Comparison of Biomaterials and Extracellular Matricesas a Culture Platform for Multiple, Independently

Derived Human Embryonic Stem Cell Lines

Heidi Hakala, M.Sc.,1 Kristiina Rajala, M.Sc.,1 Marisa Ojala, B.Sc.,1

Sarita Panula, M.Sc.,1,2 Sami Areva, Ph.D.,3 Minna Kellomaki, D.Tech.,4

Riitta Suuronen, M.D., D.D.S., Ph.D.,1,4,5 and Heli Skottman, Ph.D.1

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires afibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESCproduction for potential application in regenerative medicine. Replacing feeder cells with defined human ex-tracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production ofclinical-grade hESCs. We tested and compared different feeder cell–free hESC culture methods based on dif-ferent human ECM proteins, human and animal sera matrices, and a Matrigel� matrix. Also selected bioma-terials were tested for feeder cell–free propagation of undifferentiated hESCs. The matrices were tested togetherwith conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium,chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneicMatrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serummatrices, and the biomaterials tested. A long-term, feeder cell–free culture system was successful on Matrigel incombination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell–freehESC culture method still remains to be developed.

Introduction

Human embryonic stem cells (hESCs) are traditionallycultured in vitro on mitotically inactivated mouse em-

bryonic fibroblast (MEF) or human neonatal or fetal fibro-blasts feeder cell layers.1–5 The function of the feeder cells inthe hESC coculture system is still not fully understood. Thefeeder cells provide hESCs with appropriate cell–cell contactsand also secrete soluble factors necessary to maintain theundifferentiated hESC status. This coculture system, how-ever, presents several challenges. The production of feedercells is highly laborious and limits the large-scale productionof hESCs for future clinical applications. Also, the risk ofincorporating animal pathogens and immunogenic animalproteins into hESCs limits the use of xeno materials such asfetal bovine serum (FBS) commonly used for fibroblast fee-der cell propagation.6,7

The development of feeder cell–free hESC culture condi-tions has been an important focus of recent hESC research. In

2001, Xu et al. described the first feeder cell–free hESC cultureconditions using Matrigel�, a complex mouse sarcoma cellbasement membrane extract comprising various extracellularmatrix (ECM) proteins and growth factors, in combinationwith a culture medium conditioned by MEFs (MEF-CM).8

Conditioning, that is, incubating the hESC culture mediumon a layer of MEF feeder cells before using the medium inhESC culture, allows the fibroblasts to secrete the necessarygrowth and attachment factors into the culture medium. Inaddition, FBS coating has been used as a hESC culture matrixcombined with a chemically defined culture medium (CDM),first for mouse ESC culture9 and later for hESC culture.10

Stojkovic et al. reported successful maintenance of undiffer-entiated hESCs on human serum (HS) coating together withculture medium conditioned by fibroblast-like cells derivedfrom hESCs.11 Such culture systems are important steps for-ward, but are still xenogeneic and undefined.

Various human ECM proteins in combination with a va-riety of more or less defined culture media have also been

1REGEA, Institute for Regenerative Medicine, University of Tampere, Tampere, Finland.2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Palo Alto, California.3Turku Biomaterials Centre, University of Turku, Turku, Finland.4Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland.5Department of Eye, Ear, and Oral Diseases, Tampere University Hospital, Tampere, Finland.

TISSUE ENGINEERING: Part AVolume 15, Number 00, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=ten.tea.2008.0316

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used. Amit and Itskovitz-Eldor described a feeder cell–freeand serum-free hESC culture system on human fibronectincoating and culture medium containing Knock-Out SerumReplacement (ko-SR) together with transforming growthfactor b (TGFb) and basic fibroblast growth factor (bFGF).12

Commercial ko-SR has mostly replaced FBS as an hESCculture medium supplement as it is a more defined, serum-free alternative but still contains animal proteins such asbovine serum albumin (BSA).13 In 2006, Ludwig et al. pub-lished the first feeder cell–free and xeno-free derivationof two hESC lines using a combination of four humanECM proteins as an attachment matrix and a defined, xenocomponent–free culture medium called TeSR1.14 Both of thederived hESC lines, however, were found to be karyotypi-cally abnormal. A modified, more economical xeno protein–containing version of the medium (mTeSR1) combined withMatrigel matrix was reported and became commerciallyavailable later the same year.15

Human sourced or recombinant ECM components arevery expensive and vary batch to batch, whereas a synthetic

biomaterial would offer a fully defined, consistent hESCculture platform. Most of the work done so far on synthe-tic biomaterials and scaffolds for hESC culture has involvedthe promotion of differentiation16 and transplantation ap-plications as well as cell encapsulation strategies.17,18 In ad-dition, most of the studies have been performed using mouseor nonprimate ESCs19–23; thus, the results are not usable forhESCs due to the different cell characteristics and cell growthbehavior between the species.24 Recently, undifferentiatedpropagation of hESCs has been studied using different bio-material substrates, but these studies involved only relativelyshort-term hESC culture, and the methods were based on theuse of MEFs or MEF-CM in a coculture system.25,26

In the present study, we aimed to find a sustainable feedercell–free hESC culture method by systematically testing andcomparing selected culture methods reported by other re-search groups to support their hESC lines in the absence of afeeder layer. We chose methods based on human ECMproteins (i.e., collagen IV, vitronectin, fibronectin, and lami-nin), human and animal sera matrices, and Matrigel as cul-

Table 1. Summary of the Feeder Cell–Free hESC Culture Methods Analyzed

Matrix=biomaterial Medium hESC line and passage used Max passage

Ti hES=hES-CM HS293 p42–p59 1TiO2 hES=hES-CM HS293 p42–p59 1ZrO2 hES=hES-CM HS293 p42–p59 1PDTEC hES=hES-CM HS293 p54 1

