-proline, 100 mg/mL piruvate, 10 ng/mL TGF-b, 50mML-ascorbic acid and insulin-transferrin-selenium (ITS) 5 mg/mL. For theadipogenic differentiation, the basal medium was supplementedwith 1mM dexametasone, 0.5 mM 3-Isobutyl-1-methylxanthine and ITS5 mg/mL. Finally, for the neurogenic induction, basal medium wassupplemented with 20 nM valproic acid, 1 mM of butylated hydroxynisole(BHA), 50 mM potassium chloride, 10 nM forskolin, 1 nM hydrocortisoneand 5 mg/mL ITS.

All cells and bioengineered tissues were cultured in the differentinductive media for 20 days and the transdifferentiation processwas analyzed after 24 h, 10 days and 20 days of the addition of theinductive media.




As noninduced controls, 2D-ACC and 3D-FAH were cultured in basalnoninductive media. As positive controls of cell differentiation, tissuesections of human bone, cartilage, adipose tissue, and brain were used.

Histological Analysis

After the different induction periods, all samples were washed in PBSand fixed in 4% formaldehyde (Sigma-Aldrich). Staining and analysis of2D-ACC samples was carried out directly on the culture chambers,whereas tissue sections of paraffin-embedded tissues were used for3D-FAH.

For detection of the osteoblastic phenotype and to identify any signs ofmineralization, samples were stained with 2% Alizarin Red S for 5 minat room temperature. Chondrogenesis was confirmed by mucopolysac-charide staining using Alcian Blue solution (1% alcian blue 8GX and 3%glacial acetic acid, pH adjusted to 2.5). Adipogenic differentiation wasidentified by Oil Red O staining (0.7 mg in 100 mL of propylene glycol)for 5 min at 608C. Morphological analysis of all samples was carried outby conventional hematoxylin and eosin staining (H&E). Finally, allsamples were analyzed using a light microscope, and the percentage ofcells stained per field was determined using NIS-Elements imagingsoftware (Nikon, Tokyo, Japan).

As controls, tissue sections of formaldehyde-fixed, paraffin-embeddedhuman bone, cartilage, adipose tissue, and brain were processed andstained following the same protocols (Figure 1).

Statistical comparison of the percentage of positive cells between 2D-ACC and 3D-FAH was performed using the exact test of Fisher,considering all p values below 5% as statistically significant.

Immunofluorescence

After 2D-ACC were induced in the slide chambers, the cells were washedfor 10 min in PBS and fixed in 70% ethanol for 30 min. For immunofluor-escence, the cells were washed in distilled water, preincubated in 2.5% horseserum and hybridized for 2 h with one of the following primary antibodies:anti-alkaline phosphatase (1 : 200 dilution, Sigma-Aldrich), anti-collagentype II (1 : 200 dilution, Santa Cruz Biotechnology, CA, USA), anti-leptin-OB(1 : 200 dilution, Sigma-Aldrich) and anti-nestin (1 : 250 dilution, abcam,Cambridge, UK). Then, samples were washed in PBS and incubated for30 min in specific anti-mouse FITC-conjugated secondary antibodies (anti-collagen type II, anti-leptin-OB), anti-goat Cy3-conjugated secondaryantibodies (anti-alkaline phosphatase) or anti-rabbit Cy3-conjugated




secondary antibodies (anti-nestin). Finally, samples were washed in PBS,coversliped with DAPI mounting medium (Vector Laboratories,Peterborough, UK) and analyzed using fluorescence light microscopy.

On the other hand, control human tissues and transdifferentiated3D-FAH were washed in PBS and fixed in metacarn

Figure 1. Human native tissues as positive controls: (A) and (B) human bone tissue.

(A) Alizarin red S stain, (B) alkaline phosphatase protein by immunofluoresecence. (C) and(D) Human native cartilage. (C) alcian blue stain. (D) type II collagen. (E) and (F) Human

adipose tissue controls. (E) H&E staining. (F) leptin marker. (G) and (H) Native human

brain tissue. (G) H&E staining. (H) nestin immunofluorescence.




(methanol : chloroform : glacial acetic acid, 60 : 30 : 10) for 6 h at 48C.Then, samples were washed twice in ethanol 100%, dehydrated,paraffin-embedded and sectioned to 5 mm and mounted on microscopeslides.

After this, all samples were deparaffinized in xylene, rehydrated inethanol series (100%, 95%, 70%, 50%) and distilled water, preincubatedin 2.5% horse serum and hybridized for 2 h with one of the followingprimary antibodies: anti-alkaline phosphatase (1 : 200 dilution, Sigma-Aldrich), anti-collagen type II (1 : 200 dilution, Santa Cruz Antibodies,USA), anti-leptin-OB (1 : 200 dilution, Sigma-Aldrich), anti-nestin(1 : 250 dilution, abcam, UK). Then, samples were washed in PBS andincubated for 30 min in specific anti-mouse FITC-conjugated secondaryantibodies: anti-collagen type II and anti-leptin-OB (1 : 500 dilution,Sigma-Aldrich) or anti-goat Cy3-conjugated secondary antibodies: anti-alkaline phosphatase (1 : 500 dilution, Sigma-Aldrich) or anti-rabbit Cy3-conjugated secondary antibodies: anti-nestin (1 : 500 dilution, Sigma-Aldrich). Finally, samples were washed in PBS, coversliped with DAPImounting medium and analyzed using fluorescence light microscopy(Nikon Co. Eclipse i90, Japan). As positive controls, sections of normalhuman bone, cartilage, adipose tissue, and brain were processedfollowing the same immunofluorescence protocols (Figure 1).

Statistical comparison of the percentage of positive cells between 2D-ACC and 3D-FAH was performed using the exact test of Fisher,considering all p values below 5% as statistically significant.

Genome-wide Gene Expression Analysis of 2D-ACC usingOligonucleotide Microarrays

Total RNA corresponding to control noninduced ASCs and induced2D-ACC was extracted using the Qiagen RNeasy System (Qiagen,Mississauga, Ontario, Canada), according to the manufacturers’recommendations. RNA concentration was determined by absorbencyat 260 nm, and quality was verified by using a Bioanalyzer (Agilent).Total cDNA was synthesized with a T7-polyT primer and reversetranscriptase (Superscript II, Life Technologies, Inc., Carlsbad, CA)before in vitro transcription with biotinylated UTP and CTP (EnzoDiagnostics, Farmingdale, NY). Labeled nucleic acid target washybridized (458C for 16 h) to Affymetrix Human Genome U133 plus2.0 oligonucleotide arrays. After automated washing and staining,absolute values of expression were calculated and normalized from thescanned array by using Affymetrix Microarray Suite.




In this work, we have selected 95 genes with relevant functions relatedto each transdifferentiated cell type (osteocyte-like, adipocyte-like,chondrocyte-like, and neuron-like cells). To identify genes that becameupregulated after the induction process, we first calculated the averageexpression of control noninduced ASCs and of the different types oftransdifferentiated 2D-ACC. Then, the fold-change relative expression ofnoninduced controls versus transdifferentiated 2D-ACC was calculatedfor each cell type. At this step, all genes with a relative fold-change of atleast 1.2 (i.e., genes whose average expression became upregulated by atleast 20% in comparison with the control ASCs) were selected.

