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Biomaterials 35 (2014) 2816e2826

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

A defined xeno-free and feeder-free culture system for the derivation,expansion and direct differentiation of transgene-free patient-specificinduced pluripotent stem cells

Hong Fang Lu a,*, Chou Chai b, Tze Chiun Lim a, Meng Fatt Leong a, Jia Kai Lim a,Shujun Gao a, Kah Leong Lim b,c, Andrew C.A. Wan a,*

a Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, SingaporebDuke-NUS Graduate Medical School, Singapore 169857, SingaporecNational Neuroscience Institute, Singapore 308433, Singapore

a r t i c l e i n f o

Article history:Received 5 December 2013Accepted 19 December 2013Available online 9 January 2014

Keywords:Xeno-free cultureiPSCReprogrammingExpansionDirect differentiationLaminin 521

* Corresponding authors. Tel.: þ65 6824 7134; fax:E-mail addresses: hflu@ibn.a-star.edu.sg (H.F. Lu),

A. Wan).

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.12.050

a b s t r a c t

A defined xeno-free system for patient-specific iPSC derivation and differentiation is required fortranslation to clinical applications. However, standard somatic cell reprogramming protocols rely onusing MEFs and xenogeneic medium, imposing a significant obstacle to clinical translation. Here, wedescribe a well-defined culture system based on xeno-free media and LN521 substrate which supportedi) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages intohiPSCs with a 15e30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisablepolycistronic vector and optimized culture conditions, we achieved up to 0.15%e0.3% reprogrammingefficiencies. Subsequently, transgene-free hiPSCs were obtained by Cre-mediated excision of thereprogramming factors. The derived iPSCs maintained long-term self-renewal, normal karyotype andpluripotency, as demonstrated by the expression of stem cell markers and ability to form derivatives ofthree germ layers both in vitro and in vivo. Importantly, we demonstrated that Parkinson’s patienttransgene-free iPSCs derived using the same system could be directed towards differentiation intodopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robustplatform for the generation of patient-specific iPSCs and derivatives for clinical and translationalapplications.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Human induced pluripotent stem cells (hiPSCs) provide aunique platform for treatment of various human diseases withoutethical issues. The ability of patient-specific iPSCs to differentiateinto relevant autologous tissues or organs highlights their potentialas a cell source for exploration of disease mechanisms, to identifynovel therapeutic targets, and ultimately, for possible autologouscellular therapy [1,2]. In parallel with the rapid progress in iPSCresearch, the number of tissue banks set up using patient-specificfibroblasts has been steadily increasing, while the therapeuticrelevance and potential of iPSCs has been recognized [3e5].

þ65 6478 9082.awan@ibn.a-star.edu.sg (A.C.

All rights reserved.

Derivation, expansion and differentiation of hiPSCs under xeno-free conditions are an essential prerequisite towards realizing iPSC-based clinical application. Generally, the somatic cell reprogram-ming process requires initial cell proliferation, after which a frac-tion of the cell progeny successfully converts into an embryonicstem cell (ESC)-like state with different time latencies [6,7]. Due tothe poor efficiency of existing reprogramming methods, it is oftennecessary for reprogramming to be performed in the presence ofmouse embryonic fibroblast (MEF) feeder cells along with the useof serum and xeno-containing products, to maximize colony for-mation [8]. To overcome this safety issue, xeno-free iPSC derivationusing human feeder cells has recently been reported [9,10]. How-ever, the use of human feeder cells adds complexity and set up timeto the reprogramming procedure, introduces technical variability,and interferes with the monitoring and analysis of the reprog-ramming process. In the search for methods of hiPSC derivationunder feeder-free condition, several research groups have reported

Table 1Generation of hiPSCs by pOSKM vector under chemically defined condition, listingdetails of fibroblasts and annotations used for the fibroblasts and derived iPSC celllines.

Human adultfibroblasts

Cellsource

Sex Age Reprogrammingefficiency*

iPSC Symbol

Beforeexcise

Post excise

HDF-A ATCC Female 25 0.26%** A-iPSC EX-A-iPSCHDF-C Cascade

BiologicsFemale 31 0.30%** C-iPSC EX-C-iPSC

HDF-L Lonza Female 32 0.18%*** L-iPSC EX-L-iPSCHDF-PD(ND30116)

Coriell Female 67 0.23%*** PD-iPSC EX-PD-iPSC

Reprogramming efficiencies were calculated on the number of AP expressing col-onies normalized to the number of cells seeded onmatrigel- or LN521-coated plates.*: Reprogramming experiment was conducted in xeno-free E8 medium. **: Matrigelas substrates. ***: Xeno-free condition, and xeno-free LN521 as substrates.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e2826 2817

reprogramming studies based on Matrigel- or Vitronectin-coatedsubstrates along with the use of chemically defined media [11e13]. However, a big challenge still exists in developing well-defined and safe xeno-free systems which can integrate efficientderivation, expansion and direct relevant tissue differentiation ofdisease-specific iPSCs for transplantation or disease modeling.