HS237 p71 1PLDLA hES=hES-CM HS237 p63 1

Fibronectin mhES HS360 p62 2HS401 p50 1

CDM HS401 p40–p45 2HS360 p53–p56 2

B&D BioCoat,Human fibronectincellware

mhES HS360 p61 2HS401 p49 2

CDM HS360 p59–p61 2

HS401 p49 2

Human ECM mixture:Collagen IVVitronectinFibronectinLaminin

hES=hES-CM HS237 p79 1=2HS360 p79 2=2HS401 p48 2=2

TeSR1 HS360 p54–p56 6HS401 p41–p42 7

HS coating hES=hES-CM HS293 p50–p51 1=1HS237 p62–p80 1=2HS401 p32–p35 1=3

CDM HS401 p39 2HS360 p52 1

H237 p94 (46X, abnormal X) 14 (9þ 5)

FBS coating CDM HS360 p52–p57 10HS401 p39 3

HS237 p94 (46X, abnormal X) 13

Matrigel hES=hES-CM HS401 p48 2=5mTeSR1 HS401 p48–p56 > 30

Regea 06=015 p71 > 30

Ti, titanium; TiO2, titanium dioxide–coated titanium; ZrO2, zirconium dioxide–coated titanium; PDTEC, poly(desaminotyrosyl-tyrosine-ethyl ester carbonate); PLDLA, poly-L,D-lactide; ECM, extracellular matrix; HS, human serum; FBS, fetal bovine serum; hES, standard hESCculture medium consisting of ko-DMEM, 20% ko-SR, 2 mM GlutaMax, 1% nonessential amino acids, 0.5% penicillin=streptomycin, 0.1 mM b-mercaptoethanol, and 8 ng=mL bFGF; hES-CM, human foreskin fibroblast-conditioned hES medium; TeSR1, chemically defined xeno-freehESC culture medium; mTeSR1, modified TeSR1 containing xeno-derived components; CDM, chemically defined medium; mhES, modifiedhES medium: 15% ko-SR, 0.12 ng=mL TGFb, and 4 ng=mL bFGF.

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ture matrices, to seek the true potential of the various ma-trix–media combinations to support hESC attachment andgrowth. The matrices were tested together with conventionalhESC culture medium (hES medium), modified hES (mhES)medium, hES medium conditioned with human foreskin fi-broblasts (hES-CM), and three different chemically definedmedia (CDM, TeSR1, and mTeSR1).

In addition, selected biomaterials, pure titanium (Ti), ti-tanium dioxide (TiO2)– and zirconium dioxide (ZrO2)–coated titanium, poly-L,D-lactide (PLDLA), and poly(desaminotyrosyl-tyrosine-ethyl ester carbonate) (PDTEC),were tested as hESC culture substrates. Ti and TiO2-coated Tihave been used in tissue engineering applications as seedingscaffold for bone marrow stromal cells27 and Ti dishes asculture substrate for mesenchymal stem cells with excellentattachment and proliferation.28 The PLDLA and PDTECwere chosen as potential hESC culture substrates becausepolylactic acid has been used for hESC differentiation29 andtyrosine-derived polycarbonate for guided bone regenerationin animal models.30

Many of the published feeder cell–free culture methodshave not been verified to maintain undifferentiated hESCculture of different hESC lines. In addition, it has been sug-gested that feeder cell–free culture methods may even causechromosomal abnormalities.31 Thus, we used several inde-pendently derived hESC lines in our experiments as well as akaryotypically abnormal hESC line to evaluate the differenthESC culture methods.

Materials and Methods

The culture matrix and media combinations tested and thehESC lines and passages used are summarized in Table 1.

hESC lines and culture

Five karyotypically normal hESC lines were used in theexperiments: HS237 (46, XX), HS293 (46, XY), HS360 (46, XY),HS401 (46, XY), and Regea 06=015 (46, XY). In addition, kar-yotypically abnormal HS237 (46X, abnormal X) hESC line wasused. All hESC lines except Regea 06=015 were derived at theKarolinska Institutet in Stockholm, Sweden, by Professor Outi

Hovatta’s research group and characterized as describedpreviously.1,32 The Regea 06=015 line was derived and char-acterized in our laboratory similarly to the other hESC lines.The Ethics Committee of Pirkanmaa Hospital District ap-proved the study to culture the hESC lines used. All hESClines were cultured on irradiated (40 Gy) human foreskin fi-broblast (CRL-2429; American Type Culture Collection[ATCC], Manassas, VA) feeder cells (hFF) and using hESCsculture medium prior the transfer to feeder cell–free cultureconditions. All hESC lines are regularly characterized forexpression of markers of undifferentiated hESCs (Nanog,OCT-3=4, SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60) by im-munocytochemical stainings, pluripotency by embryoid bodyformation and RT-PCR for markers of the three embryonicgerm layers, and karyotypic stability by standard G-banding.The typical morphology and hESC marker expression of un-differentiated hESCs on hFF feeder cells are shown in Figure 1.

The hESCs were passaged manually to all feeder cell–freeculture systems tested, culture media were changed, andgrowth was monitored daily. The cells were passaged eithermanually or with a combination of manual and enzymatictechniques using 0.5–1 mg=mL dispase or 1–5 mg=mL colla-genase IV (both from Invitrogen, Carlsbad, CA) every 3–7days when the colonies reached an appropriate size withoutexcessive differentiation.