RESULTS

Induction to the Osteogenic Lineage

As shown in Figure 2 and Table 1, histological analysis of 2D-ACCinduced to osteoblast-type cells revealed that the production of a calcifiedextracellular matrix was absent for the first 24 h of induction (0% of areastained per field) for both alizarin red S staining and alkaline phosphataseimmunofluorescence. Then, when the cells were cultured in the osteo-inductive medium for 10 days, an initial generation of calcified extra-cellular matrix was detected in some scattered areas of the culture (5.24%of cells stained per field), with alkaline phosphatase being expressed by6.26% of the cells. Finally, cells incubated in the osteoinductive mediumfor 20 days showed large areas of calcification scattered all along the cellculture corresponding to 47.29% of stained cells per field and 40.6% of thecells showing alkaline phosphatase expression.

Then, the histological analysis of the 3D tissue constructs analyzed inthis study revealed that some of the cells in the 3D-FAH became trans-differentiated into osteoblast-like cells after 10 and 20 days of induction(7.60% positive cells by alizarin red S staining and 93.8 by alkalinephosphatase after 10 days of induction and 74.56% and 100%,respectively after 20 days of induction) (Figure 2 and Table 2). Asshown in Table 3, the percentage of positive cells was significantly higherin the 3D-FAH model than in 2D-ACC for alkaline phosphatase after 10days and for alkaline phosphatase and alizarin red S staining after 20 days( p¼ 0.0000).

Induction to the Chondrogenic Lineage

First, our results revealed that induction of 2D-ACC to thechondrogenic lineage during 24 h was not able to generate any




Figure 2. 2D-ACC and 3D-FAH transdifferentiation to the osteogenic lineage. (A)–(C)Alizarin red staining of 2D-ACC cultured for 24 h, 10 days and 20 days, respectively.

(G)–(I): Alkaline phosphatase immunofluorescence in 2D-ACC induced for 24 h, 10 days

and 20 days, respectively. Orange signals correspond to positive alkaline phosphatase

expression. (D)–(F) Alizarin red staining of 3D-FAH cultured for 24 h, 10 days and 20 days,respectively. (J)–(L): Alkaline phosphatase immunofluorescence in 3D-FAH induced for

24 h, 10 days and 20 days, respectively.

Table 1. Percentage of positive transdifferentiated ASCs on the 2D-ACCmodel when induced to the osteogenic, chondrogenic, adipogenic and

neurogenic lineages as determined by histological staining for alizarin red S,alcian blue, oil red O, and hematoxylin and eosin, and by

immunofluorescence for alkaline phosphatase, type II collagen,leptin-OB and nestin after 24 h, 10 days, and 20 days of induction.

Osteogenicinduction

Chondrogenicinduction

Adipogenicinduction

Neurogenicinduction

2D-ACCAlizarinred S

Alkalinephosphatase

Alcianblue

Type IIcollagen

Oilred O Leptin-OB H&E Nestin

24 h 0.00 0.00 0.00 0.00 0.00 0.00 31.00 43.4210 days 5.24 6.26 84.08 92.34 0.00 90.50 44.00 57.3420 days 47.29 40.6 94.94 93.00 33.33 98.10 44.00 85.71




detectable levels of cell transdifferentiation as determined by alcian bluestaining and type II collagen expression (0% of cells stained per field)(Figure 3 and Table 1). However, 2D-ACC cultured for 10 days inchondrogenic medium resulted in a high percentage of cells transdiffer-entiated to chondrocyte-like cells (84.08% of alcian blue positive cellsand 92.34% of cells showing collagen II expression). This percentage waseven higher after 20 days of induction (94.94% by alcian blue and 93.00%by immunofluorescence).

Similarly, the analysis of 3D-FAH induced to the chondrogenic lineageshowed that the process of cell transdifferentiation was not effectiveafter 24 h, with 0% of cells transdifferentiated (Figure 3 and Table 2).Nevertheless, when these bioengineered tissues were induced during 10

Table 2. Percentage of ASCs for the 3D-FAH model transdifferentiated positivelyto the osteogenic, chondrogenic, adipogenic, and neurogenic lineages as

determined by histological staining for alizarin red S, alcian blue, oil red O andhematoxylin and eosin, and by immunofluorescence for alkaline phosphatase, type

II collagen, leptin-OB and nestin after different induction times.

Osteogenicinduction


Adipogenicinduction

Neurogenicinduction

3D-FAHAlizarinred S

Alkalinephosphatase

Alcianblue

Type IIcollagen

Oilred O leptin-OB H&E Nestin

24 h 0.00 0.00 0.00 0.00 0.00 0.00 50.00 78.8010 days 7.60 93.80 14.80 80.00 0.00 87.00 55.60 85.3020 days 74.56 100.00 98.70* 86.70 92.30 87.90 60.00 95.74

Table 3. Differentiation analysis between 2D-ACC and 3D-FAH models induced tothe osteogenic, chondrogenic, adipogenic, and neurogenic lineages as deter-

mined by alizarin red S, alcian blue, oil red O and hematoxylin and eosin (H&E) andby immunofluorescence for alkaline phosphatase, type II collagen, leptin-OB and

nestin after 24 h, 10 days, and 20 days of induction process. Significantdifferences at p50.05 are considered as statistically significant.

2D-ACC

Osteogenicinduction


Adipogenicinduction

NeurogenicInduction

vs 3D-FAH(FET)

Alizarinred S

Alkalinephosphatase

Alcianblue

Type IIcollagen

Oilred O Leptin-OB H&E Nestin

24 h 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.0093 0.000010 days 0.5679 0.0000 0.0000 0.0236 1.0000 0.5149 0.1196 0.000020 days 0.0000 0.0000 0.2116 0.2380 0.0000 0.0101 0.0335 0.0238




days, 14.8% of the cells became positive by alcian blue staining, with80% of cells expressing collagen type II by immunofluorescence.The statistical analysis demonstrated that these percentages weresignificantly lower than those of the 2D-ACC model ( p¼ 0.0000 for thealcian blue and p¼ 0.0236 for the immunofluorescence) (Table 3).Finally, tissues induced to the chondrogenic lineage during 20 daysshowed 98.7% of the tissue area strongly stained by alcian blue and86.7% of the cells expressing collagen type II.

Induction to the Adipogenic Lineage

Histological analysis of 2D-ACC induced for 24 h or 10 days to theadipogenic lineage did not show the presence of any intracellular lipid

Figure 3. 2D-ACC and 3D-FAH chondrogenic differentiation: (A)–(C) Alcian blue

staining of ASCs induced after 24 h, 10 days and 20 days, respectively. (G)–(I) Type IIcollagen immunofluorescence in 2D-ACC chondroinduced for 24 h, 10 and 20 days,

correspondingly. (D)–(F) Alcian blue staining of 3D-FAH cultured for 24 h, 10 days and 20

days, respectively. (J)–(L) Type II collagen immunofluorescence in 3D-FAH induced for

24 h, 10 days and 20 days, respectively.




bodies by oil red O staining, with 0% of the cells expressing leptin-OB byimmunofluorescence after 24 h of induction. However, 90.5% of the cellsincubated for 10 days in the adipogenic medium showed detectableleptin-OB expression by immunofluorescence (Figure 4 and Table 1).Finally, one third of the cells induced for 20 days became stained by oilred O histochemistry, whereas 98.1% of cells expressed leptin-OB.

