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Biomaterials 34 (2013) 5336e5343

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Biomaterials

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

Delivery of reprogramming factors into fibroblasts for generation ofnon-genetic induced pluripotent stem cells using a cationicbolaamphiphile as a non-viral vector

Majad Khan 1, Karthikeyan Narayanan*,1, Hongfang Lu, Yang Choo, Chan Du,Nikken Wiradharma, Yi-Yan Yang, Andrew C.A. Wan*

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669

a r t i c l e i n f o

Article history:Received 8 March 2013Accepted 23 March 2013Available online 16 April 2013

Keywords:Protein transductionIntracellular deliveryStem cellsInduced pluripotent stem cellsCationic bolaamphiphileReprogramming of fibroblasts

* Corresponding authors. Tel.: þ65 6824 7134; fax:E-mail addresses: karthikeyan@ibn.a-star.edu.sg

star.edu.sg (A.C.A. Wan).1 These authors contributed equally.

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.03.072

a b s t r a c t

Protein delivery allows a clinical effect to be directly realized without genetic modification of the hostcells. We have developed a cationic bolaamphiphile as a non-viral vector for protein delivery application.The relatively low toxicity and efficient protein delivery by the cationic bolaamphiphile prompted us totest the system for the generation of induced pluripotent stem cells (iPSCs) as an alternative to theconventional vector-based genetic approach. Studies on the kinetics and cytotoxicity of the proteindelivery system led us to use an optimized cationic bolaamphiphile-protein complex ratio of 7:1 (wt/wt)and a 3 h period of incubation with human fibroblasts, to ensure complete and non-toxic protein deliveryof the reprogramming proteins. The reprogrammed cells were shown to exhibit the characteristics ofembryonic stem cells, including expression of pluripotent markers, teratoma formation in SCID mice, andability to be differentiated into a specific lineage, as exemplified by neuronal differentiation.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Proteins are considered as modulators of cell function, involvingprocesses ranging from cell differentiation to cell death. The de-livery of proteins with specific functions in an efficient and simplemanner can potentially give rise to huge benefits with regards torealizing the goals of certain therapeutic and non-therapeutic ap-plications (reviewed in Refs. [1] and [2]). One particular applicationof protein delivery that could have a far reaching influence withinthe fields of tissue engineering and bioengineering is the genera-tion of induced pluripotent stem cells (iPSCs) from fibroblasts. Thegeneration of iPSCs from fibroblasts, as demonstrated initially byYamanaka [3], has opened up a new chapter in the field of regen-erative medicine and brought it to a new level. The forcedexpression of Oct4, Klf4, Sox2 and c-Myc (Yamanaka factors, [3]) orOct4, Sox2, Nanog and Lin28 (Thomson factors, [4]) were shown toinduce the transformation of adult fibroblasts to iPSCs. However,beyond the success of reprogramming somatic cells, there is still

þ65 6478 9082.(K. Narayanan), awan@ibn.a-

All rights reserved.

some concern on the method used to introduce the exogenousgenetic material into fibroblasts. This certainly remains an issuewhen applying these cells to translational applications [5,6]. Ofparticular concern is that the process of introducing these factorsmight bring about detrimental genetic modifications, such as thepotential insertional mutagenesis [7]. To overcome this, severalmethods have been designed such as transposon based Cre-Loxtechnology [8], adenovirus [9], Sendai virus [10], the piggyBacsystem [11], minicircle DNA vectors [12], episomal vectors [13],protein-based methods [14,15], synthetic RNA [16] and microRNA[17]. The iPSCs developed by these methods have been shown to besimilar to hES, both biologically and functionally. For example, theiPSCs have been demonstrated to differentiate in vitro and alsopossess the ability to form chimera and teratoma.