Maintaining genomic integrity is critical for therapeutic appli-cation of hiPSCs. hiPSCs were first derived in 2007 from humanfibroblasts through viral introduction of a set of stemness factors:Oct3/4, Sox2, Klf4 and c-Myc with about 0.01% efficiency [14]. Dueto its reliability and relatively high efficiency at delivering thereprogramming factors to the cells, the viral-based approach re-mains the most widely used method for iPSC derivation [15], andpatient-specific iPSCs from a variety of diseases, including amyo-trophic lateral sclerosis, Parkinson’s disease (PD), type 1 diabetesmellitus, Huntington’s disease, and Down’s syndrome have beenreported [16,17]. However, viral vector-mediated reprogrammingresults in integration of both the vector backbone and transgenesinto the genome [14,18], presenting a formidable obstacle to ther-apeutic use of iPSCs. In the search for methods to induce pluripo-tency without incurring genetic change, several integration-freemethods have been employed to reprogram murine cells orneonatal normal human cells only, by using purified proteins,mRNAs, or plasmids and xenogeneic culture conditions [19e25].Although these methods have the potential to generate geneticallyunmodified iPSC lines, their main disadvantage lie in the low effi-ciency of reprogramming, preventing reliable application forreprogramming disease-specific adult human somatic cells [22e25]. To improve the reprogramming efficiency, several studies re-ported iPSC derivation on MEF layer using reprogrammingenhancing molecules [26,27]. Of note, many of these moleculeshave pleiotropic effects that could result in transient or permanentepigenetic or genetic alterations, hindering the use of chemicallyinduced iPSCs for therapeutic purposes [28,29]. Thus, an efficientand safe reprogramming method with a compliant xeno-free cul-ture system is still needed in order to achieve the widespreadderivation of disease-specific transgene-free iPSCs from humanswith inherited or degenerative diseases.

The goal of our study is to develop a defined xeno-free andfeeder-free culture system for efficient transgene-free patient-specific iPSC derivation, expansion and subsequent differentiation.To achieve this, we chose a single, excisable polycistronic lenti-virus vector for human adult fibroblast reprogramming [30]. Thisapproach allows efficient introduction of the reprogramming fac-tors into human adult fibroblasts, followed by easy excision of thetransgenes from the iPSCs when treated with Cre recombinase. Inaddition, we chose xeno-free recombinant human laminin 521(LN521) for the fibroblast reprogramming substrate because of therole of laminins in early embryonic development [25,31]. Lamininsare the central components of basement membranes, containing15 different isoforms in human tissues [32]. Recent studiesrevealed a striking difference in the effects of various lamininisoforms on ESC culture. Isoform LN411 was not capable of sup-porting ESC adhesion or survival, and LN111 supported ESCattachment, while LN511 and laminin fragment 8 supported hu-man pluripotent stem cell culture in the chemically defined me-dium [19,33,34]. Similar to LN511, LN521 is normally expressedand secreted by human embryonic stem cells (hESCs) [35]. Wehypothesized that LN521 would support fibroblast growth andreprogramming. Chemically defined xeno-free media including E8,NutriStem and TeSR2 were tested in the reprogramming experi-ments, and compared with the xenogeneic media, mTeSR1 andMEF conditioned medium (CM). Four adult fibroblast lines fromhuman individuals of different ages, including one PD patientfibroblast line, were reprogrammed using this culture system, and

tested for long-term expansion as well as directed differentiationunder totally defined xeno-free conditions.

2. Materials and methods

2.1. Culture of human adult fibroblasts (HDF)

Four human adult fibroblast lines (Table 1) were used in this study: HDF-A (PCS-201, ATCC, USA), HDF-L (CC-2511, Lonza, USA), HDF-C (Cascade Biologics, LifeTechnologies) and PD patient fibroblasts HDF-PD (ND30116, Coriell, USA). Humanfibroblast lines were cultured in standard culture media containing DMEM sup-plemented with 100 IU/mL penicillin-streptomycin (Life Technologies) and 10% fetalbovine serum (DMEM-FBS, Life Technologies) or human serum (DMEM-HS, ATCC,USA) and maintained at 37 �C in a 5% CO2 incubator. Cells were allowed to expandto 80%e90% confluence before passaging with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA, Life Technologies). Early-passage HDF cellswere used for virus transduction experiment.

2.2. Culture of human pluripotent stem cell lines

Human embryonic stem cell line HUES7 was obtained from Dr. Douglas Melton’sLab at Harvard University. The preparation of conditioned medium (CM) and theprocedure for maintenance of HUES7 under feeder-free condition were essentiallythe same as described previously [36]. Briefly, HUES7 cells were cultured onMatrigel(BD Biosciences, San Jose, CA, USA)-coated culture wells in mTeSR1 (StemcellTechnologies, Vancouver, BC, Canada) at 37 �C in a 5% CO2 incubator with dailymedium changes. Cells were subcultured every 5e6 days with 1 mg/mL dispase(STEMCELL Technologies, Vancouver, Canada). The newly generated pre-excised andpost-excised iPSCs were cultured under defined feeder-free culture conditions, asdescribed below.

2.3. Production and infection of lentiviral vectors

Polycistronic reprogramming lentivirus vector Lenti-OSKM (pOSKM) wasmodified from pKP332 (Addgene #21627) by the insertion of a IRES-cMyc fragmentat a unique PacI site. This modification dramatically improved the reprogrammingefficiency of pKP332. Supplementary Fig. 1 shows the schematic representation ofLenti-OSKM vector. As a negative control for reprogramming, cDNA encodingcopepod GFP (maxGFP, Lonza) was subcloned into pLenti6/V5-D-TOPO (Life Tech-nologies) to give pLenti6maxGFP. For preparation of lentivirus particles, viralpackaging was performed in 293FT cells using reagents and protocol from theViraPower� Lentiviral Packaging Kit (Life Technologies). Lentivirus particles wereconcentrated from cell culture supernatant according to the protocol of DeiserothLab (http://www.stanford.edu/group/dlab/resources/lvprotocol.pdf).