Similarly, our analysis of 3D-FAH showed that the adipogenic trans-differentiation process was not efficient at 24 h, and that 0% of cellsbecame stained by oil red O after 10 days in culture. In addition, 87% ofcells induced for 10 days expressed high amounts of leptin-OB. When thebioengineered 3D-FAH tissue constructs were induced during 20 days, thepercentage of oil red O-positive cells was significantly higher than that ofthe 2D-ACC (92.3% in 3D-FAH and 33.33% in 2D-ACC; p¼ 0.0000),

Figure 4. 2D-ACC and 3D-FAH adipogenic induction: (A)–(C) Oil red O staining of

induced ASCs to the adipogenic lineage for 24 h, 10 days and 20 days, respectively. (G)–(I)

Leptin-OB immunofluorescence in ASCs cultured for 24 h, 10 and 20 days, correspond-ingly. (D)–(F) Oil red O staining of 3D-FAH cultured for 24 h, 10 days and 20 days,

respectively. (J)–(L) Leptin OB immunofluorescence in 3D-FAH induced for 24 h, 10 days

and 20 days, respectively.




although the proportion of cells that expressed leptin-OB was signifi-cantly lower in comparison to the 2D-ACC (87.9% in 3D-FAH and 98.1%in 2D-ACC; p¼ 0.0101) (Table 3).

Induction to the Neurogenic Lineage

H&E staining of 2D-ACC induced to the neurogenic lineage revealedthat the transdifferentiation process initiated after 24 h of induction,with 31% of the cells developing a cytoplasmic cell prolongation thatresembled a rudimentary axon-like process and 43.42% showing nestinexpression by immunofluorescence (Figure 5 and Table 1). When thecells were incubated in the neurogenic medium for 10 and 20 days, 44%of the cells showed large axon-like prolongations as well as some small

Figure 5. 2D-ACC and 3D-FAH induction to the neurogenic lineage: (A)–(C) 2D-ACC

induced in neurogenic medium for 24 h, 10 days and 20 days, respectively; and stained with

H&E. (G)–(I) Nestin immunofluorescence in neuroinduced ASCs for 24 h, 10 and 20 dayscorrespondingly. (D)–(F) H&E staining of 3D-FAH cultured for 24 h, 10 days and 20 days,

respectively. (J)–(L) Nestin immunofluorescence in 3D-FAH induced for 24 h, 10 days and

20 days, respectively.




dentritiform cellular extensions, with 57.34% of the cells being positivefor nestin at day 10 and 85.71% of cells at day 20.

The analysis of the induced bioengineered 3D-FAH tissues revealedthat 50% of the cells in the tissues displayed significant morphologicalmodification compatible with the process of neuron-like transdiffer-entiation after 24 h of induction, with 78.8% of the cells expressingnestin protein by immunofluorescence (Figure 5 and Table 2). Then,induction during 10 days resulted in 55.6% of neuron-like cells and85.3% of cells with nestin expression, whereas the incubation of thetissues in the inductive medium for 20 days was able to generate 60% ofneuron-like cells and 95.74% of cells expressing nestin protein. All thesenestin expression percentages were significantly higher in 3D-FAH thanin 2D-ACC, with the morphological changes being higher only after20 days of induction (Table 3).

Gene Expression Analysis of Transdifferentiated 2D-ACC

Gene expression analysis of 2D-ACC induced to the osteogenic lineagefor 20 days showed significant upregulation of several genes related toosteogenic differentiation and bone function (more than 20% expressionincrease in comparison with controls), including ALPL, BGLAP, BMP15,BMP3, BMP6, BMP7, BMP8B and BMPR1B (Table 4). In addition,BMP8A and B, BMPER and BMPR1A showed a gene expressionincrement that was lower than 20%. Moreover, the expression of somegenes decreased after osteogenic induction, including BMP1, BMP10,BMP2, BMP4, BMP5, and BMP8A. The gene expression analysis of 2D-ACC induced to the chondrogenic lineage after 20 days of inductionshowed upregulation of the genes BGN, CHAD, CHSY1, CHSY3, CILP,CILP2, COL10A1, COL11A1, COL2A1, CSGALNACT2, HAS1, andHAS2 (Table 4), whereas COL11A2, COL6A1, and COL6A3 showed agene expression increment that was lower than 20% and some genes(ACAN, COL6A2, COL6A6, COL9A1, COL9A2, COL9A3, and HAS3)decreased after chondrogenic induction. Similarly, 2D-ACC incubated inadipogenic medium showed clear upregulation (more than 20% overcontrols) of the genes ACACB, ADFP, ADIPOQ, ADIPOR1, ADIPOR2,CFD, DGAT2, FAB1, FAB4, FAB7, FADS1, FADS2, FADS3, GPAM,GPD1, LBP, LEP, LEPROT, LEPROLT1, LPIN1, LPIN3, LPL, PLIN,and PNPLA3, with two genes downregulated after the induction processLPIN2 and RETN (Table 4). Finally, after induction of 2D-ACC to theneurogenic lineage, the microarray gene expression analysis revealedthat several genes related to neurogenic development and differentiationbecame upregulated by 20%, including DDN, HNT, NAV1, NAV2, NAV3,




Tab

le4

.G

en

eex

pre

ssio

nan

aly

sis

of

co

ntr

olA

SC

san

dA

SC

str

an

sdiffe

ren

tiate

dto

the

ost

eo

ge

nic

,ad

ipo

ge

nic

,c

ho

nd

rog

en

ic,

an

dn

eu

rog

en

iclin

eag

es

as

de

term

ine

db

ym

icro

arr

ay.

Ge

ne

ID:

Aff

yme

trix

refe

ren

ce

of

the

spe

cifi

cg

en

eo

rp

rob

e-s

et.

FC

CA

RT

/CT

R:

fold

-ch

an

ge

exp

ress

ion

of

ce

llsin

du

ce

dto

the

ch

on

dro

ge

nic

line

ag

evs

co

ntr

olA

SC

s.F

CB

ON

E/C

TR

:fo

ld-c

han

ge

exp

ress

ion

ofA

SC

sin

du

ce

din

too

ste

oc

yte

-lik

ec

ells

vsc

on

tro

lAS

Cs.

FC

NE

UR

O/C

TR

:fo

ld-c

han

ge

exp

ress

ion

ofc

ells

ind

uc

ed

toth

en

eu

rog

en

iclin

eag

evs

co

ntr

olA

SC

s.F

CA

DIP

O/C

TR

:fo

ld-c

han

ge

exp

ress

ion

ofc

ells

ind

uc

ed

toth

ead

ipo

ge

nic

line

ag

evs

co

ntr

ol

AS

Cs.

Ge

ne

ssh

ow

ing

co

nsi

de

rab

leove

rexp

ress

ion

(mo

reth

an

20

%in

cre

ase

)afte

rth

ein

du

ctio

nto

the

diffe

ren

tc

ell

line

ag

es

we

rese

lec

ted

.