Another possible method of delivering proteins is via non-viralsystems such as synthetic polymers and liposomes/micelles/am-phiphiles. These offer several advantages over viral based carrierssuch as the promise of lower immunogenicity, ease of tailoring thedelivery vehicle to target certain cells over others, ability to carrytwo types of cargo (chemical drug and protein) and ease ofmanufacture on a large scale. In addition, researchers can easilyalter the synthetic biomaterial’s physical and chemical propertiesin order to generate a tailor made carrier for a specific group of

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proteins. As a result, over the past decade in particular, there hasbeen a rapid surge in research activity in utilizing various syntheticdelivery systems to act as protein delivery vehicles. Researchershave shown that one can employ various cationic and anionicpolymers/micelles as carriers of protein cargo into cells [18e24]. Itis generally believed that charged polymers/micelles are able tocondense proteins via electrostatic interactions, hydrogen bonding,hydrophobic interactions and van der Waals interactions[18,25,26]. For example, Li et al. [27] have demonstrated thatmaltoside-based bolaamphiphiles were able to stabilize membraneproteins such as Escherichia coli diacylglycerol kinase (DAGK). Withthis in mind, we employed a simple 1,12-diaminododecane basedcationic bolaamphiphile to complex and deliver various recombi-nant (reprogramming factor) proteins into human fibroblasts so asto generate iPSCs in an efficient manner. We characterized theprotein-based iPSCs by various biochemical and molecularmethods. We further examined the potential of these cells to formmultiple phenotypes under in vitro and in vivo conditions, includingdifferentiation of the iPSCs into the neural lineage.

2. Materials and methods

2.1. Synthesis and characterization of cationic bolaamphiphile

The detailed synthesis and characterization for the cationic bolaamphiphile hasbeen shown elsewhere [28]. This symmetrical bolaamphiphile was synthesizedusing a three-step, two-pot procedure where pentaethylenehexamine was used asthe hydrophilic amine unit, whilst 1,12-diaminododecane was used as the hydro-phobic unit. In brief, the bolaamphiphile precursor molecule was prepared byreacting 1,12-diaminododecane with methyl-3-mercaptopropionate thiol ester vianucleophilic substitution in a one-pot procedure at 80 �C for 24 h. The precursormolecule was then reacted with epichlorohydrin (glycidyl) to form the linker unitthat was then connected to the hydrophilic pentaethylenehexamine unit vianucleophilic substitution to give rise to the final cationic bolaamphiphile. Cationicbolaamphiphile was characterized by IR, 1H NMR and 13C NMR spectroscopy (BrukerAVANCE 400 at 400 MHz) using the indicated deuterated solvent. Bolaamphiphile:ymax/cm�1 3360 strong (sharp) [y(NeH)]; 3310 strong (sharp) [y(NeH)]; 2960 me-dium (sharp) [y(CeH)]; 2830 medium (sharp) [y(CeH)]; 1660 strong (sharp) [y(C]O)]; 1120 weak (sharp) [y(CeOH)]. dH (400 MHz, D2O) 3.70 (1H, m, eCH2eCH(OH)CH2e); 3.30e2.20 (2H, t, NH2eCH2eCH2eNHe, eCH2eCH2eCH2eNHeC(]O)eCH2eCH2eSe, eCH2eCH(OH)CH2e) and 1.60e1.00 ppm (e(O])CeNHeCH2e

(CH2)10eCH2eNHeC(]O)e).

2.2. Protein complex formation

Cationic bolaamphiphile-protein was complexed at various ratios (wt/wt).Briefly, the cationic bolaamphiphile and protein solutions were mixed together andleft undisturbed for 30 min at room temperature. Serum free media was added tobring the volume to 150 ml. The complex was added to cells seeded in a 24 well dishand incubated for 3 h at 37 �C. The mediumwas removed from the cells, which weresubsequently washed with PBS and processed for downstream applications.

2.3. Particle size and zeta potential analyses

The particle size and zeta potential of the bolaamphiphile-protein complexeswere measured by dynamic light scattering (Brookhaven Instrument Corp., Holts-ville, NY, U.S.A.) and Zetasizer (Malvern Instrument Ltd., Worchestershire, UK),respectively. In brief, bolaamphiphile-protein complexes were formed in serum freeDMEM (colorless) (Invitrogen, CA, USA) at room temperature at a mass ratio of 7:1.All the particle size measurements were performed with a HeeNe laser beam at658 nm and a scattering angle of 90� . The particle size and zeta potential mea-surements were repeated for 5 runs for each sample, and the data were reported asthe average of 5 readings.