For determining the titer of the lentivirus, w0.1 � 106 human adult fibroblastswere plated in each well of a 6-well plate (BD falcon) in DMEM-FBS. On the day oftransduction, 0 ml, 50 ml, 100 ml and 150 ml of freshly prepared lentivirus vectors weremixed with 1mL of DMEM-FBS containing 8 mg/mL polybrene (Millipore) and addedto the cells. The culture wells were gently shaken, and placed at 37 �C in a 5% CO2

incubator overnight. The next day, virus-containing supernatant was replaced with2 mL of DMEM-FBS. The cell numbers were assessed after 3 days transduction byphase contrast microscopy. After a pre-test, 100 ml of vectors/well was used for ourstudy. For each reprogramming experiment, the transduced cells were trypsinized48 h post-transduction, transferred to Matrigel-coated tissue culture plates, andcultured in stem cell culture media on the same well for 4 weeks with daily mediachanges. The stem cell media used in this study include MEF conditioned medium(CM), mTeSR1 (medium containing BSA), TeSR2 (xeno-free medium) (Stemcelltechnologies, Vancouver, BC, Canada), NutriStem (xeno-free medium, Stemgent, SanDiego, CA, USA), and Essential 8 medium (E8, xeno-free medium, Life Technologies).Putative hiPSC colonies started to appear after 1e2 weeks. These colonies were

H.F. Lu et al. / Biomaterials 35 (2014) 2816e28262818

individually picked and cultured on Matrigel-coated plate for expansion andanalysis.

2.4. Reprogramming of human adult fibroblasts under feeder-free, xeno-free cultureconditions

ND30116 and HDF-L fibroblasts cultured in DMEM-HS were seeded in the wellsof 6-well plate and transduced with 100 ml of lentivirus vector per well. Mediumwere replaced with fresh DMEM-HS after overnight culture. The transduced cellswere harvested 48 h post-transduction, plated to a 12-well plate, precoated withxeno-free human recombinant protein LN521 (Biolamina, Sweden), and cultured inE8 for 4weeks with dailymedia changes.Matrigel-coated culturewells served as thecontrols. Putative iPSC colonies started to appear after 1e2 weeks. These colonieswere individually picked and cultured on LN521- or Matrigel-coated plate forexpansion and analysis. iPSCs were passaged every 4e5 days with 0.05 mM EDTA(Life Technologies).

2.5. Generation of transgene-free hiPSCs

The excision of OSKM transgene was achieved by the transient transfection of aplasmid mixture expressing an estrogen-inducible Cre recombinase (pCAG-ERT2-CreERT2, Addgene # 13777) and GFP (EX-EGFP-Lv105, Genecopoeia) into theparental pre-excised iPSC line. hiPSCs were plated on LN521-coated plate. Cre andGFP plasmids (1:1 ratio) and Lipofectamine 2000 (Life Technologies) were mixedwith E8 medium and added to the culture well. After 3 h, the mediumwas replacedwith fresh E8. After 48 h, the cells were detached using 0.05 mM EDTA and sus-pended into single cells in E8 containing 10 mM ROCK inhibitor (Stemcell Technol-ogies, Vancouver, BC, Canada). The GFP-expressing cells were obtained by FACS andplated in LN521-coated 96-well plates at w1 cell/well in E8 containing ROCK in-hibitor. The next day, the medium was replaced with E8 containing 500 nM 4-hydroxytamoxifen (4-OHT, SigmaeAldrich, St Louis, MO, USA) to induce ERT2Creexpression and allow for the excision of the transgenes. After 2 days, the cells werecultured in E8 mediumwith daily media change. Individual colonies were observedunder microscopy and subcultured as described in Section 2.4 for expansion andcharacterization. The excision of the construct of transgene in the hiPSCs was probedby genomic PCR.

2.6. Long-term culture of hiPSCs on LN521 under xeno-free culture conditions

Long-term xeno-free culture of hiPSCs was conducted with E8 or NutriStem onLN521-coated 12-well culture plates. Cells were passaged every 4e5 days with0.05 mM EDTA. Cell attachment and self-renewal were assessed from three inde-pendent cultures and cells cultured on Matrigel served as the controls. Stem cellphenotypewas assessed by alkaline phosphatase staining, PCR, immunostaining andflow cytometry.

2.7. Assessment of pluripotency of hiPSCs in vitro

To assess whether the generated iPSCs maintained pluripotency in vitro, iPSCscultured in stem cell culture media were detached with dispase from culture wells,plated in ultra-low attachment culture plates (Corning) and cultured with DMEMmedium supplemented with 20% (v/v) FBS, 1� Glutamax (Life Technologies), 1%penicillin/streptomycin for 2e3 weeks. RNAwas extracted using Trizol method (LifeTechnologies). The representative gene markers of the three embryonic germ layerswere analyzed by PCR, using the primer sequences listed in Supplementary Table 1.

2.8. Assessment of pluripotency of hiPSCs in vivo

In vivo pluripotency of the cells was assessed by their ability to form teratoma, asdescribed previously [36]. The hiPSCs (w1e5 � 106 cells) detached from culturewells were resuspended in PBS, and injected subcutaneously into SCID mice toinduce teratomas. After 8 weeks, mice were euthanized; explants were isolated andprocessed for histological analysis at Histopathology Unit (Biopolis Shared Facilities,Singapore). All animal experimental procedures were approved and performedfollowing the guidelines of the National Advisory Committee on Laboratory AnimalResearch (NACLAR).

2.9. Directed differentiation of EX-iPSCs into dopaminergic neurons on LN521substrate under xeno-free culture conditions

The dopaminergic neuronal differentiation of EX-iPSCs cultured on LN521 wascarried out using the protocol reported by Kriks et al. with modification [37].Typically, the EX-iPSCs were plated on LN521 in E8 medium for 4 days to reach nearconfluence. The cells were washed with PBS, and exposed to medium consisting ofDMEM/F12 supplemented with N2 (N2 Supplement CTS, Life Technologies),1� Glutamax, LDN193189 (100 nM, Stemgent), SB431542 (10 mM, Tocris), CHIR99021(3 mM, added starting on day 3), SHH (100 ng/ml, R&D), FGF8 (100 ng/ml, R&D) for 10days with media change every 2 days. On day 11, the medium was changed toDMEM/F12 supplemented with 1� Glutamax, N2, B27, BDNF (brain-derived neu-rotrophic factor, 20 ng/ml; R&D), ascorbic acid (0.2 mM, SigmaeAldrich), GDNF (glialcell-line-derived neurotrophic factor, 20 ng/ml; R&D), DAPT (10 mM, Tocris), TGFb3(transforming growth factor type b3, 1 ng/ml; R&D), dibutyryl cAMP (0.5 mM;

SigmaeAldrich) for 9 days. Then the cells were dissociated using Accutase andreplated under high cell seeding density conditions on dishes precoated with pol-yornithine/laminin (SigmaeAldrich) in differentiation medium for characterization.