Ge

ne

ID

FC

CA

RT

/C

TR

FC

BO

NE

/C

TR

FC

NE

UR

O/

CT

R

FC

AD

IPO

/C

TR

Ge

ne

Sym

bo

lG

en

eT

itle

1557924_s

_at/

//215783_s

_at

0.7

11.5

51.3

31.3

8A

LPL

Alk

alin

ep

ho

sph

ata

se,

liver/

bo

ne/k

idn

ey

206956_a

t0.9

61.2

90.8

01.0

6B

GLA

PB

on

eg

am

ma-c

arb

oxy

glu

tam

ate

(gla

)p

rote

in1570383_a

t///

202701_a

t///

1569002_

x_at/

//205574_x

_at/

//206725_x

_at/

//207595_s

_at/

//1569001_a

t

1.0

00.6

20.8

80.7

9B

MP

1B

on

em

orp

ho

gen

etic

pro

tein

1

208292_a

t1.0

10.5

91.5

50.8

1B

MP

10

Bo

ne

mo

rph

og

en

etic

pro

tein

10

221332_a

t2.3

71.8

91.7

41.5

2B

MP

15

Bo

ne

mo

rph

og

en

etic

pro

tein

15

205290_s

_at/

//205289_a

t0.5

00.2

60.6

30.2

2B

MP

2B

on

em

orp

ho

gen

etic

pro

tein

2208244_a

t4.9

01.9

00.6

09.9

0B

MP

3B

on

em

orp

ho

gen

etic

pro

tein

3211518_s

_at

0.0

70.0

50.0

80.0

6B

MP

4B

on

em

orp

ho

gen

etic

pro

tein

4205431_s

_at/

//205430_a

t0.3

80.8

30.7

10.5

9B

MP

5B

on

em

orp

ho

gen

etic

pro

tein

5241141_a

t///

206176_a

t///

215042_a

t1.1

82.5

01.3

41.2

6B

MP

6B

on

em

orp

ho

gen

etic

pro

tein

6211260_a

t///

209591_s

_at

1.4

22.2

61.5

11.8

7B

MP

7B

on

em

orp

ho

gen

etic

pro

tein

7207866_a

t///

220203_a

t///

220204_s

_at

1.4

40.7

70.8

21.1

0B

MP

8A

Bo

ne

mo

rph

og

en

etic

pro

tein

8a

221615_a

t0.8

81.0

10.9

01.0

5B

MP

8A

///

BM

P8B

Bo

ne

mo

rph

og

en

etic

pro

tein

8a

///

bo

ne

mo

rph

og

en

etic

pro

tein

8b

235275_a

t///

207865_s

_at

0.8

22.5

50.6

80.7

6B

MP

8B

Bo

ne

mo

rph

og

en

etic

pro

tein

8b

241986_a

t0.1

11.1

30.4

11.0

8B

MP

ER

BM

Pb

ind

ing

en

do

thelia

lre

gu

lad

or

(co

ntin

ue

d)




Tab

le4

.C

on

tinu

ed

.