2.4. Generation of iPSCs with recombinant proteins (Pro-iPSCs)

The respective recombinant proteins for Sox2, Nanog, Klf4 and NR5A2were usedin the generation of iPSCs. Sox2 and Nanogwere purchased fromPeprotech (NJ, USA)and Klf4 and NR5A2 were purchased from Abnova (Taipei, Taiwan, R.O.C.). Valproicacid (VPA, SigmaeAldrich, MO, USA), a small molecule (histone deactylase inhibitor)was used at 1 mM. Human foreskin fibroblasts (ATCC, VA, USA) were seeded into 3wells at 5 � 104 cells per well in a 24 well plate. The cells were cultured in fibroblastgrowth media (ATCC, VA, USA) to attain confluence. The individual cationicbolaamphiphile-protein complexes were mixed and added to fibroblasts. After 3 h ofincubation, the cells were washed with PBS and were supplemented with MEF-

conditioned media. The media was changed everyday, and the fibroblasts wereobserved for colony formation. At 14 days of culture, the colonies appeared, andthese colonies were isolated and cultured onto MEF layer. We picked about 6 col-onies and expanded them on MEF layer using MEF-conditioned media. Subse-quently, we randomly chose 3 colonies by picking under amicroscope, for expansionon hESC qualified Matrigel. These cells were characterized as described below.

2.5. Alkaline phosphatase assay

Alkaline phosphatase expression is a conventional assay for the characterizationof stem cell populations. The pro-iPSC colonies grown on the MEF layer were testedfor alkaline phosphatase (AP) activity using the Alkaline Phosphatase kit (MerckMillipore, Darmstadt, Germany) as instructed by the manufacturer.

2.6. Flow cytometry

Flow cytometry was used to analyze the population of pro-iPSCs with pluripo-tent markers. The pro-iPSCs were dissociated with accutase to a single-cell sus-pension. The cells were used for flowcytometry as described earlier [29]. The stainedcells were analyzed with a BD LSR II Flow Cytometer.

2.7. Immunostaining assay

Immunostaining of the cells was carried out as described earlier [29]. Thesamples were visualized under a Zeiss LSM 510 laser scanning microscope anddigital photographs were processed with either LSM Image Browser software orImageJ (freeware from NIH).

2.8. RNA extraction, real-time-PCR and microarray

Total RNA was isolated using RNeasy kit (Qiagen, CA, USA) according to themanufacturer’s protocol. The RNAwas used for the real time PCR (qRT-PCR) analysisand DNA microarray.

Taqman assays for human specific OCT3/4, Nanog, Sox2, and GADPH werepurchased from Invitrogen (CA, USA). Amplification was carried out by TaqManUniversal PCR Master Mix in a iQ� 5 Real-time PCR System (Bio-Rad, CA, USA).GAPDH was used as a housekeeping gene for normalization.

2.9. In-vitro and in-vivo differentiation of pro-iPSC

We examined the in vitro differentiation potential by generating embryoidbodies on ultra low attachment plates. The cell aggregates were allowed to differ-entiate for 15 days. Total RNAwas isolated and screened for various marker genes forthe three germ layers by RT-PCR.

The in vivo differentiation potential was examined by teratoma assays. Typically,1 � 107 cells were injected subcutaneously into the back of NOD/SCID mice andallowed to grow for 6 weeks. The formalin fixed sections of the explants wereanalyzed using H&E stain at Histopathology Unit (Biopolis Shared Facilities,Singapore). Experiments involving animals were approved by the institute basedIACUC.

2.10. Differentiation into neural lineage

The cells were differentiated to neural lineage by adapting the published pro-tocol [30]. The colonies of pro-iPSCs grown on Matrigel were isolated and grown onultra low attachment plates (Corning, NY, USA) in DMEM/F12 containing B27 (1:50),2 mM glutamine, 20 ng/ml EGF and 20 ng/ml (Invitrogen, CA, USA) of bFGF (Invi-trogen, CA, USA). The neural progenitor clusters were cultured for 20 days and platedonto poly-D-lysine laminin coated plates. The attached cells were stained with an-tibodies against nestin and beta III tubulin.