2.10. Alkaline phosphatase (AP) assay

To determine the number of hiPSC colonies and hiPSC AP activity, culture plateswere stained with a Vector�Red alkaline phosphatase substrate kit (Vector Labo-ratories, Inc. California, USA) according to the manufacturer’s instructions. Thestained cell colonies were observed under microscope. The reprogramming effi-ciencies were calculated on the number of AP-positive colonies normalized to thenumber of cells seeded on Matrigel- or LN521-coated plates.

2.11. Genomic and reverse transcription-polymerase chain reaction analysis

Genomic DNAwas isolated from pre- and post-excised hiPSCs by DNAeasy Bloodand Tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s in-structions. PCR was performed by using DreamTaq Green PCR Master kit (ThermoScientific) with a five-step PCR protocol as follows: initial denaturation at 95 �C for3 min; 45 cycles of each of the following: denaturation at 95 �C for 30 s, primerannealing at 55 �C for 35 s, and extension at 72 �C for 3 min; followed by a singlecycle final extension at 72 �C for 30 min. Primers specific for exogenous integrationsof the OSKM lentivirus (PT) are listed in Supplementary Table 1.

2.12. Karyotype analysis

The hiPSCs were plated on Matrigel-coated glass coverslips. The samples weresubmitted for karyotyping analysis by G-banding (Parkway Health Laboratory Ser-vices Ltd, Singapore). At least 20 metaphase spreads were screened and evaluatedfor chromosomal rearrangement.

2.13. Flow cytometry and FACS

The iPSCs cultured on different substrates were dissociated with Accutase(Stemcell technology) for 6e10 min at 37 �C, followed by gentle trituration to asingle-cell suspension. The cells were processed for stainingwith antibodies listed inSupplementary Table 2 and analyzed with a BD LSR II.

In order to obtain the transgene-free hiPSCs, the Cre/GFP plasmid-transfectedhiPSCs cultured on LN521 were dissociated with EDTA for 6e10 min at 37 �C, fol-lowed by gentle trituration to a single-cell suspension in E8 medium containing10 mM ROCK inhibitor. The single GFP-positive cell was obtained by sorting single-cellsuspension using Beckman Coulter Moflo XDP Cell Sorter (Beckman), plated inLN521-coated 96-well plates, and cultured in E8 containing ROCK as described in2.4.

2.14. RNA extraction, reverse transcription and polymerase chain reaction

Total RNA was isolated using Trizol reagent (Life Technologies) according tomanufacturer’s instruction as reported previously [36]. Before reverse transcription,RNA samples were treated with DNase I (Life Technologies) to remove contami-nating genomic DNA. cDNA was synthesized using SuperScript III Reverse Tran-scriptase and Oligo (dT)18 primers according to the manufacturer’s instructions (LifeTechnologies). PCR was carried out using Taq DNA Polymerase (Life Technologies)with gene specific primers. The primers utilized for the reactions are listed inSupplementary Table 1.

2.15. Immunocytochemistry

hiPSCs or their differentiated derivatives were plated on Matrigel-coated cov-erslips, fixed with 4% paraformaldehyde and immunostained with the antibodies asreported previously [38]. Appropriate fluorescence (Alexa-Fluor-488/568)-taggedsecondary antibodies were used for visualization (molecular probes, Eugene, USA).4,6-diamidino-2-phenylindole (DAPI) counterstain was used for nuclear staining.The samples were observed under a Zeiss LSM510 laser scanning microscope andphotographed and processed with LSM Image Browser software. The antibodiesused in this study are listed in Supplementary Table 2.

2.16. Statistical analysis

Data are expressed as mean � s.d. Statistical significance between two groupswas determined by the unpaired Student’s t-test. Results for more than twoexperimental groups were evaluated by one-way ANOVA to specify differencesbetween groups. P < 0.05 was considered significantly different.

3. Results

3.1. Optimizing the reprogramming of human adult fibroblastsunder feeder-free condition

Our initial efforts focused on developing a simplified approachfor the efficient derivation of transgene-free hiPSCs from adult

H.F. Lu et al. / Biomaterials 35 (2014) 2816e2826 2819

fibroblasts, without the need for concurrent feeder cells andreprogramming enhancing molecules. Towards this end, we usedMatrigel-coated substrates to optimize the reprogramming exper-iment, by exploring a matrix of conditions encompassing virustransduction strategies, culture medium and fibroblast cell sourceproperties.

Previously, a single polycistronic lentiviral vector expressingthree transgenes (Oct4, Sox2 and Klf4) was reported for transgene-free iPSC derivation from humanized mouse fibroblasts (16). Inorder to use this strategy for human adult fibroblast reprogram-ming, we modified this three-transgene vector into a four-transgene vector by the insertion of a IRES-cMyc fragment at aunique PacI site. c-Myc has been reported to play a key role in thereprogramming of human fibroblasts [39]. This modified vector,named pOSKM, dramatically improved the reprogramming effi-ciency (data not shown). A schematic displaying the various ele-ments of pOSKM vector is shown in Supplementary Fig. 1. Titrationof lentivirus was performed following the generation and concen-tration of the virus, and finally 100 ml of viral vector per well of 6-well plate was chosen for our study (refer to Materials andMethod).