Culture media

hES medium. The conventional hESC culture medium (hESmedium) consisted of ko-DMEM (Invitrogen) supplementedwith 20% ko-SR (Invitrogen), 2 mM GlutaMax (Invitrogen),1% MEM Eagle nonessential amino acid solution (CambrexBio Science, Walkersville, MD), 0.5% penicillin=streptomycin(Cambrex Bio Science), 0.1 mM b-mercaptoethanol (Invitro-gen), and 8 ng=mL bFGF (R&D Systems, Minneapolis, MN).

Human foreskin fibroblast-conditioned hES medium. The hFF-conditioned hES-CM was produced by adding conventionalhES medium to a culture dish containing irradiated (40 Gy)hFF cells as a confluent monolayer of approximately 4�104

cells=cm2. The hES medium was incubated at 378C, 5% CO2

FIG. 1. Morphology of an undifferentiated colony of Regea 06=015 hESC line cultured in standard culture conditions on hFFfeeders in hES medium, and the expression of markers of undifferentiated hESCs (Nanog, OCT-3=4, TRA-1-60, SSEA-3, andSSEA-4). Scale bars¼ 200mm except for (A)¼ 500 mm.

BIOMATERIALS AND ECMS AS A CULTURE PLATFORM FOR HESCS 3

incubator for 24 h and thereafter used as culture medium forhESCs. An additional 8 ng=mL bFGF was freshly added tothe CM before using in feeder cell–free hESCs culture, exceptfor media used for the biomaterial testing.

Modified hES medium. The modified hES medium (mhES)was prepared as described by Amit and Itskovitz-Eldor.12

Modified hES medium consisted of ko-DMEM (Invitrogen),supplemented with 15% ko-SR (Invitrogen), 2 mM GlutaMax(Invitrogen), 1% NEAA (Cambrex Bio Science), 0.1 mM b-mercaptoethanol (Invitrogen), 0.12 ng=mL TGFb (Sigma–Aldrich, St. Louis, MO), and 4 ng=mL bFGF (R&D Systems).

Chemically defined medium. The CDM culture medium, amodification of the medium used by Vallier et al.,10 consistedof 50% IMDM=50% F12þGlutamax I (Invitrogen) supple-mented with 5 mg=mL HS albumin (HSA; Sigma–Aldrich),1% chemically defined lipid concentrate (Invitrogen), 450mMmonothioglycerol (Sigma–Aldrich), 7mg=mL hr-insulin (In-vitrogen), 15 mg=mL human holo-transferrin (Sigma–Aldrich), 10 ng=mL bFGF (R&D Systems), and 12 ng=mLActivin A (R&D Systems).

TeSR1 medium and modified TeSR1 medium. The TeSR1medium was prepared according to the original publication byLudwig et al.14 with the modification of adding antibiotics.DMEM=F12 (Invitrogen) was supplemented with 2mg=mLglutathione (Sigma–Aldrich), 45mg=mL L-ascorbic acid2-phosphate (Sigma–Aldrich), 10.4mg=mL transferrin (Sigma–Aldrich), 0.014 mg=L selenium (Sigma–Aldrich), 6 mg=L thia-mine (Sigma–Aldrich), 12.9 mg=mL HSA (Sigma–Aldrich),1:1000 Trace elements B (Cellgro, Herndon, VA), 1:1000 Traceelements C (Cellgro), 22.8 mg=L insulin (Invitrogen), 1 mMGlutamax (Invitrogen), 1% nonessential amino acid solution100�(Invitrogen), 0.5% penicillin=streptomycin (Cambrex BioScience), 0.1 mM b-m-EtOH (Cambrex Bio Science), 100 ng=mLbFGF (R&D Systems), 0.6 ng=mL TGFb (Sigma–Aldrich),0.127 mg=L pipecolic acid (Sigma–Aldrich), 101mg=mL gammaamino butyric acid (Sigma–Aldrich), 1:500 chemically definedlipid concentrate 100�(Invitrogen), and 41.54 mg=L LiCl(Sigma–Aldrich).

The mTeSR1 medium containing xeno proteins was pur-chased from StemCell Technologies (http:==www.stemcell.com) and handled according to the manufacturer’s instructions.

Culture matrices

Biomaterials. Different biomaterials were tested for undif-ferentiated hESC culture together with hES medium andhES-CM. Pure Ti metal plates were compared with TiO2-coated Ti plates (Vivoxid, Turku, Finland) and ZrO2-coatedTi plates (Turku Biomaterials Centre, University of Turku,Finland). The coatings were produced using sol-gel dip-coating method. About 10�10 mm pieces were disinfectedwith 70% ethanol, allowed to air dry, and placed in a four-chamber slide (Nalge Nunc International, Rochester, NY).Chambers without biomaterial were used as a control. Theexperiment was repeated five times for all three biomaterials.In addition, five different TiO2 coating modifications in re-spect to sol composition and calcination temperatures wereproduced on glass slides and tested accordingly (TurkuBiomaterials Centre).

The biodegradable tyrosine-derived polymer PDTEC wasobtained as sterile plates (Institute of Biomaterials, TampereUniversity of Technology, Finland) and cut with sterile scis-sors to fit into culture dishes. The plate pieces were either usedas matrix as such or attached to the dish with sterile tissueglue (Tisseel� Duo Quick Iþ II; Baxter, Deerfield, IL).The poly-L,D-lactide 96=4 (PLDLA) 3D scaffold (Institute ofBiomaterials) was disinfected with 70% ethanol, allowed toair dry, and placed on a tissue culture dish. Human ESCswere plated directly on the scaffolds and cultured for up to 7days.