3. Results and discussion

The focus of this study is to develop a simple non-viral basedprotein delivery system for obtaining human iPSCs without geneticmodifications. The theme of the paper is illustrated in the sche-matic shown in Fig. 1a. We have recently reported on the synthesisand characterization of a 1,12-diaminododecane based cationicbolaamphiphile (Fig. 1b), which when complexed with plasmids,gave rise to nanoparticles that delivered the plasmids into variouscell lines in an efficientmanner [28]. The advantages of employing acationic bolaamphiphile as a protein delivery vehicle is that notonly does it provide amines to encourage electrostatic interactionsand hydrogen bonding between the carrier and the cargo proteinbut it also has a central hydrophobic core (1,12-diaminododecane)

Fig. 1. Cationic bolaamphiphile mediated encapsulation of proteins. (a) Schematic representation of the entry of encapsulated cationic bolaamphiphile-protein complex into the celland their transformation of the cells to iPSCs. The cationic bolaamphiphile-protein complex enters the cell via the cell membrane (1). The cationic bolaamphiphile-protein dis-sociates and releases the proteins into the cytoplasm of the cell (2). The released proteins get translocated to the nucleus by the well studied pathway involving nuclear localizationsequence (3). In the nucleus the proteins bind to their respective response elements and trigger the transcription of the genes associated with reprogramming (4). (b) Chemicalstructure of the diaminododecane based cationic bolaamphiphile used in the study. (c) The cationic bolaamphiphile-protein complex formation and protein transduction efficiencywas analyzed at different mass ratios of cationic bolaamphiphile. FITC-BSA was complexed and transduced to human fibroblasts and the fluorescence was measured after 3 h.Results indicate that the complexation of cationic bolaamphiphile with the protein saturates at 7:1 mass ratio (cationic bolaamphiphile-protein). (d) A time lapse study was carriedout with live cells in order to estimate the time required to transduce protein. FITC-BSA was used at 7:1 ratio (bolaamphiphile:protein). The cells were imaged using confocalmicroscope for every 2 min at 37 �C under 5%CO2. The fluorescence intensity of a selected region was measured for different time frames using ImageJ (NIH freeware). The in-tensities were plotted against time with a regression (dotted line). The intensity of fluorescence was seen to increase along with time, attaining saturation at 2 h.

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to facilitate hydrophobic interaction between the carrier and thecargo protein so as to ensure that the carriereprotein complex isstable. In this work we have employed the cationic bolaamphiphileas a non-viral vector to deliver various reprogramming factors inorder to generate iPSCs from human fibroblasts.

3.1. Protein transfection

Initial screening of the protein delivery was carried out usingfluorescein isothiocynate (FITC) labeled bovine serum albumin(BSA). The efficiency of the cationic bolaamphiphile to form acomplex and deliver proteins was tested at different mass ratios ofcationic bolaamphiphile:protein, where the amount of protein waskept constant. The different ratios tested were 3:1, 5:1, 7:1, 9:1 and11:1 [cationic bolaamphiphile:protein (wt/wt)]. We observed asteady state increase in the detection of the transfected protein,which led to saturation of the capacity of the protein delivery by thecationic bolaamphiphile (Fig. 1c). The cationic bolaamphiphilecontains a mixture of primary and secondary amines. The former

provide positive charges for aiding complexation to take place be-tween the carrier and cargo protein via electrostatic interactionsand hydrogen bonding, and the latter secondary amines help withbuffering capacity to aid the endosomal escape of cationicbolaamphiphile-protein complexes. Studies done with non-viralbased plasmid carriers have shown that both types of amines areessential for high transfection efficiency [31e33]. In addition, webelieve that the presence of the hydrophobic 1,12-diaminodoecanecomponent within the bolaamphiphile promoted cellular uptake ofthe cationic bolaamphiphile-protein complexes, leading to hightransfection.

Themaximumefficiency of the transfectionwas observed at 11:1(wt/wt) ratio of cationic bolaamphiphile to protein. However,cytotoxicity studies suggested that the concentration at amass ratioof 7:1 presents a safer ratio for the cell viability (Fig. S1A). The timerequired for the cationic bolaamphiphile-protein complex to enterthe cell wasmeasured using a live cell imaging system. Imagesweretaken every 2 min and the fluorescent intensity of the selected areawas measured in the stack of images using the imageJ software. The

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time taken for the delivery of FITC-BSA was observed to increasewith time and saturated after 2 hof cationic bolaamphiphile-proteinaddition (Fig. 1d). For these reasons, cationic bolaamphiphile-protein complexes formed at 7:1 (wt/wt) ratio were used, andwhich were incubated with the cells for 3 h to ensure complete andnon-toxic protein delivery. We also analyzed the toxicity of thebolaamphiphile when used consecutively for three times andobserved approximately 15% decrease in the viability of the fibro-blasts (Fig. S1B). We did not observe any uptake of protein (BSA) bythe cells when supplied in the medium (Fig. S1C), which was astriking contrast to the uptake of cationic bolaamphiphile-proteincomplex (Fig. S1D).