Ge

ne

ID

FC

CA

RT

/C

TR

FC

BO

NE

/C

TR

FC

NE

UR

O/

CT

R

FC

AD

IPO

/C

TR

Ge

ne

Sym

bo

lG

en

eT

itle

204832_s

_at/

//213578_a

t1.0

91.1

21.1

30.7

6B

MP

R1A

Bo

ne

mo

rph

og

en

etic

pro

tein

rece

pto

r,ty

pe

IA229975_a

t///

242579_a

t1.8

32.8

32.3

50.7

7B

MP

R1B

Bo

ne

mo

rph

og

en

etic

pro

tein

rece

pto

r,ty

pe

IB1554950_a

t///

217161_x

_at/

//207692_s

_at/

//205679_x

_at

0.6

30.5

40.9

01.1

1A

CA

NA

gg

reca

n

201262_s

_at/

//213905_x

_at/

//201261_x

_at

13.7

00.4

23.4

70.4

5B

GN

Big

lyca

n

206869_a

t2.1

50.3

01.0

00.8

5C

HA

DC

ho

nd

road

herin

205567_a

t///

203044_a

t1.8

51.7

11.3

73.0

6C

HS

Y1

Ch

on

dro

itin

sulfa

tesy

nth

ase

1242100_a

t1.6

70.8

21.3

51.6

9C

HS

Y3

Ch

on

dro

itin

sulfa

tesy

nth

ase

3206227_a

t5.4

30.4

81.4

01.0

6C

ILP

Cart

ilag

ein

term

ed

iate

laye

rp

rote

in,

nu

cleo

tide

pyr

op

ho

sph

oh

ydro

lase

1552289_a

_at/

//1552288_a

t11.2

70.5

51.1

50.7

7C

ILP

2C

art

ilag

ein

term

ed

iate

laye

rp

rote

in2

217428_s

_at/

//205941_s

_at

24.6

91.8

21.6

90.6

6C

OL1

0A

1C

olla

gen

,ty

pe

X,

alp

ha

1229271_x

_at/

//37892_a

t///

204320_a

t10.7

22.3

74.5

36.0

4C

OL1

1A

1C

olla

gen

,ty

pe

XI,

alp

ha

1213870_a

t///

216993_s

_at

1.0

81.0

31.0

31.0

6C

OL1

1A

2C

olla

gen

,ty

pe

XI,

alp

ha

2217404_s

_at/

//213492_a

t6.3

21.8

12.0

92.2

2C

OL2

A1

Co

llag

en

,ty

pe

II,alp

ha

1212940_a

t///

214200_s

_at/

//212938_a

t///

212937_s

_at/

//213428_s

_at/

//212091_s

_at/

//216904_a

t///

212939_a

t

1.0

41.3

90.9

61.0

9C

OL6

A1

Co

llag

en

,ty

pe

VI,

alp

ha

1

209156_s

_at/

//213290_a

t0.9

01.1

70.9

30.8

4C

OL6

A2

Co

llag

en

,ty

pe

VI,

alp

ha

2201438_a

t1.0

20.9

40.9

90.8

3C

OL6

A3

Co

llag

en

,ty

pe

VI,

alp

ha

3230867_a

t0.2

50.4

30.4

40.1

8C

OL6

A6

Co

llag

en

,ty

pe

VI,

alp

ha

61555527_a

t///

222008_a

t0.8

81.0

52.0

91.2

4C

OL9

A1

Co

llag

en

,ty

pe

IX,

alp

ha

1232542_a

t///

213622_a

t0.4

60.5

31.0

90.7

8C

OL9

A2

Co

llag

en

,ty

pe

IX,

alp

ha

2




204724_s

_at

0.8

50.3

50.3

00.4

5C

OL9

A3

Co

llag

en

,ty

pe

IX,

alp

ha

3222235_s

_at/

//218871_x

_at/

//239077_a

t1.5

00.8

51.2

40.8

0C

SG

ALN

AC

T2

Ch

on

dro

itin

sulfa

teN

-ace

tylg

ala

cto

sam

inyl

tran

sfera

se2

207316_a

t8.6

30.7

01.7

35.1

5H

AS

1H

yalu

ron

an

syn

thase

1230372_a

t///

206432_a

t23.6

04.1

91.6

59.2

8H

AS

2H

yalu

ron

an

syn

thase

2223541_a

t///

228179_a

t///

1552980_a

t0.9

00.9

00.7

00.3

8H

AS

3H

yalu

ron

an

syn

thase

3214788_x

_at

0.7

31.5

31.7

31.3

3D

DN

Den

drin

227566_a

t///

222020_s

_at/

//241934_a

t10.7

40.5

52.8

81.3

4H

NT

Neu

rotr

imin

233567_a

t///

233870_a

t///

224774_s

_at/

//227584_a

t///

224771_a

t///

224770_

s_at/

//224773_a

t///

224772_a

t

2.8

71.6

61.6

91.2

8N

AV

1N

eu

ron

navi

gato

r1

1567357_a

t///

222599_s

_at/

//1556606_

at/

//1567358_a

t///

218330_s

_at/

//222598_s

_at

0.9

31.6

81.2

21.7

1N

AV

2N

eu

ron

navi

gato

r2

1552658_a

_at/

//1562234_a

_at/

//204823_a

t///

216632_a

t///

216466_a

t1.5

51.0

41.5

21.8

1N

AV

3N

eu

ron

navi

gato

r3

214952_a

t///

229799_s

_at/

//231532_a

t///

212843_a

t///

217359_s

_at/

//227394_a

t///

209968_s

_at

0.8

40.7

71.0

10.8

0N

CA

M1

Neu

ral

cell

ad

hesi

on

mo

lecu

le1

205669_a

t///

232390_a

t0.3

00.9

50.8

31.1

2N

CA

M2

Neu

ral

cell

ad

hesi

on

mo

lecu

le2

205143_a

t0.7

82.5

62.6

15.0

6N

CA

NN

eu

roca

n204412_s

_at/

//33767_a

t1.2

73.0

41.5

42.7

2N

EF

HN

eu

rofil

am

en

t,h

eavy

po

lyp

ep

tide

221805_a

t///

221801_x

_at/

//221916_a

t///

2.0

64.6

32.8

78.5

2N

EF

LN

eu

rofil

am

en

t,lig

ht

po

lyp

ep

tide

205113_a

t///

223902_a

t0.8

20.9

90.8

41.3

8N

EF

MN

eu

rofil

am

en

t,m

ed

ium

po

lyp

ep

tide

1553194_a

t///

229461_x

_at/

//243357_a

t///

239548_a

t3.1

96.3

92.7

28.0

7N

EG

R1

Neu

ron

al

gro

wth

reg

ula

tor

1

1556057_s

_at/

//206282_a

t0.6

22.6

42.8

41.0

2N

EU

RO

D1

Neu

rog

en

icd

iffere

ntia

tion

1210271_a

t///

1552953_a

_at

6.2

11.4

42.1

02.1

7N

EU

RO

D2

Neu

rog

en

icd

iffere

ntia

tion

2221318_a

t1.3

40.3

11.1

40.8

0N

EU

RO

D4

Neu

rog

en

icd

iffere

ntia

tion

4220045_a

t1.5

00.5

01.2

12.4

3N

EU

RO

D6

Neu

rog

en

icd

iffere

ntia

tion

6208497_x

_at

2.8

87.1

25.3

58.9

4N

EU

RO

G1

Neu

rog

en

in1

(co

ntin

ue

d)




Tab

le4

.C

on

tinu

ed

.

Ge

ne

ID

FC

CA

RT

/C

TR

FC

BO

NE

/C

TR

FC

NE

UR

O/

CT

R

FC

AD

IPO

/C

TR

Ge

ne

Sym

bo

lG

en

eT

itle

215632_a

t1.1

71.1

72.1

72.6

7N

EU

RO

G2

Neu

rog

en

in2

207965_a

t1.3

70.2

40.3

10.3

1N

EU

RO

G3

Neu

rog

en

in3

206814_a

t0.6

10.1

50.7

60.4

9N

GF

Nerv

eg

row

thfa

cto

r(b

eta

po

lyp

ep

tide)

205858_a

t3.6

041.6

04.4

0108.4

0N

GF

RN

erv

eg

row

thfa

cto

rre

cep

tor

(TN

FR

sup

erf

am

ily,

mem

ber

16)

217963_s

_at

0.8

61.2

30.9

91.1

1N

GF

RA

P1

Nerv

eg

row

thfa

cto

rre

cep

tor

(TN

FR

SF

16)

ass

oci

ate

dp

rote

in1

204105_s

_at/

//216959_x

_at

2.0

25.4

43.1

213.6

9N

RC

AM

Neu

ron

al

cell

ad

hesi

on

mo

lecu

le240532_a

t0.7

06.9

00.8

01.1

0S

LC32A

1S

olu

teca

rrie

rfa

mily

32

(GA

BA

vesi

cula

rtr

an

spo

rter)

,m

em

ber

11554724_a

t9.2

01.4

01.2

01.8

0S

LC6A

11

So

lute

carr

ier

fam

ily6

(neu

rotr

an

smitt

er

tran

spo

rter,

GA

BA

),m

em

ber

11

237058_x

_at

1.0

50.6

70.8

20.7

8S

LC6A

13

So

lute

carr

ier

fam

ily6

(neu

rotr

an

smitt

er

tran

spo

rter,

GA

BA

),m

em

ber

13

217213_a

t///

215715_a

t///

217214_s

_at/

//210353_s

_at/

//2.1

80.6

91.4

82.4

3S

LC6A

2S

olu

teca

rrie

rfa

mily

6(n

eu

rotr

an

smitt

er

tran

spo

rter,

no

rad

ren

alin

),m

em

ber

2207519_a

t2.7

12.0

62.4

60.9

4S

LC6A

4S

olu

teca

rrie

rfa

mily

6(n

eu

rotr

an

smitt

er

tran

spo

rter,

sero

ton

in),

mem

ber

4210854_x

_at/

//202219_a

t///

213843_

x_at/

//2.1

21.2

70.9

13.0

8S

LC6A

8S

olu

teca

rrie

rfa

mily

6(n

eu

rotr

an

smitt

er

tran

spo

rter,

creatin

e),

mem

ber

8207043_s

_at

1.0

80.2

61.2

50.4

9S

LC6A

9S

olu

teca

rrie

rfa

mily

6(n

eu

rotr

an

smitt

er

tran

spo

rter,

gly

cin

e),

mem

ber

9214584_x

_at/

//221928_a

t///

49452_a

t///

1552616_a

_at/

//1552615_a

t///

43427_a

t0.8

11.8

91.7

951.5

0A

CA

CB

Ace

tyl-C

oen

zym

eA

carb

oxy

lase

beta

215895_x

_at/

//209122_a

t0.4

20.7

30.9

51.6

7A

DF

PA

dip

ose

diff

ere

ntia

tion

-rela

ted

pro

tein

///A

dip

op

hili

n




207175_a

t0.3

09.0

26.3

6556.2

1A

DIP

OQ

Ad

ipo

nect

in,

C1Q

an

dco

llag

en

do

main

con

tain

ing

217748_a

t1.2

81.1

20.8

81.3

3A

DIP

OR

1A

dip

on

ect

inre

cep

tor

1201346_a

t1.3

51.0

30.6

41.7

6A

DIP

OR

2A

dip

on

ect

inre

cep

tor

2205382_s

_at

0.1

01.5

70.9

33.5

4C

FD

Co

mp

lem

en

tfa

cto

rD

(ad

ipsi

n)