Purified human ECMs. Human fibronectin coating wastested as a hESC culture matrix with two types of culturemedia: mhES and CDM. The culture plates were coatedwith 5 mg=cm2 fibronectin from human foreskin fibroblasts(Sigma–Aldrich) at room temperature (RT) for 2 h and testedwith the mhES medium. The fibronectin coating was re-moved after 2 h, and hESCs were plated without washing.12

Culture plates coated at 48C overnight with 5 or 20mg=cm2

fibronectin from hFF (Sigma–Aldrich) were tested with theCDM. The fibronectin was removed, and the wells werewashed once with phosphate-buffered saline (PBS) (CambrexBio Science) before plating the hESCs. BD BioCoat� HumanFibronectin Cellware 12-well plates (BD Biosciences, FranklinLakes, NJ) were also tested with both media types by directlyplating the hESCs in the respective culture media on theplate brought from 48C to RT.

A mix of four human ECM components (hECM mix)consisting of 10 mg=cm2 collagen type IV from human pla-centa, 0.2 mg=cm2 vitronectin from human plasma, 5mg=cm2

fibronectin, and 5 mg=cm2 laminin from human placenta (allfrom Sigma–Aldrich) was tested in combination with dif-ferent culture media. Culture plates were coated with a mixof the four human ECM proteins in PBS at least overnight at48C or first with collagen IV for 2 h at RT, followed bywashing with PBS and overnight incubation at 48C with theother three components. The coating mix was removed,surfaces were washed once with PBS, and hESCs were platedin appropriate culture medium. The hECM mix was testedwith hES, hES-CM, and TeSR1.

Human or bovine serum coatings. HS coating was tested ashESC culture matrix together with hES medium and hES-CM. The culture plates were coated with sterile filtered HS(H1388; Sigma–Aldrich). The culture plates were first incu-bated with HS for 1 h at RT followed by 1 h drying in thesterile hood.11 As this coating method was not successful, thecoating time was increased to overnight at 378C in a 5% CO2

incubator. The HS was removed, replaced with culture me-dia, and hESCs plated. The feeder cell–free culture on the HSmatrix was performed as described by Stojkovic et al.,11 witha modification of using fresh hES-CM as culture mediuminstead of conditioned medium recovered from fibroblast-like cells derived from hESCs (hES-dF-CM).

Both 10% HS and 10% FBS coatings were tested togetherwith CDM. Culture plates were coated with 10% heat in-activated HS (type AB; PAA Laboratories GmbH, Pasching,Austria) or 10% heat inactivated FBS (Invitrogen) in IMDM(Invitrogen) and 0.5% penicillin=streptomycin (Cambrex BioScience). The coated plates were incubated at 378C, 5% CO2

4 HAKALA ET AL.

incubator from 1 to 7 days and washed once with PBS beforeplating the hESCs using CDM.

Matrigel. BD Matrigel hESC-qualified Matrix (BD Bio-sciences) was tested in combination with hES medium, hES-CM, and commercial mTeSR1. Culture plates were coated withMatrigel at 48C at least overnight, as instructed by the manu-facturer. The plate was brought to RT, coating was removed,and hESCs were plated using the appropriate medium.

Characterization of cells

Morphologic characterization. The hESC growth on the dif-ferent matrices was judged primarily by colony attachmentand morphology. The growth was monitored daily under aNikon Eclipse TE2000-S phase contrast microscope (NikonInstruments Europe B.V. Amstelveen, The Netherlands). Co-lonies were judged as undifferentiated if the colony had aneven form and structure with defined borders (Fig. 2L, O). Theloss of defined borders and the emergence of other cell typeswere judged as differentiation. The colony differentiation infeeder cell–free hESC culture was typically manifested as theemergence of mesenchymal-like (fibroblast-like) cells, first atthe colony borders (Fig. 2K) and then progressively throughoutthe whole colony (Fig. 2H). This phenomenon is also referred toas autologous feeder formation and leads to loss of hESC col-onies in the culture plate. The morphologic characterization ofthe undifferentiated hESC colonies was confirmed with im-munocytochemical staining and fluorescence-activated cellsorter analysis (FACS).

Immunocytochemical staining. The hESCs cultured in feedercell–free conditions were characterized by immunocyto-chemical staining with antibodies specific for undifferenti-ated hESCs (Nanog, OCT-3=4, SSEA-3, SSEA-4, TRA-1-81,and TRA-1-60) and an antibody specific for differentiatedhESCs (SSEA-1). The hESC colonies were fixed with 4%paraformaldehyde for 20 min at RT and permeabilizedwith 0.1% Triton X-100 (Sigma–Aldrich), 1% BSA (Sigma–Aldrich), and 10% normal donkey serum (Sigma–Aldrich) inPBS for 45 min at RT. Following are the primary antibodiesthat were incubated at 48C over night: Nanog 1:200 (R&DSystems), Oct-3=4 1:300 (R&D Systems), SSEA-3 1:300(Novus Biologicals, Littleton, CO), SSEA-4 1:200 (Santa CruzBiotechnology, Santa Cruz, CA), TRA-1-81 1:200 (Santa CruzBiotechnology), TRA-1-60 1:200 (Millipore, Billerica, MA),and SSEA-1 1:200 (Santa Cruz Biotechnology). The cells wereprobed with secondary antibodies for 1 h in the dark at RT.Alexa Fluor 568–conjugated donkey anti-goat IgG, goat anti-mouse IgM, and goat anti-mouse IgG antibodies; Alexa Fluor488–conjugated donkey anti-goat IgG and goat anti-mouseIgM; and FITC-conjugated anti-Rat IgM antibodies at a di-lution of 1:800 were used as secondary antibodies (all fromInvitrogen, except anti-Rat IgM, which was from NovusBiologicals). Vectashield mounting medium containing 40,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Bur-lingame, CA) was used for nuclei counter staining. Thelabeled cells were photographed using an Olympus IX51phase contrast microscope with fluorescence optics andOlympus DP30BW camera (Olympus Corporation, Tokyo,Japan). Human ESC labeled only with secondary antibodiesand hFF cells were used as negative controls.