3.2. Complexation with reprogramming factors and delivery intothe nucleus

The versatility of the cationic bolaamphiphile as a protein de-livery vehicle is evident, with its ability to form complexes withproteins having different physico-chemical properties (Table-S1).Complexations with reprogramming factors were performed asdescribed in the methods section. Initially using BSA as a modelprotein, the particle size and zeta potential of the bolaamphiphile-

Fig. 2. Transduction of reprogramming factors into primary human fibroblasts. The reprogrMaterials and methods section. The complexes were incubated with the cells for 3 h. Theramming factors. Panel a shows the immunostaining with antibodies against KLF4, Nanog, Nwith FITC conjugated phalloidin (green, panel b) and the nucleus was labeled with DAPI (bluthe immunostaining. The presence of these transduced proteins in the nucleus (colocalizattransduced proteins to their native cellular compartment. Scale bar ¼ 100 mm. (For interpretversion of this article.)

protein complexes in colorless serum free medium at the optimalmass ratio of 7:1 were evaluated using dynamic light scattering andzeta sizer. The findings show that the particle size was approxi-mately 908 nm while the surface charge of the bolaamphiphile-protein complex was slightly positive (þ1.0 mV). The less than 1micron size of the bolaamphiphile-protein complexes at theoptimal mass ratio of 7:1 aids in delivering the protein cargo in anefficient manner via cellular uptake, while the extremely lowcationic charge minimizes the cytotoxicity of the complexes. Theparticle size and zeta potential of the bolaamphiphile-proteincomplexes at the optimal mass ratio of 7:1 for the other deliveredproteins was also measured in the same manner, the values ob-tained are as follows: KLF4 ¼ 606 nm (þ3.5 mV), NR5A2 ¼ 231 nm(þ1.1 mV) and SOX2 ¼ 177 nm (�1.4 mV). The data obtained fromthe particle size and zeta sizer measurements proves that theproteins to be delivered can form complexes with the non-viralbased cationic bolaamphiphile. Even though some of the deliv-ered proteins possess an overall positive charge, it is still possiblefor such proteins to form complexes with cationic systems such asthe bolaamphiphile in this study, because some of the domains inthese proteins are also negatively charged, which would allowcomplexation to take place. In addition, complexes can form

amming factors were complexed with the cationic bolaamphiphile as described in thecells were stained with corresponding primary antibodies against the specific reprog-R5A2 and Sox2 (red, top to bottom). The cellular microtubule architecture was stainede, panel c). The delivery of the respective proteins into the fibroblasts was confirmed byion of protein and DAPI staining in panel d) asserts the nuclear transportation of theation of the references to colour in this figure legend, the reader is referred to the web

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between the bolaamphiphile and the delivered proteins viahydrogen bonding, van der Waals forces and hydrophobicinteractions.

The factors that are commonly used in iPSC generation can begrouped into 2 groups as Yamanaka factors (developed in Yama-naka’s lab) and Thomson factors (developed in Thomson’s lab).Here we have used recombinant proteins of Thomson factors, butwe have replaced OCT3/4 with orphan nuclear receptor (NR5A2)instead. Although OCT3/4 is an essential component of thereprogramming cocktail, NR5A2 acts as an upstream regulator forOCT3/4, and has been shown to replace the necessity of OCT3/4[35]. The four reprogramming factors that we chose include KLF4,Nanog, NR5A2 and Sox2. These transcription factors are known totransform fibroblasts to iPSCs when introduced as the respectivegenes [35]. We tested the delivery of these proteins into humanfibroblasts. The proteins were complexed with cationic bolaam-phiphile individually and incubated with fibroblasts for 3 h. Thecells were washed, fixed and examined with respective antibodies.By immunostaining, the proteins could be detected in the nucleusof the fibroblasts, indicating that the proteins were released fromthe complex and transported to their native compartment i.e. nu-cleus (Fig. 2). Further orthogonal sections of the z-stacks of imagesobtained from confocal microscopy suggest the presence of theproteins in the nuclear compartment (Fig. S2A). Identification of thetransfected proteins in the nucleus supports the concept that theseproteins are able to dissociate themselves from the polymere