We infected human fibroblasts derived from two individuals atdifferent ages (HDF-A; HDF-C; Table 1) with pOSKM or GFP (as acontrol). As expected in control groups, more than 98% cellsexpressed strong GFP florescence after 48 h infection, suggestingthe efficient lentiviral transduction efficiency (SupplementaryFig. 2). The cells were harvested 48 h post-infection, plated onMatrigel-coated plates and cultured with stem cell culture mediumfor w4 weeks. Experiments were carried out using both xeno-freemedium (E8, NutriStem, TeSR2) and conventional animalcomponent-containing medium (CM and mTeSR1). Fig. 1B showsthe cell morphology changes during fibroblast reprogramming.When cultured with CM, the infected fibroblasts proliferated fast,but no obvious cell colonies were observed. In contrast, from day 7post-transduction, pOSKM treated fibroblasts cultured in E8 andNutriStem media retracted their widespread processes andassumed a compact, epithelioid morphology. On day 12, colonieswith hESC-like morphology were observed in these wells, but notin the GFP wells treated with the same medium (Fig. 1B,Supplementary Fig. 2C). These colonies grew rapidly in thefollowing week, and by day 16, they were large enough to beselected manually. On the other hand, wells cultured with mTeSR1and TeSR2 showed few ES-like colonies.

In order to quantify the efficiency of the reprogramming, cellscultured with the different stem cell media were analyzed foralkaline phosphatase (AP) activity 20 days post-transduction.Fig. 1C shows representative AP staining results from reprog-rammed cells cultured in 24-well Matrigel-coated plates. Consis-tent with our observation under the microscope, about 1e3 AP-positive colonies were obtained in the wells cultured withmTeSR1 and TeSR2, corresponding to a reprogramming efficiencyof w0.01%. No AP-positive colonies were found in cells culturedwith CM. In contrast, a significant increase in AP-positive colonieswas observed in cells cultured with E8 and NutriStem (Fig. 1C).Notably, E8 culture yielded the highest reprogramming efficiency of0.3% (Fig. 1D), which is 30 fold higher than those typically reportedfor viral vector-based reprogramming of human adult fibroblasts[14,27]. Thus, using our optimized protocol, the reprogrammingefficiency of human adult fibroblasts greatly exceeds that of con-ventional retroviral approaches.

We next evaluated the contribution of cell seeding density to theefficiency of hiPSC derivation. We plated the transduced fibroblastson Matrigel-coated substrates at 0.005, 0.01, 0.02, and 0.03 � 106

cells/cm2, and cultured in E8 medium for 3e4 weeks. By screeningfor AP-positive colonies, we observed optimal iPSC generation

efficiency when the cell density was between 0.01 and 0.02 � 106

cells/cm2, but significantly reduced at very low or high cell den-sities (Fig. 1E). Similar results were observed for reprogramming ofHDF-C. Thus we chose a cell density of 0.02 � 106 cells/cm2 for ourreprogramming study.

To study the developed colonies in more detail, we manuallypicked up 6e10 individual colonies from cells cultured with E8 andNutriStem, between 16 and 24 days post-transduction, and platedthem on Matrigel-coated plates. The derived colonies were easilyexpanded, with notably very few clones failing to establish. TheseiPSC colonies robustly proliferated in E8 or NutriStem, exhibitingtypical ESC-like morphology (e.g. compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). Immunostaining ofiPSC colonies revealed the expression of pluripotency markers, AP,OCT4, NANOG, SSEA4 and TRA-1-81 (Fig. 2A). Flow cytometryconfirmed that more than 98% of these iPSCs were positive for thestem cell markers OCT4, NANOG, SOX2 and SSEA4 (Fig. 2B). Kar-yotype analysis indicated a normal human karyotype for all of theclones tested (Fig. 2C, Supplementary Fig. 3).

To further assess whether the hiPSCs had been reprogrammedto pluripotency, we tested the developmental potential of thegenerated hiPSCs. We differentiated iPSCs in vitro using standardembryonic body (EB) culture approach, where three independentiPSC colonies were cultured with DMEM-FBS for an additional twoweeks. Eight lineage-specific gene markers including endoderm(AFP, PC2), ectoderm (SOX1, PAX6), mesoderm (GATA2, Myosin)and stem cell pluripotent markers (NANOG, OCT4) were measuredfor differentiated cells and undifferentiated control cells. As shownin Fig. 3A, the differentiated cells express all of the selected lineage-specific genes. Furthermore, immunostaining confirmed the pres-ence of differentiated cells from the three germ layers, includingendoderm (a-fetoprotein), ectoderm (bIII-tubulin) and mesoderm(a-actin) (Fig. 3B). Finally, to assess the potential of hiPSCs to formteratoma and differentiate to ectoderm, mesoderm and endodermin vivo, two clones of each hiPSC were implanted subcutaneously inSCID mice. Teratoma formation was observed in all mice receivinghiPSC cell clones. Histological analyses showed that derivativesfrom all three embryonic germ layers were observed in these ter-atomas, confirming the trilineage differentiation potential of hiPSCsin vivo (Fig. 3C, Supplementary Fig. 4). Together, these datademonstrate that the hiPSCs generated in our feeder-free approachwere morphologically, phenotypically and functionally identical topluripotent stem cells.