Fluorescence-activated cell sorter analysis. The HS401 hESCscultured for 28 passages on Matrigel in mTeSR1 mediumwere analyzed using FACS for SSEA-4 and TRA-1-81 ex-pression. The cells were dissociated with TrypleTM Select(Invitrogen) and counted with a hemocytometer using try-pan blue exclusion. For SSEA-4 analysis 0.5% BSA in PBSand for TRA-1-81 analysis 2% FBS in PBS were used as FACSbuffers. Cells (0.1�106) were probed for 45 min at 48C with0.5 mL of phycoerythrin-conjugated anti-human=mouse-SSEA-4 antibody (R&D Systems) or for 30 min at 48C with

FIG. 2. Typical morphology of hESCs on different culturematrices. (A) HS293 cultured on Ti in hES-CM for one pas-sage. (B) HS293 cultured on PDTEC in hES-CM for onepassage. (C) Unattached colony of HS237 floating on PLDLAscaffold in hES-CM (arrow). (D) HS360 cultured on hFF fi-bronectin in mhES medium for two passages. (E) HS360cultured on BD BioCoat Human Fibronectin plate in CDMfor two passages. (F) HS401 cultured on hECM mix in hESmedium and (G) in hES-CM for two passages. (H) HS401cultured on hECM mix in xeno-free TeSR1 medium for sixpassages. (I) HS401 cultured on HS coating in hES-CM forone passage. (J) HS360 cultured on 10% HS coating in CDMfor one passage and (K) on 10% FBS coating in CDM for fivepassages. (L) HS237 (46X, abnormal X) cultured on 10% FBScoating in CDM for seven passages. (M) HS401 cultured onMatrigel in hES medium and (N) in hES-CM for two pas-sages. (O) HS401 cultured on Matrigel in mTeSR1 mediumfor 20 passages. Scale bars¼ 500 mm, except for (G) and(O)¼ 200 mm. For abbreviations, see Materials and Methodssection.

BIOMATERIALS AND ECMS AS A CULTURE PLATFORM FOR HESCS 5

1:200 dilution of TRA-1-81 (Santa Cruz Biotechnology). ForTRA-1-81 analysis, the cells were probed with 1:500 dilutionof R-phycoerythrin–conjugated anti-mouse IgM secondaryantibody (Invitrogen) at 48C for 20 min. The cells were ana-lyzed using BD FACSAria� (BD Biosciences). The sampleswere analyzed in triplicate, and acquisition was set for 20,000events per sample. R-Phycoerythrin–conjugated goat-antimouse IgG antibody (Invitrogen) was used as an isotypecontrol, and R-phycoerythrin–conjugated anti-mouse IgMantibody (Invitrogen) as a secondary control. The data wereanalyzed using FACSDiva Software version 4.1.2 (BD Bios-ciences, San Jose, CA).

Karyotype analysis. Karyotype analysis was performed onHS401 hESC lines cultured on Matrigel matrix in mTeSR1medium for 24 passages. The hESCs were transferred back tohFF feeders and cultured using hES medium on hFF feedercells for 2 to 4 passages before the karyotype analysis. Acytogenetic analysis of 20 metaphase cells was performedusing G-banding at Medix Laboratories (Espoo, Finland).

Quantitative RT-PCR. The expression of Oct-4 was analyzedover time in three of the tested culture systems with quanti-tative RT-PCR (q-RT-PCR). Total RNA was extracted fromHS360 cells cultured on Matrigel in mTeSR1 for 1, 2, and 3passages and Regea 06=015 cells cultured for 10, 21, and 32passages. RNA samples were also collected from HS360 cellscultured on hECM mix on TeSR1 for 1, 2, and 3 passages andon 10% FBS coating in CDM for 1 and 2 passages. RNA fromthree different passages (p83, p84, and p85) of HS360 hESCscultured on hFF feeder cells in hES medium was collected toserve as a reference level of gene expression. The hESCs werecollected from the culture plates, lysed to RLT plus buffer(Qiagen, Valencia, CA), and stored at �708C until RNA wasextracted with Qiagen RNeasy Plus Mini kit according tomanufacturer’s instructions. The RNA concentration andquality was assessed with NanoDrop 1000 spectrophotometer(NanoDrop Technologies, Wilmington, DE).

Fifty nanogram of RNA was transcribed to cDNA in atotal volume of 20mL with Sensicript Reverse Transciriptionkit (Qiagen) according to manufacturer’s instructions for 1 hat 378C. The cDNA was stored at �208C, until used in q-RT-PCR analyses. The q-RT-PCR was performed with AppliedBiosystems (Foster City, CA) Gene Expression Assays:POU5F1 (Hs00999632_g1) and GAPDH (Hs99999905_m1).GAPDH, known to have constant expression in our hESClines (data not shown), was used as a housekeeping control.The PCR reaction consisted of 3mL of cDNA in 1:10 dilution,7.5 mL of 2� TaqMan Universal PCR Master Mix (AppliedBiosystems), and 0.75 mL of assay. All samples, dH2O con-trols from cDNA synthesis, and no template controls wereanalyzed as three replicates. The q-RT-PCR was carried outwith Applied Biosystems 7300 Real-time PCR system: 2 minat 508C, 10 min at 958C, and 40 cycles of 0.15 min at 958C and1 min at 608C. The data were analyzed with 7300 System SDSSoftware (Applied Biosystems).

Ct values were determined for every reaction and quali-fied for analysis if the standard deviation of the three repli-cate values was <0.5. Relative quantification was calculatedwith the 2�DDCt method.33 The data were normalized withthe expression of the housekeeping gene GAPDH, and the

expression level of Oct-4 in HS360 hESCs cultured in stan-dard culture conditions on hFF feeder cells in hES mediumwas used as reference level. The data are presented as meanfold change values as compared to the reference level.Standard deviation is presented as error bars. For deter-mining statistical significance, the Mann–Whitney U-test forunmatched pairs was used. p-Value <0.05 was consideredstatistically significant.