224327_s

_at/

//226064_s

_at

0.1

81.3

21.3

152.0

7D

GA

T2

Dia

cylg

lyce

rol

O-a

cyltr

an

sfera

seh

om

olo

g2

(mo

use

)205892_s

_at/

//231693_a

t0.7

81.3

71.8

71.2

8F

AB

P1

Fatty

aci

db

ind

ing

pro

tein

1,

liver,

mR

NA

(cD

NA

clo

ne

IMA

GE

:4712653)/

//fa

tty

aci

db

ind

ing

pro

tein

1,

liver

235978_a

t///

203980_a

t0.9

716.0

58.2

2924.0

5F

AB

P4

Fatty

aci

db

ind

ing

pro

tein

4,

ad

ipo

cyte

205029_s

_at/

//205030_a

t///

216192_a

t1.7

21.5

52.6

910.8

4F

AB

P7

Fatty

aci

db

ind

ing

pro

tein

7,

bra

in208964_s

_at/

//208963_x

_at/

//208962_s

_at

0.7

51.5

91.2

95.7

2F

AD

S1

Fatty

aci

dd

esa

tura

se1

202218_s

_at/

//243953_a

t1.8

02.2

61.3

43.4

4F

AD

S2

Fatty

aci

dd

esa

tura

se2

204257_a

t///

216080_s

_at

1.2

91.5

11.2

23.4

3F

AD

S3

Fatty

aci

dd

esa

tura

se3

225420_a

t///

225424_a

t3.2

31.6

62.0

344.3

2G

PA

MG

lyce

rol-3-p

ho

sph

ate

acy

ltran

sfera

se,

mito

cho

nd

rial

204997_a

t///

213706_a

t1.0

07.4

52.2

0997.9

3G

PD

1G

lyce

rol-3-p

ho

sph

ate

deh

ydro

gen

ase

1(s

olu

ble

)214461_a

t0.1

63.6

90.2

592.4

1LB

PLi

po

po

lysa

cch

arid

eb

ind

ing

pro

tein

207092_a

t13.2

070.2

06.1

019.8

0LE

PLe

ptin

227095_a

t///

202378_s

_at

0.8

21.2

41.3

01.4

4LE

PR

OT

Lep

tinre

cep

tor

ove

rlap

pin

gtr

an

scrip

t202595_s

_at

1.9

01.1

51.3

41.4

9LE

PR

OTL1

Lep

tinre

cep

tor

ove

rlap

pin

gtra

nscr

ipt-

like

1212276_a

t///

212274_a

t///

212272_a

t0.3

60.9

30.9

82.8

2LP

IN1

Lip

in1

202460_s

_at/

//202459_s

_at

0.8

81.0

90.8

10.9

5LP

IN2

Lip

in2

232966_a

t1.2

51.3

51.1

51.7

5LP

IN3

Lip

in3

203549_s

_at/

//203548_s

_at

0.2

19.5

24.0

8438.9

5LP

LLi

po

pro

tein

lipase

205913_a

t0.8

224.2

45.8

21879.9

4P

LIN

Peril

ipin

220675_s

_at/

//233030_a

t0.4

57.8

21.7

459.9

5P

NP

LA3

Pata

tin-lik

ep

ho

sph

olip

ase

do

main

con

tain

ing

3220570_a

t0.4

00.3

10.1

90.1

3R

ETN

Resi

stin




NCAN, NEFH, NEFL, NEGR1, NEUROD1, NEUROD2, NEUROD6,NEUROG1, NEUROG2, NGFR, NRCAM, SLC6A2, SLC6A4, andSLC6A9. In contrast, NCAM1, NEUROD4, and SLC6A11 showed agene expression increment that was lower than 20% and the genesNCAM2, NEFM, NEUROG3, NGF, NGFRAP1, SLC32A1, SLC6A13,and SLC6A8 decreased their expression after induction towards theneurogenic lineage (Table 4).

DISCUSSION

Generation of artificial tissues using adult stem cells and biocompa-tible biomaterials is one of the main objectives of current biomedicalresearch. Although these bioengineered tissues could be potentiallyuseful for the clinical substitution of damaged tissues, harvesting ofnative cells with proliferation and differentiation capabilities is notalways possible from these damaged tissues. For that reason, the searchof alternative cell sources for use as substitutes of the native cells is oneof the current challenges of the field.

In this context, ASCs are one of the most promising sources of humanadult mesenchymal stem cells [16,23] with high proliferative anddifferentiation capabilities [16,17]. One of the main properties of thesecells is their accessibility and the possibility of obtaining different types ofcells from a single pluripotential cell source. Although the potentiality ofASCs cultured on 2D systems was previously established [24,25], verylittle is so far known about the phenotype and differentiation potentialityof cells in 3D culture systems [26,27], in which cells resemble the naturalstructure of native tissues. For that reason, in this work we havecompared the behavior of ASCs cultured on 2D and 3D conditions usingfibrin-agarose biomaterials previously described by our research group[8,12–14]. Ideally, a good biomaterial should be suitable to act as a cellcarrier [28–37], guiding cell differentiation and proliferation [28].However, the influence of fibrin-agarose biomaterials on the differentia-tion capabilities of ASCs has not been elucidated to the date.

In this regard, our results demonstrated that 2D-ACC can beefficiently transdifferentitated to several tissue types, which wasconfirmed not only by histological analysis, but also by histochemistry,immunofluorescence, and high-throughput mRNA expression as deter-mined by microarray. In addition, our analyses confirmed the cellularplasticity of ASCs when subjected to the osteogenic, chondrogenic,adipogenic, and neurogenic induction media on 3D culture conditions(3D-FAH) as well. Strikingly the transdifferentiation potential of ASCssubjected to 3D cultures was comparable or even higher than that of 2D




cultures, except for the 10 days chondrogenic induction. These resultsimply that the fibrin-agarose 3D culture model could support theefficient transdifferentiation capability of ASCs, suggesting that thegeneration of 3D human tissue substitutes using ASCs is a feasibletechnique in the laboratory.

When ASCs induced to the osteogenic lineage were analyzed, weobserved that a high percentage of the cells acquired the osteogenicphenotype after 20 days of induction, especially in the 3D culturesystem. In addition to the histological analysis, the gene expressionanalysis of 2D-ACC revealed that a high number of genes with a role inbone development and function became overexpressed at day 20 ofinduction. These results imply that transdifferentiated osteocyte-likecells not only resembled the morphology of bone cells, but they alsobehave like bone cells from the genetic point of view, supporting thepotential clinical use of these cells. In contrast, other relevantosteogenic-related genes did not show considerable levels of upregula-tion, likely due to the fact that the cells have been induced to theosteogenic lineage for only 20 days or to a lack of specific in situ bonesignals that would develop in an in vivo environment. Further in vivoexperiments could give new information of genes expression of inducedand transdifferentiated ASCs.

On the other hand, when we evaluated ASCs transdifferentiation to thechondrogenic lineage, we found that both the 2D-ACC and the 3D-FAHwere able to synthesize high amounts of mucopolysaccharides andcollagen type II at day 10 of induction, suggesting that the chondrogenicphenotype could be acquired before the osteogenic phenotype. Also,microarray gene expression of 2D-ACC confirmed that several genesrelated to the cartilage lineage were overexpressed upon induction,including BGN, CHAD, CHSY1, CHSY3, CILP, CILP2, COL10A1,COLL11A1, CSGALNACT2, HAS1, HAS2, and COL2A1. This latterhas been reported in previous studies as an important gene expressed inchondroinduced stem cells [24,25,38]. Quite the opposite, some genesencoding for some collagen isoforms and the gene HAS3 showeddownregulation after chondrogenic induction. This reduced gene expres-sion could be due to the fact that an in vivo environment or a higherex vivo induction period could be necessary for the proper upregulation ofcertain types of genes. All these data support the idea that ASCs inducedto the chondrogenic lineage have a very similar behavior to the nativecartilage and may have a potential usefulness in regenerative medicine.