Fig. 3. Generation of iPSCs using recombinant proteins. The recombinant proteins were coschematic illustration of the pro-iPSC generation. Briefly, human fibroblasts were transducedThe positive colonies were isolated and transferred to feeder layer (MEF). The cells were fur(b) The colonies expanded on feeder layer were immunostained with pluripotent markercolonies identified as pro-iPSCs. Scale bar ¼ 200 mm.

protein complex by amechanismwhich has yet to be identified. It iswell known that the reprogramming factors are transcription fac-tors that bind to the DNA of host cells to trigger the stem cellspecific transcription network. Initiation of the transcription ma-chinery and network is sufficient to activate the pathway towardsthe generation of iPSCs. For the successful generation of the iPSCs, itis understood that the transfected proteins should be present in thenuclear compartment. We analyzed the cytosol and nuclear com-partments of the transfected cells for the presence of proteins after6 days of transfection. Dot-blot analysis of the cytosol and nuclearextracts with specific antibodies showed that the proteins wereindeed present for 6 days after transfection in the nucleus(Fig. S2B). Additionally the amounts of the proteins transfectedwere similar to the expression level of these proteins in hESCs.However, transfection of SOX2 indicated a slightly lower efficiencyof transfection (Fig. S2C).

3.3. Reprogramming, colony formation and expansion

Human foreskin fibroblast of low passage number (passage 2)was used for the generation of pro-iPSCs. The proteinswere allowedto complex with the cationic bolaamphiphile and were incubatedwith the fibroblasts as mentioned earlier. The cells were cultured inthe MEF-conditioned media. The morphological changes of thehuman fibroblasts after the delivery of the reprogramming proteinscan be observed between day 3 and day 5. The morphological

mplexed with the cationic bolaamphiphile and transduced to human fibroblasts. (a) Awith reprogramming factors for 3 times and allowed to transform to iPSCs for 20 days.ther expanded on conventional hES qualified Matrigel and grown with mTeSR1 media.s. The antibody markers suggested the presence of immuno-positive cells within the

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changes include shrinkage of the cells towards epithelialmorphology. However we were able to identify the emergence ofcolonies between 15 and 20 days of protein transduction. Controlfibroblasts cultured in the presence of MEF-conditioned mediaremained as fibroblasts with fibroblast morphology, and we did notobserve any colonies (Fig. S3). We picked three colonies from threedifferent plates and subcultured themonMEF layer. Initial growthofthe colonieswas not observed to be robust, however the subsequentsubcultures onMatrigel and culturewith commercial growthmedia(mTeSR1, Stem cell technologies) (Fig. 3a) showed an increasedproliferation rate, similar to that of hESCs (doubling time w22 h).

The iPSCs generated by the viral method contain large segmentsof integrated viral vectors which could bring unpredictable geneticmalfunction [34]. Ourmethod of using recombinant proteins for thegeneration of iPSCs largely eliminates the risk of genomic integra-tion and insertion associated mutagenesis. The kinetics and effi-ciency of iPSC generation fluctuates with many factors orconditions. The virus based system can generate iPSCs at an effi-ciency of 0.04%, while efficiency of RNA based iPSCs can reach up to1.4% [16]. Apart from this, oxygen was shown to play an important

Fig. 4. The pro-iPSCs were further characterized by FACS to determine the percentage of cefeeder layers with antibodies specific for OCT3/4, Nanog and SSEA4. The percentage of posiplates with media containing 20% fetal bovine serum. Total RNA was extracted from the embThe expression level was compared with undifferentiated pro-iPSCs using a scatter plotmesoderm) were identified to be up-regulated while stemness specific genes were down-regsubcutaneously into immune-compromised mice (SCID) to perform teratoma assay. The explas blood vessel (i), chondrocytes (ii), glandular tissue (iii), neural rosettes (iv), muscle (v) a

role in the generation of iPSCs. Low oxygen (5%) was shown toinduce reprogramming at an efficiency of up to 2.28% [36]. In thecase of the virus based system, the initial colony was observed after24 days of reprogramming. However RNA based methods showed afaster kinetics in reprogramming; the colonieswere noticed after 16days of reprogramming. The protocol described here shares thesimilar kinetics as that of the RNA based system. However, the ef-ficiency of the reprogramming resembles that of the virus system(0.05%).