Results

The maximum passages for which each culture matrix andmedia combination supported hESC culture are summarizedin Table 1.

hESC culture on selected biomaterials

Ti, TiO2, ZrO2, PDTEC plate, and PLDLA scaffold weretested as hESC attachment and culture matrices togetherwith hES and hES-CM media. The hESCs did not attach toany of the biomaterials in the presence of unconditioned hESmedium, but some attachment occurred on Ti, TiO2, ZrO2,and PDTEC when hES-CM was used (Fig. 2A, B). The hESCsdid not attach to the PLDLA scaffold (Fig. 2C) or to theuncoated chamber glass slide used as a control material evenwith hES-CM. The hESC colonies were very fragile andeasily detached from the biomaterials. The TiO2 was alsotested as five modifications with similar results (data notshown). The hFF cells routinely used as feeder cells, how-ever, attached to and grew on PDTEC and on all of the Timaterials (data not shown).

hESC culture on purified human ECMs

Human fibronectin was tested as a culture matrix forundifferentiated hESCs according to the method describedby Amit and Itskovitz-Eldor12 using mhES media as well asthe CDM used for hESC propagation by Vallier et al.10 Hu-man FF fibronectin and BD BioCoat Human FibronectinCellware were tested. Neither type of fibronectin coatingsupported undifferentiated hESC culture beyond the secondpassage (i.e., 8 days) with either of the media types tested(Fig. 2D, E and Fig. 3A, B). The hESCs quickly underwentdifferentiation and attached poorly after passaging. Increas-ing the concentration of hFF fibronectin to 20 mg=cm2 did notyield any better results.

The hECM mix was tested together with hES me-dium, hES-CM, and defined xeno-free TeSR1 medium. Hu-man ESC colonies attached to the hECM mix in all mediatypes tested. In hES and hES-CM media, the hESC colonieswere thin, fragile, and quickly differentiated toward amesenchymal-like phenotype within two passages (8 days)(Fig. 2F, G). In TeSR1 medium, the hESCs underwent pro-gressive differentiation and poorer attachment in subse-quent passages (Fig. 2H). The hESCs were cultured formaximum of 7 passages under these conditions, after whichall cells had a differentiated morphology and lost the ex-pression of OCT-3=4, a marker of undifferentiated hESCs(Fig. 3C, D).

hESC culture on serum coatings

On HS coating the hESC colony pieces either did not at-tach at all or grew poorly and easily detached during culture

6 HAKALA ET AL.

with hES medium. In hES-CM medium, the colonies attachedmore readily compared to hES medium, and the observedcolony growth and morphology was generally better in thefirst passage. Even in hES-CM medium, the hESC coloniesunderwent excessive differentiation (Fig. 2I) and could onlybe cultured for the maximum of three passages, after whichthe colonies had completely differentiated and expressedSSEA-1, a marker of differentiated hESCs, with only mod-erate expression of Nanog, a marker of undifferentiatedhESCs (Fig. 3E). No difference in attachment or hESC growthwas observed when 1% insulin, transferrin, and seleniumsupplement was added to the hES-CM medium (data notshown), as described by Stojkovic et al.11

We also tested culture dishes coated with cell culturemedium containing 10% HS or 10% FBS together with CDM.Again, neither of the matrices supported long-term undif-ferentiated hESC culture together with the CDM. On 10% HScoating, the hESCs (Fig. 2J) could only be cultured for upto two passages in CDM, after which the cells either differ-entiated or detached and were lost. On 10% FBS coating,

the hESCs progressively differentiated (Fig. 2K), and after10 passages, autologous feeder cells had taken over the en-tire surface of the culture dish and hESCs had lost theirexpression of Nanog (Fig. 3F, G). The abnormal HS237hESC line (46X, abnormal X) was cultured for 13 passages on10% FBS coating with comprehensive attachment andeven, round colonies with defined borders (Fig. 2L). ThehESCs showed no differentiation toward other cell typesafter the first passages and strongly expressed the surfacemarkers of undifferentiated hESCs (Fig. 3H). The HS237(46X, abnormal X) performing ideally on 10% FBS coatingwas transferred to 10% HS coating at the ninth passage andcultured for five more passages on this substratum, againwith ideal behavior. After a total of 14 passages, the culturewas aborted.

hESC culture on Matrigel

The combination of Matrigel and hES medium could notsupport undifferentiated hESC culture beyond the second

FIG. 3. Human ESC colonies immunostained with markers of undifferentiated hESCs (Nanog, OCT-3=4, SSEA-4, TRA-1-81,and TRA-1-60) and a marker for differentiated hESCs (SSEA-1). (A) HS360 cultured on fibronectin derived from humanforeskin fibroblasts in mhES medium for two passages. Note the autologous feeders around the colony that have lost theexpression of Nanog. (B) HS360 cultured on B&D BioCoat fibronectin plate in CDM for one passage showing ragged colonymorphology and only moderate Nanog expression. (C) HS360 cultured on hECM mix in TeSR1 medium for two passagesshowing strong expression of OCT-3=4 and (D) HS401 for seven passages showing the loss of colony morphology and OCT-3=4 expression. (E) HS237 cultured on HS coating in hES-CM for two passages showing expression of both Nanog and SSEA-1 in a double staining. (F) HS360 cultured on 10% FBS coating in CDM for five passages showing strong expression ofNanog. (G) HS360 cultured on 10% FBS coating in CDM for 10 passages have lost the expression of Nanog. (H) HS237 (46X,abnormal X) cultured on 10% FBS coating in CDM for 13 passages showing strong expression of SSEA-4. (I–L) HS401cultured on Matrigel in mTeSR1 for 23 passages showing strong expression markers of undifferentiated hESCs. Scalebars¼ 200 mm.