Similarly, ASCs induced to adipocyte-like cells displayed high levels ofmRNA gene expression for most of the genes related to the adiposelineage. Indeed, 92% of the genes related to adipose tissue that were




analyzed in this work, became overexpressed after induction. Mostlikely, this is due to the fact that ASCs reside in adipose tissue and evenafter being isolated and expanded in culture, they could maintain a highamount of growing factors or intra/extracellular signals which couldreact once the adipogenic induction medium was applied ex vivo to thesecells. Also, our microarray analysis revealed that this gene over-expression was very high for several genes such as ADIPOQ, FABP4,GPD1, LPL, and PLIN, which are essential for adipocyte differentiationand function [39–42]. Nevertheless, cell staining with oil red O showedthat 2D-ACC and 3D-FAH did not show any cytoplasmatic droplets withtriglycerides content until the 20 day of adipoinduction, although asignificant leptin-OB expression was found from day 10. All these resultssuggest that ASCs have a high potential to differentiate into adipocyte-like cells ex vivo, which could be potentially useful in medicine.

In respect to the neurogenic induction, 2D-ACC overexpressed 63% ofthe related genes to the neural lineage after 20 days of neurogenicinduction, including among others, NEUROD6, NEUROG1, NEUROG2,and the nerve growth factor receptor NGFR. Interestingly, induced cellson both the 2D and the 3D culture models showed importantmorphological changes, with the formation of axon-like cell prolonga-tions, and rudimentary dendritic extensions at the cell membrane afteronly 24 h of induction. These findings coincide with the upregulation ofgenes that are specific of neuron development, including two types ofneurofilaments (the light and the heavy polypeptides), the neural celladhesion molecule NRCAM and the gene encoding for dendrin, with animportant role in the development of neuronal dendrites [43]. Accordingto the nestin protein expression determined by immunofluorescence,both the 2D-ACC and the 3D-FAH resulted in most of the cells (485%)expressing nestin after 20 days of induction. The potential clinical use ofautologous neuron-like cells developed on both models from adult stemcells is unlimited.

One of the main advantages of the methods described here is theirsimplicity, the rapidness of the transdifferentiation induction and thefact that a single cell type has been used for the generation of severaladult tissue types. Thus, by using specific conditioning culture media,ASCs can be efficiently differentiated to adipocyte-like cells in 2D-ACCand 3D-FAH models after 20 days in culture, transdifferentiated toneurons and chondrocytes-like cells after 10 and 20 days, respectivelyand into osteocytes-like cells after 20 days. Another advantage of thismethodology is that it does not imply any genetic manipulation of thecells. Therefore, the possibility of inducing a severe genomic alterationwith oncogenic potential is very low.




In summary, in this work we have demonstrated that highly accessiblestem cells obtained from the adipose tissue (ASCs) are an efficientsource of cells for use in tissue engineering with high differentiationpotentiality on both 2D and 3D culture systems. The use of highlybiocompatible fibrin-agarose scaffolds results in comparable or evenhigher efficiencies of cell transdifferentiation ex vivo.

ACKNOWLEDGEMENTS

This work was supported by grants P06-CTS-2191 and SAS PI0135/2007 of Junta de Andalucia. This study was approved by the localResearch and Ethics committees of the University of Granada (number2006/2191).

REFERENCES

1. Holcomb, J.D. and Gentile, R.D. Aesthetic Facial Surgery of MalePatients: Demographics and Market Trends, Facial Plast. Surg., 2005: 21:223–231.

2. Housman, T.S., Hancox, J.G., Mir, M.R. et al. What Specialties Perform theMost Common Outpatient Cosmetic Procedures in the United States?Dermatol. Surg., 2008: 34: 1–7.

3. Chiu, R.C. Msc Immune Tolerance in Cellular Cardiomyoplasty, Semin.Thorac. Cardiovasc. Surg., 2008: 20: 115–118.

4. Baillie, D.R., Stawicki, S.P., Eustance, N., Warsaw, D. and Desai, D. Use ofHuman and Porcine Dermal-derived Bioprostheses in Complex AbdominalWall Reconstructions: A Literature Review and Case Report, Ostomy WoundManage., 2007: 53: 30–37.

5. Kim, I.K., Bedi, D.S., Denecke, C., Ge, X. and Tullius, S.G. Impact of Innateand Adaptive Immunity on Rejection and Tolerance, Transplantation, 2008:86: 889–894.

6. Atala, A. Advances in Tissue and Organ Replacement, Curr. Stem Cell Res.Ther., 2008: 3: 21–31.

7. Priya, S.G., Jungvid, H. and Kumar, A. Skin Tissue Engineering for TissueRepair and Regeneration, Tissue Eng. Part B Rev., 2008: 14: 105–118.

8. Alaminos, M., Del Carmen Sanchez-Quevedo, M., Munoz-Avila, J.I. et al.Construction of a Complete Rabbit Cornea Substitute Using a Fibrin-agarose Scaffold, Invest. Ophthalmol. Vis. Sci., 2006: 47: 3311–3317.

9. Alaminos, M., Sanchez-Quevedo, M.C., Munoz-Avila, J.I. et al. Evaluation ofthe Viability of Cultured Corneal Endothelial Cells by Quantitative ElectronProbe X-ray Microanalysis, J. Cell. Physiol., 2007: 211: 692–698.

10. Stevens, B., Yang, Y., Mohandas, A., Stucker, B. and Nguyen, K.T. A Reviewof Materials, Fabrication Methods, and Strategies Used to Enhance BoneRegeneration in Engineered Bone Tissues, J. Biomed. Mater. Res. B Appl.Biomater., 2008: 85: 573–582.




11. Fuchs, S., Ghanaati, S., Orth, C. et al. Contribution of Outgrowth EndothelialCells from Human Peripheral Blood on In Vivo Vascularization of BoneTissue Engineered Constructs Based on Starch Polycaprolactone Scaffolds,Biomaterials, 2009: 30: 526–534.

12. Alaminos, M., Garzon, I., Sanchez-Quevedo, M.C. et al. Time-course Study ofHistological and Genetic Patterns of Differentiation in Human EngineeredOral Mucosa, J. Tissue Eng. Regen. Med., 2007: 1: 350–359.

13. Sanchez-Quevedo, M.C., Alaminos, M., Capitan, L.M. et al. Histological andHistochemical Evaluation of Human Oral Mucosa Constructs Developed byTissue Engineering, Histol. Histopathol., 2007: 22: 631–640.

14. Garzon, I., Sanchez-Quevedo, M.C., Moreu, G. et al. In Vitro and In VivoCytokeratin Patterns of Expression in Bioengineered Human PeriodontalMucosa, J. Periodontal Res., 2009: 44(5): 588–597.