3.2. Generation of transgene-free patient-specific iPSCs on LN521under xeno-free culture condition

Our intent was to develop a simple xeno-free culture system forfacilitating patient-specific transgene-free iPSC derivation. Thus,we further optimized our culture substrate to fulfill this purpose.We transduced one PD patient fibroblast line (ND30116) and oneadult fibroblast line (HDF-L) with pOSKM in DMEM-HS. After 2 daysof transduction, cells were transferred to 12-well plates that hadbeen precoated with LN521 and Matrigel (as a control), andcultured in E8. Similar to what we had been observed in the earlieroptimization experiments, colonies with hESC-like morphologyfirst became visible 12 days after transduction both in LN521- andMatrigel-coated culture wells (Fig. 4A). AP staining revealed thatsimilar reprogramming efficiency was obtained for both cultureconditions, whereas no colonies were observed in GFP-lentivirustransduced controls (Fig. 4B, C). We next picked 6e10 cell col-onies from individual groups and expanded them on LN521-coatedsubstrates for further characterization. All these cell colonies couldbe expanded and expressed genes and cell-surface markers char-acteristic of hESCs, including NANOG, OCT4, SSEA4 and TRA-1-81

Fig. 1. Reprogramming of human adult fibroblasts by using a single polycistronic lentiviral vector pOSKM. (A) Schematic diagram of the reprogramming protocol used. The platedhuman fibroblasts were transduced with lentiviral polycistronic vector in HDF culture medium (day-0), and replated on Matrigel-coated tissue culture wells on day-2 in differentstem cell culture medium: CM, mTeSR1, TeSR2, E8 and Nutristem. The transduced cells were cultured for 3e4 weeks with daily media changes. Emergence of colonies was

H.F. Lu et al. / Biomaterials 35 (2014) 2816e28262820

Fig. 2. Characterization of hiPSCs generated from human adult fibroblasts using pOSKM under feeder-free condition. (A) Immunofluorescence staining of alkaline phosphataseactivity (AP) and pluripotency markers (OCT4, NANOG, SSEA4, and TRA-1-81) in A-iPSCs derived from human fibroblasts HDF-A. Cells were counterstained for nuclei with DAPI.Scale bar: i and ii: 200 mm; others: 50 mm (B) FACS analysis of pluripotency markers expressed in A-iPSCs. (C) G-banding karyotype analysis of A-iPSCs.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e2826 2821

(Supplementary Fig. 5). These data suggest that LN521 supportshiPSC derivation from human adult fibroblasts under the presentxeno-free culture conditions.

Next, we excised out the reprogramming transgenes fromhiPSCs by transient expression of inducible Cre recombinase by co-transfecting a plasmid expressing Cre-ER with GFP plasmid in a 1:1ratio. After one day of transfection, we observed GFP florescence inhiPSC colonies, indicating successful plasmid transfection (Fig. 4Di,ii). Single GFP-positive cells were selected using FACS and plated onLN521-coated 96-well plates, at 1 cell per well. Subsequently, in-dividual colonies were picked and expanded. To prove the excisionof transgenes, we performed PCR on genomic DNA to detect specificregions of the transgene with genomic GAPDH as a control. Asshown in Fig. 4E, a target band of w2.3 kb, was amplified fromgenomic DNA before vector excision (PD-iPSC), but was absent inDNA extracted from the selected individual colonies after vectorexcision (Ex-PD-iPSC1, Ex-PD-iPSC2). This result demonstrates thatOSKM transgenes were successfully removed in these selected

monitored daily until discernible hESC-like colonies were picked (days 16e24). (B) Black-whstaining conducted at day 20 for reprogrammed cells cultured in 24-well plate. (D) The reramming efficiency using different cell seeding densities. *: P < 0.05.

hiPSCs colonies. The excised iPSC clones cultured on LN521-coatedplates demonstrated typical ESC-like compact colony morphologycomposed of small rounded cells with a large prominent nucleus,and expressed strong alkaline phosphatase activity (Fig. 4Diii, iv).

To further confirm the transgene-excised iPSC status, semi-quantitative RT-PCR was performed with OCT4, SOX2, NANOG,LIN28, DAPP5, GDF3 and REX1 primers. HUES7 was used as apositive control. As shown in Fig. 4F, the excised clones were pos-itive for pluripotent stem cell markers. Flow cytometry analysisconfirmed that more than 98% of these EX-iPSCs were positive forthe stem cell markers OCT4 and NANOG (Supplementary Fig. 6).

3.3. LN521 supports long-term transgene-free iPSCs self-renewalunder xeno-free condition

We next tested whether the generated EX-iPSCs could bepropagated on LN521 substrates in E8 medium for long-term cul-ture. EX-iPSCs were maintained with E8 on LN521-coated

ite images of cells during reprogramming. Scale bar: 200 mm. (C) Alkaline phosphataseprogramming efficiency by pOSKM, with various media as indicated. (E) The reprog-

Fig. 3. In vitro and in vivo differentiation of hiPSCs. (A, B) In vitro differentiation of A-iPSCs using embryonic body approach. (A) RT-PCR analysis of gene marker expression of threegerm layers in differentiated cells. NC: negative control. (B) Micrographs of EBs generated from hiPSCs in vitro differentiation. Ectodermal, mesodermal, and endodermal cell typeswere revealed by antibodies specific for markers bIII-tubulin, SMA, and AFP, respectively. (C) In vivo differentiation assay by teratoma formation. hiPSCs were injected subcuta-neously in SCID mice and teratomas were dissected after 8 weeks. Sections from excised tumors were stained by hematoxylin and eosin for histology analysis. Scale bar: Bi, Bii, Ci,Civ : 100 mm; Biii: 10 mm; Biv, Cii, Ciii: 50 mm.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e28262822

substrates for 10 sequential passages. Cells robustly proliferatedon LN521 culture plates with E8 medium, and were passaged every4e5 days by the EDTA method. Fig. 5 shows the characterization ofEX-PD-iPSC1 cultured on LN521 for 10 passages. Throughout 10passages, PCR analysis revealed similar expression levels ofpluripotent gene markers (Fig. 5A). The cells exhibited typicalhESC-like morphology and expressed the stem cell nuclear markers(SOX2, OCT4 and NANOG) and surface markers (SSEA4, TRA-1-60and TRA-1-81) (Fig. 5B). Flow cytometry assay revealed that >95%of the cells expressed OCT4 throughout 10 passages (Fig. 5C). WhenEX-PD-iPSC1 differentiated in vitro using EB approach, immuno-staining of differentiated cells showed marker protein expressionfor all three embryonic germ layers (data not shown). Two-monthafter being subcutaneously injected into immunodeficient mice,EX-PD-iPSCs formed teratomas consisting of derivatives of all threeembryonic germ layers (Fig. 5D). Similarly, the cells from the EX-L-iPSC clones displayed stable self-renewal phenotypewhen culturedon LN521 after 10 sequential passages. Taken together, these datademonstrated that LN521 supports long-term transgene-free hiPSCself-renewal under xeno-free culture conditions.