BIOMATERIALS AND ECMS AS A CULTURE PLATFORM FOR HESCS 7

passage. With hES-CM the hESCs could be cultured for fivepassages, after which the colonies were completely lost dueto inadequate attachment or because there were only one ortwo partially undifferentiated colonies left in the well and nofurther passaging was feasible. Compared to culture onhECM mix, the colonies were, however, much thicker andmore solid on Matrigel (Fig. 2G, N).

With commercial mTeSR1 medium the hESCs underwentdifferentiation in the beginning of the culture, but after a fewpassages of selection most colonies had an undifferentiatedmorphology. The HS401 hESCs were successfully culturedover 30 passages with an undifferentiated morphology (Fig.2O). The colonies attached properly and showed little dif-ferentiation and strong expression of markers of undiffer-entiated hESCs in both immunocytochemical stainings (Fig.3I–L), and as confirmed with FACS analysis. According toFACS analysis, 97% of HS401 hESCs were SSEA-4 positiveand 95% were TRA-1-81 positive after 28 passages on Ma-trigel in mTeSR1 (Fig. 4A, B). The karyotype of the cell linewas confirmed to be normal diploid (46, XY) after 24 pas-sages in the culture system (Fig. 4C). The Regea 06=015 hESCline has to date also been cultured using Matrigel andmTeSR1 over 30 passages with similar undifferentiatedmorphology and protein expression in immunocytochemicalstainings and FACS as well as normal karyotype after long-term feeder-free culture (data not shown).

Expression of Oct-4 in hESCs cultured with differentfeeder cell–free culture systems

The relative gene expression of Oct-4 was studied inhESCs cultured with three matrix–media combinations:Matrigel combined to mTeSR1 medium that supportedlong-term undifferentiated hESC culture and two non-supportive culture methods based on the hECM mix to-gether with TeSR1 and 10% FBS coating together withCDM. The constant expression of Oct-4 in hESCs culturedin the standard conditions on hFF was used as a referencelevel. The lower expression of Oct-4 in reference sample ascompared to the feeder cell–free samples is explained bythe feeder cell RNA present in the reference sample. Theexpression of Oct-4 remained constant throughout the long-term culture on Matrigel between the samples of thetwo hESC lines from passages 1, 2, 3, 10, 21, and 32 (Fig. 5).As anticipated, on hECM in TeSR1 medium and on FBScoating in CDM, the Oct-4 expression level decreased ascolonies differentiated and detached from early on.On hECM mix in TeSR1 medium the expression of Oct-4decreased significantly ( p< 0.05) at passages 2 and 3 com-pared to culture on Matrigel. Also on 10% FBS coat-ing, the expression level decreased from first passage tothe second although the difference was not statisticallysignificant.

FIG. 4. Characterization of HS401hESCs cultured on Matrigel inmTeSR1 medium. FACS analysisconfirmed that (A) 97% of hESCswere SSEA-4 positive and (B) 95%were TRA-1-81 positive after 28passages. (C) The karyotype of thecell line was confirmed to be normaldiploid (46, XY) after 24 passagesin the culture system.

8 HAKALA ET AL.

Discussion

Eliminating the feeder cells from the hESC culture systemwould substantially reduce the cost and labor of hESC cul-ture, offer more defined culture systems that could be moreeasily reproduced, and enable the scale-up of hESC pro-duction for potential clinical use. The human ECM proteins,synthetic biomaterials, or their combinations would offer axeno-free alternative to feeder cells that could be validated tocorrespond to GMP-quality requirements.

Feeder cell–free hESC culture is extremely demanding. Themaintenance of hESC pluripotency is interplay between thematrix and the soluble factors provided by the culture me-dium. Especially in the absence of CM, the feeder cell–freeculture conditions described so far rely on high concentrationsof growth factors, which reflects the currently insufficientknowledge about the maintenance of self-renewal and plur-ipotency of hESCs. The published culture methods have alsobeen difficult to reproduce in different laboratories with dif-ferent cell lines. It has been speculated that the origin of thehESC line as well as the derivation and culture conditions,media, matrix, and passage numbers used in different studieshave a major effect on the results of feeder cell–free and otherexperiments conducted with hESC lines. The hESC lines arealso genetically different, and it is possible that some cell linesare better suited for special purposes and more prone to growon anything than other lines.34,35 This makes undefined cul-ture systems highly undesirable, especially if different hESClines show different responses to the culture conditions.

To date, biomaterials as culture matrices have not beenadequately tested for undifferentiated propagation of hESCculture. The hESCs in general do not easily attach to stan-

dard cell culture plastics; if they do attach, the colonies un-dergo spontaneous differentiation. The biomaterials testedhere did not support hESC attachment or growth as such,even though they were nontoxic to cells because the hFF cellswere able to attach to and grow on them.

The hESC attachment was clearly better on the hECMproteins tested compared to the synthetic biomaterials. Thisstrongly emphasizes the role of ECM in both attachment andhESC self-renewal. Also, the importance of the soluble fac-tors secreted by the fibroblast feeder cells for the attachmentand growth of hESCs was evident as the hES medium wasinferior to hES-CM in combination with both the biomateri-als and hECM proteins tested. Nevertheless, the hES-CMcollected from hFF cells did not support undifferentiatedhESC culture on any of the matrices tested, in contrast toMEF-CM, that is widely used.8,36,37 Other CM types suc-cessfully used in hESC culture are usually collected fromfibroblasts of fetal origin or from fibroblast-like cells derivedfrom hESCs.11,38,39 In our experiments, the hES-CM wascollected from confluent monolayers that at the same densitysupport undifferentiated hESC culture as feeder layers. Thisfurther emphasizes the role of the cell–ECM protein and cell–cell interactions in the maintenance of hESC pluripotency.