15. Kinikoglu, B., Auxenfans, C., Pierrillas, P. et al. Reconstruction of a Full-Thickness Collagen-based Human Oral Mucosal Equivalent, Biomaterials,2009: 30: 6418–6425.

16. De Ugarte, D.A., Alfonso, Z., Zuk, P.A. et al. Differential Expression of StemCell Mobilization-associated Molecules on Multi-lineage Cells from AdiposeTissue and Bone Marrow, Immunol. Lett., 2003: 89: 267–270.

17. Schaffler, A. and Buchler, C. Concise Review: Adipose Tissue-derivedStromal Cells – Basic and Clinical Implications for Novel Cell-basedTherapies, Stem Cells, 2007: 25: 818–827.

18. Weinzierl, K., Hemprich, A. and Frerich, B. Bone Engineering with AdiposeTissue Derived Stromal Cells, J. Craniomaxillofac. Surg., 2006: 34:466–471.

19. Xu, Y., Malladi, P., Wagner, D.R. and Longaker, M.T. Adipose-derivedMesenchymal Cells as a Potential Cell Source for Skeletal Regeneration,Curr. Opin. Mol. Ther., 2005: 7: 300–305.

20. Wei, Y., Sun, X., Wang, W. and Hu, Y. Adipose-derived Stem Cells andChondrogenesis, Cytotherapy, 2007: 9: 712–716.

21. Lin, C.S., Xin, Z.C., Deng, C.H., Ning, H., Lin, G. and Lue, T.F. RecentAdvances in Andrology-related Stem Cell Research, Asian J. Androl., 2008:10: 171–175.

22. Yoshimura, K., Sato, K., Aoi, N. et al. Cell-assisted Lipotransfer for FacialLipoatrophy: Efficacy of Clinical Use of Adipose-derived Stem Cells,Dermatol Surg., 2008: 34(9): 1178–85.

23. Zuk, P.A, Zhu, M., Ashjian, P. et al. Human Adipose Tissue is a Source ofMultipotent Stem Cells, Mol. Biol. Cell, 2002: 13: 4279–4295.

24. Guilak, F., Lott, K.E., Awad, H.A. et al. Clonal Analysis of theDifferentiation Potential of Human Adipose-derived Adult Stem Cells,J. Cell. Physiol., 2006: 206: 229–237.

25. Matsumoto, T., Kano, K., Kondo, D. et al. Mature Adipocyte-derivedDedifferentiated Fat Cells Exhibit Multilineage Potential, J. Cell. Physiol.,2008: 215: 210–222.

26. Duggal, S., Fronsdal, K.B., Szoke, K., Shahdadfar, A., Melvik, J.E. andBrinchmann, J.E. Phenotype and Gene Expression of Human MesenchymalStem Cells in Alginate Scaffolds, Tissue Eng. Part A, 2009: 15: 1763–1773.




27. Mauney, J.R., Nguyen, T., Gillen, K., Kirker-Head, C., Gimble, J.M. andKaplan, D.L. Engineering Adipose-like Tissue In Vitro and In Vivo UtilizingHuman Bone Marrow and Adipose-derived Mesenchymal Stem Cells withSilk Fibroin 3D Scaffolds, Biomaterials, 2007: 28: 5280–5290.

28. Hutmacher, D.W., Goh, J.C. and Teoh, S.H. An Introduction toBiodegradable Materials for Tissue Engineering Applications, Ann. Acad.Med. Singapore, 2001: 30: 183–191.

29. Bumgardner, J.D., Wiser, R., Gerard, P.D. et al. Chitosan: Potential Use as aBioactive Coating for Orthopaedic and Craniofacial/Dental Implants,J. Biomater. Sci. Polym. Ed., 2003: 14: 423–438.

30. Saadeh, P.B., Khosla, R.K., Mehrara, B.J. et al. Repair of a Critical SizeDefect in the Rat Mandible Using Allogenic Type I Collagen, J. Craniofac.Surg., 2001: 12: 573–579.

31. Seol, Y.J., Lee, J.Y., Park, Y.J. et al. Chitosan Sponges as TissueEngineering Scaffolds for Bone Formation, Biotechnol. Lett., 2004: 26:1037–1041.

32. Solchaga, L.A., Gao, J., Dennis, J.E. et al. Treatment of OsteochondralDefects with Autologous Bone Marrow in a Hyaluronan-based DeliveryVehicle, Tissue Eng., 2002: 8: 333–347.

33. Trubiani, O., Orsini, G., Zini, N. et al. Regenerative Potential of HumanPeriodontal Ligament Derived Stem Cells on Three-dimensional Biomaterials:A Morphological Report, J. Biomed. Mater. Res. A, 2008: 87: 986–993.

34. Behravesh, E., Yasko, A.W., Engel, P.S. and Mikos, A.G. SyntheticBiodegradable Polymers for Orthopaedic Applications, Clin. Orthop. Relat.Res., 1999: 367(S): 118–129.

35. Ishaug, S.L., Crane, G.M., Miller, M.J., Yasko, A.W., Yaszemski, M.J. andMikos, A.G. Bone Formation by Three-dimensional Stromal OsteoblastCulture in Biodegradable Polymer Scaffolds, J. Biomed. Mater. Res., 1997:36: 17–28.

36. Neuss, S., Apel, C., Buttler, P. et al. Assessment of Stem Cell/BiomaterialCombinations for Stem Cell-based Tissue Engineering, Biomaterials, 2008:29: 302–313.

37. Munirah, S., Kim, S.H., Ruszymah, B.H. and Khang, G. The Use of Fibrinand Poly(lactic-co-glycolic acid) Hybrid Scaffold for Articular CartilageTissue Engineering: An In Vivo Analysis, Eur. Cell. Mater., 2008: 15: 41–52.

38. Mehlhorn, A.T., Zwingmann, J., Finkenzeller, G. et al. Chondrogenesis ofAdipose-derived Adult Stem Cells in a Poly-lactide-co-glycolide Scaffold,Tissue Eng. Part A, 2009: 15: 1159–1167.

39. Suga, H., Eto, H., Inoue, K. et al. Cellular and Molecular Features of LipomaTissue: Comparison with Normal Adipose Tissue, Br. J. Dermatol., 2009:161: 819–825.

40. De Rosa, A., De Francesco, F., Tirino, V. et al. A New Method for theCryopreserving ASCs: An Attractive and Suitable Large-scale and Long-termCell Banking Technology, Tissue Eng. Part C Methods, 2009: 15: 659–667.

41. Noer, A., Boquest, A.C. and Collas, P. Dynamics of Adipogenic PromoterDNA Methylation During Clonal Culture of Human Adipose Stem Cells toSenescence, BMC Cell. Biol., 2007: 8: 18–29.




42. Qi, L., Zhang, C., Greenberg, A. and Hu, F.B. Common Variations inPerilipin Gene, Central Obesity, and Risk of Type 2 Diabetes in US Women,Obesity (Silver Spring), 2008: 16: 1061–1065.

43. Neuner-Jehle, M., Denizot, J.P., Borbely, A.A. and Mallet, J.Characterization and Sleep Deprivation-induced Expression Modulation ofDendrin, a Novel Dendritic Protein in Rat Brain Neurons, J. Neurosci. Res.,1996: 46: 138–151.




Journal of Biomaterials Applications

Documents

Transcript of Journal of Biomaterials Applications