3.4. Directed differentiation of transgene-free PD patient iPSCs onLN521 under xeno-free culture condition

We further investigated whether EX-PD-iPSCs expanded on LN-521 could be directed to differentiate into dopaminergic neuronsunder xeno-free culture conditions. To perform differentiation,nearly confluent EX-PD-iPSCs cultured in E8 on LN521-coatedculture wells were treated with xeno-free dopaminergic neuraldifferentiation media (refer to Materials and Methods) [37]. After 3weeks of culture, the differentiated cells were harvested for char-acterization. Fig. 6A showed the changes in cell morphology duringdifferentiation experiment. RT-PCR results confirmed that the

differentiated cells expressed the mid brain markers, includingLMX1B, NURR1, TH and GIRK2 (Fig. 6B). Immunostaining confirmedthat the majority of cells expressed the mid brain marker TH, alongwith GIRK2 and FOXA2 (Fig. 6C). Taken together, these in vitrodifferentiation data demonstrate that transgene-free PD patientEX-PD-iPSCs can be directed to differentiate into dopaminergicneurons efficiently on LN521 under xeno-free culture condition.

4. Discussion

Since the first report by Takahashi et al. describing human iPSCderivation in 2007 [14], substantial interest has been focused ondeveloping a safe culture system towards the clinical application ofiPSCs. In this study, we devised a simple, highly reproducibledefined xeno-free and feeder-free culture system which supportsrobust iPSC derivation, expansion and direct differentiation.Compared with other reprogramming approaches, our facile cul-ture system provides several important clinically compliant ad-vantages: 1) By obviating the need to perform reprogrammingexperiments using feeder cells and animal-containing medium asrequired for the current approaches, use of xeno-free LN521 sub-strates and E8 medium should make xeno-free reprogrammingeasily accessible to a wider community of researchers. 2) With theoptimized reprogramming conditions, our approach providesrobust reprogramming efficiency from human adult fibroblastscompared to the conventional viral vector-based method, withoutthe use of reprogramming enhancing molecules. Furthermore, ourmodified excisable polycistronic lentiviral vector eliminates the riskof transgene integration in the generated iPSC genome andconsequent problems. 3) Our system provides a platform to bothefficiently reprogram patient-specific fibroblasts to a pluripotentstate and direct the fate of such cells to terminally differentiatedsomatic cell types under totally xeno-free culture conditions. We

Fig. 4. Generation of transgene-free iPSCs under defined xeno-free and feeder-free condition. (A) Microscopic images of cells cultured on LN521 during reprogramming and alkalinephosphatase staining image of reprogrammed fibroblasts. (B) Alkaline phosphatase staining of reprogrammed cells cultured in 12-well plate, at day 20. Matrigel-coated substratesserved as control. (C) The reprogramming efficiency of cells cultured on LN521- and Matrigel-coated substrates in E8 medium. (D) Microscopic images of PD-iPSCs transientlytransfected with Cre/GFP plasmid (i, ii), post-excision iPSC colonies (iii) and AP staining (iv). (E) Genomic PCR confirmed transgene excision in two iPSC clones: Pre-(PD-iPSC) andpost-excision (EX-PD-iPSC1, EX-PD-iPSC2). (F) RT-PCR analysis of gene marker expression of pluripotency in EX-PD-iPSC1. hESC serves as control. Scale bar: A:100 mm; B: 200 mm.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e2826 2823

demonstrated that PD patient-specific iPSCs derived using thisclinically compliant culture system could be directed toward dif-ferentiation into dopaminergic neurons. These results shouldenhance the feasibility of generating patient iPSCs and derivativesfor drug discovery and cell-based therapy.

A defined xeno-free system for hiPSC derivation, expansion anddifferentiation is ideally required for iPSC application in clinical celltherapy. Towards that end, much progress has been made indeveloping defined xeno-free substrates for long-term hESC self-renewal in defined or xeno-free media [13,33,34,40e46]. To date,however, there are limited studies reporting the use of a singlewell-defined xeno-free culture system (including xeno-free

substrate and medium) for all three processes of hiPSC derivation,expansion and direct differentiation. Currently, the poor efficiencyof existing reprogramming methods necessitates the use of MEFlayers to enhance the reprogramming efficiency [8]. Thus, ourapproach was to start with a xeno-free laminin isoform as oursubstrate, and investigate if iPSC derivation, expansion and differ-entiation could be efficiently performed using xeno-free culturemedia. Our choice of the laminin isoform, LN521, was based onrecent discovery of the effects of various laminin isoforms on ESCculture. Human laminins are the first matrix proteins expressed inthe embryo [25,31]. Among the 15 laminin isoforms, LN511, LN111and LN521 have been reported to be normally expressed and