The hECM mix tested has previously been reported tosupport undifferentiated hESC culture by Ludwig and co-workers together with xeno-free TeSR1 medium.14 In ourown experiments, different human ECM proteins were ini-tially tested individually as culture matrices using hES me-dium and hES-CM. The purified proteins alone were allinferior compared to the mix of all four (data not shown),consistent with the results of Ludwig et al. We previouslyreported that the hECM mix together with the xeno-freeTeSR1 medium was not sufficient for maintaining undiffer-entiated hESC culture.40 Here, we even tested the mediumcomposition as described in the original publication relatingto the concentrations of HSA, insulin, selenium, and trans-ferrin. The human ECM mix and xeno-free TeSR1 mediumdid not support undifferentiated hESC culture of our celllines beyond the early passages but instead led to detach-ment and loss of pluripotency markers. The decrease of Oct-4expression already in the early culture passages was con-sistent with the morphological findings. The expression levelof Oct-3=4 has been shown to be a sensitive indicator ofpluripotency status in ES cells and changes even less thantwofold to be biologically relevant.41

Human and animal sera are rich in ECM proteins andwere therefore tested as attachment bases in feeder cell–freehESC culture. The attachment of hESC colony pieces to HS-coated culture dishes was greatly improved by a longer in-cubation time at higher temperature, which allowed enoughECM proteins to attach to the culture dish surface to allowhESC attachment and growth. HS has been used as a hESCculture matrix by Stojkovic et al.11 The same HS was used forthe coating in this study. Stojkovic et al.11 used a differenthESC line (H1) that was derived and propagated underconditions (e.g., MEF feeders) that differed dramaticallyfrom those used for the hESC lines in the present study. Inaddition, the CM they used differed from the one used here,and these factors may underlie their success in culturinghESCs on HS coating. The FBS coating successfully used byVallier et al.10 was clearly better than HS as a hESC culture

FIG. 5. Relative gene expression of Oct-4 in hESCs at dif-ferent passages cultured with three different matrix–mediacombinations: on Matrigel in mTeSR1, on hECM mix inTeSR1, and on 10% FBS in CDM. The expression level ofOct-4 in HS360 hESCs cultured on hFF with hES mediumwas set as a reference baseline (*p< 0.05).

BIOMATERIALS AND ECMS AS A CULTURE PLATFORM FOR HESCS 9

matrix. The addition of serum is essential to most cell cultureprotocols. FBS is generally more suitable for cell culture, andit is likely that HS both lacks the components that are ben-eficial and contains components that are harmful to cells.Still, even FBS was an inadequate substratum for long-termhESC culture with CDM and hESC colonies differentiated orwere lost within the first culture passages. Consistently, theexpression level of Oct-4 decreased within the second pas-sage and would most likely have continued to decrease insubsequent passages if further passaging would have beenfeasible. The successful culture of the karyotypically abnor-mal HS237 cells on FBS coating with CDM indicates that thekaryotypic abnormality had a positive effect in that culturesystem. The FBS coating and CDM did not support undif-ferentiated culture of either of the karyotypically normalhESC lines HS401 or HS360. Feeder cell–free culture condi-tions favor the occurrence of karyotypic abnormalities,14,31

and even though the karyotypic abnormality of the HS237cell line was gained under standard culture conditions, ourresults indicate that karyotypically abnormal hESC lines areeasier to culture under feeder cell–free culture conditions.

All of the biomaterials and human ECM proteins as well assera coatings tested were inferior to the undefined, xeno-product Matrigel. The attachment and morphology of thehESC colonies was better on Matrigel compared to the hECMmix tested in parallel with hES and hES-CM media. Thecombination of Matrigel and mTeSR1 medium successfullysupported long-term undifferentiated hESC culture of twohESC lines with appropriate colony morphology and markerexpression. The culture on Matrigel in mTeSR1 also showedconstant Oct-4 expression levels between different passages.Unfortunately, both the Matrigel matrix and the mTeSR1medium are far from xeno-free, and thus this culture system isunsuitable for use in the derivation and culture of clinical-grade hESCs. Our systematic testing of different culture ma-trices and media combinations for long-term undifferentiatedhESC culture showed that feeder cell–free hESC culture isextremely difficult and that no globally reproducible, cost-effective, xeno-free, and feeder cell–free hESC culture methodexists. There is a growing need for development of such cul-ture conditions for hESCs as the phase of clinical trials ofhESC-based cellular therapies is fast approaching.

Acknowledgments

We would like to thank the entire staff of Regea and es-pecially Professor Outi Hovatta for providing the hESC linesused in this study. We would also like to thank Vivoxid andMika Pelto (Tampere University of Technology) for provid-ing us with biomaterials. This work was supported by theTEKES, the Finnish Funding Agency for Technology andInnovation, the Academy of Finland, the Competitive Re-search Funding of the Pirkanmaa Hospital District, and theUniversity of Tampere.

Disclosure Statement

No competing financial interests exist.

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Address reprint requests to:Heidi Hakala, M.Sc.

REGEA, Institute for Regenerative MedicineUniversity of Tampere

Biokatu 1233520 Tampere

Finland

E-mail: heidi.minna.hakala@regea.fi

Received: June 3, 2008Accepted: November 7, 2008

Online Publication Date: January 8, 2009

BIOMATERIALS AND ECMS AS A CULTURE PLATFORM FOR HESCS 11