Fig. 5. Characterization of transgene-free EX-iPSCs cultured on LN521 with E8 medium for 10 passages. (A) RT-PCR analysis of gene marker expression of pluripotency in EX-PD-iPSC1 for 10 passages. (B) Immunostaining of EX-PD-iPSC1 for stem cell markers NANOG, SSEA4, TRA-1-81, OCT4, SOX2, and TRA-1-60. Cells were counterstained for nuclei withDAPI. (C) FACS analysis of EX-PD-iPSC1 cultured on LN521 showing that >95% of cells express OCT4 at passages P1, P3, P6 and P10. (D) In vivo differentiation assay by teratomaformation. hiPSCs were injected subcutaneously in SCID mice and teratomas were dissected after 8 weeks. Sections from excised tumors were stained by hematoxylin and eosin forhistology analysis. Scale bar: B: 50 mm; D: 100 mm.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e28262824

secreted by hESCs [35]. LN511 and its E8 fragment, but not LN111,LN332 or LN411, was shown to support long-term human embry-onic stem cell culture in chemically defined medium [33,46].Through protocol optimization, we have shown that LN521 sup-ports both human adult fibroblast proliferation and reprogram-ming under defined xeno-free and feeder-free conditions.Reprogramming under these totally xeno-free conditions achieveda reprogramming efficiency comparable to that for Matrigel-coatedsubstrates. We were able to maintain all the tested hiPSC lines onLN521 for at least 10 passages under defined xeno-free culturemedium. These hiPSCs maintained high expression levels of stemcell maker genes, and were able to form derivatives of the threegerm layers both in vitro and in vivo. In addition, we were able todifferentiate hiPSCs cultured on LN521 to dopaminergenic neuronsunder xeno-free conditions. Taken together, these results demon-strated that LN521 is sufficient to support xeno-free hiPSC deriva-tion and long-term self-renewal as well as direct tissue-specificdifferentiation.

Despite the significant advances in hiPSC derivation technolo-gies frommurine fibroblasts and neonatal human normal skin cells,the efficiency of iPSC derivation from human adult fibroblasts hasremained extremely low (generally less than 0.01%) [20e25]. Whilereprogramming enhancing molecules can improve the reprog-ramming efficiency, application of these compounds may haveundesired pleiotropic effects [28,29]. For example, the use of

5-azacytidine has been shown to cause tumor in mice with globalalteration of DNA methylation level [28,47]. Thus, viral-based ap-proaches remain the most widely used method for patient iPSCderivation from a variety of diseases, including amyotrophic lateralsclerosis, PD, type 1 diabetes mellitus, Huntington’s disease, lungdisease and Down’s syndrome [16,17,48]. In our study, we chose asingle polycistronic lentiviral vector which can efficiently introducethe transgene into the fibroblasts, followed by transgene cleavageafter reprogramming. Most importantly, we simplified ourreprogramming study by applying defined culture medium andLN521- or Matrigel-coated substrates, without the exposure toreprogramming enhancing molecules. A robust increase inreprogramming efficiency was observed for all the tested HDFreprogramming studies. Interestingly, among the media we tested,we found that defined xeno-free E8 medium showed the highestreprogramming efficiency. Using E8 medium, we consistently ob-tained reprogramming efficiencies >0.18% for fibroblasts fromseveral adult individuals of different ages, which is much betterthan those typically reported for conventional virus vector-basedhiPSC derivations from human adult fibroblasts [14,27].

Autologous, patient-specific iPSCs would enable customizedtissue engineering and immunosuppression-free cell therapy forvarious diseases, employing cardiomyocytes, neuronal andpancreas islet cells, which are otherwise difficult to obtain. Forclinical applications, it is critical to derive transgene-free patient-

Fig. 6. Differentiation of EX-PD-iPSC1 into dopaminergic neurons on LN521 substrates under xeno-free culture condition. (A) Microscopic images of cells cultured on LN521 duringdifferentiation process (i) undifferentiated cells; (ii) differentiated cells on day 20; (iii) differentiated cells plated on laminin-coated coverslips after 48 h (B) RT-PCR analysis of midbrain gene marker expression in differentiated cells. (C) Immunofluorescence staining of mid brain markers in differentiated cells. Cells were counterstained for nuclei with DAPI.Scale bar: Ai, Aiii: 100 mm; Aii: 200 mm; C: 20 mm.

H.F. Lu et al. / Biomaterials 35 (2014) 2816e2826 2825

specific iPSCs and differentiate them into the relevant cell lineagesrequired to model or treat those diseases under a US Food and DrugAdministration-compliant process. In the present article wedemonstrate PD fibroblast reprogramming where transgene-freeiPSCs were generated and expanded under defined xeno-free cul-ture conditions. These EX-PD-iPSCs were then successfully differ-entiated into dopaminergic neurons, via a directed differentiationapproach under xeno-free conditions. PCR and immunostainingstudies confirmed the strong expression of mid brain markers inthese differentiated cells. The defined xeno-free culture conditions,including substrate (LN-521) and culture media (E8 and dopamineneuron differentiation medium), greatly reduces the risk of cellularcontamination with animal-derived pathogens and provides ascalable, robust platform for the cultivation of patient-specific iPSCsand derivatives for potential clinical applications.

5. Conclusions

Our results demonstrate that a defined xeno-free LN521 sub-strate can support efficient transgene-free hiPSC derivation andexpansion, as well as directed tissue-specific differentiation underxeno-free conditions. A combination of efficient non-integrationreprogramming of adult skin fibroblasts and defined xeno-freeculture conditions would allow Good Manufacturing Practice-compliant production of hiPSCs, which represents an importantstep towards clinical iPSC applications. With that, we believe thatthe optimized xeno-free culture system will be useful for bothresearch purposes and the production of transgene-free humanpatient iPSCs and derivatives for cellular therapy.

Acknowledgments

Funding was provided by the Institute of Bioengineering andNanotechnology (Biomedical Research Council, Agency for Science,Technology and Research, Singapore), Singapore National ResearchFoundation e Competitive Research Program grant (L.K.L) andNational Medical Research Council NIG grant (C.C).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.12.050.

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