3.4. Characterization of pro-iPSCs

The cells expanded from pro-iPSCs showed typical colony for-mation, which is characteristic of embryonic stem cells. The pro-iPSCs were further characterized by immunostaining for stem cellspecific proteins such as Nanog, OCT3/4, Sox2, TRA1-60 and TRA1-81. Antibodies specific to these marker proteins were able to stainpositively the pro-iPSC colonies. Immunostaining results withNanog, Sox2 and OCT3/4 antibodies indicated that these proteinswere localized in the nuclear compartment. The expressions of

lls positive for pluripotent antibody markers. FACS was carried out for cells grown ontive cells varied from 78% to 85% (a). The cells were plated onto ultra low attachmentryoid bodies and real time gene expression analysis for various genes was carried out.analysis (b). Genes related to three different germ layers (ectoderm, endoderm andulated in embryoid bodies. (c) Five million cells were mixed with Matrigel and injectedant was sectioned and H&E staining was carried out to identify different cell types suchnd skin (vi). Scale bar ¼ 100 mm.

M. Khan et al. / Biomaterials 34 (2013) 5336e53435342

these proteins were restricted to the colonies that appearedmorphologically similar to stem cells (Fig. 3b). Alkaline phospha-tase has been established as a marker for embryonic stem cells, andexpression of high level of alkaline phosphatase is directly corre-lated to the pluripotent state of the cells. We tested the presence ofalkaline phosphatase in the colonies grown on MEF layer. We wereable to observe a typical staining pattern restricted to the stem cellcolonies (Fig. S4A). A brief gene expression analysis with genespecific primers for Nanog and OCT3/4 further supports the plu-ripotency of the induced stem cells (Fig. S4B). Real-time PCRanalysis of the expression of pluripotency marker genes among thecolonies did not show significant variations. The expression ofNanog, OCT3/4, Sox2 and SSEA4 showed similar expression level (%GAPDH) (Fig. S5A). Next, when we analyzed the expression levelbetween hESCs, viral-iPSCs and Pro-iPSCs, we noticed an aberrantexpression of the OCT3/4 gene for viral-iPSCs compared to thehESCs. On the other hand, the expression levels of Nanog and OCT3/4 genes between hESCs (HUES-7) and Pro-iPSCs were similar(Fig. S5B). Further characterization of the Pro-iPSCs by flowcytometry with antibodies against OCT3/4, Nanog and SSEA4revealed that approximately 80% of the cells were positive for theseantibodies (Fig. 4a).

3.5. Pluripotency assay of the Pro-iPSCs in vitro and in vivo

The most incredible feature of the stem cell is the inherentproperty to differentiate into different cell types. This property ofthe stem cell can be assayed both in vitro and in vivo. The in vitrodifferentiation potential of the Pro-iPSCs was tested by embryoidbody formation, followed by the detection of various cell typespecific genes using PCR. The embryoid body was formed asdescribed in the methods (Fig. S6). Real time PCR analysis of thegenes specific to stemness, ectoderm, mesoderm and endodermwere carried out using the PCR Array system.We analyzed 90 genesrelated to pluripotency and differentiation. The expression levels ofgenes between the embryoid body and the undifferentiated cells

Fig. 5. Directed differentiation of pro-iPSCs to neural lineage. The pro-iPSC clusters were groclusters were plated onto poly-D-lysine laminin plates and cultured in the presence of RA. TDAPI was used to stain the nucleus of the cells (iii). Merged images (iv) Scale bar ¼ 200 mm

were represented in a scatter plot. The genes associated withectoderm, mesoderm and endoderm were up-regulated in theembryoid body. Meanwhile, the pluripotent marker genes such asNanog, Sox2 and OCT3/4 were down-regulated in the embryoidbody (Fig. 4b). This comprehensive gene expression analysis sug-gested that the Pro-iPSCs can differentiate into the three germlayers. Next, we tested the in vivo potential of the Pro-iPSCs informing teratoma. After 6 weeks the explants were fixed withformaldehyde and sectioned. The H&E staining of the sectionsshowed the presence of multiple cell types which originated fromthe three germ layers. We were able to observe the presence ofblood vessels, chondrocytes, glandular tissues, neural rosettes,muscle and skin (Fig. 4c). The in vivo results further supports theearlier observation made in vitro that the Pro-iPSCs can developinto 3 germ layers.

3.6. Directed differentiation of Pro-iPSCs to neural lineage

We demonstrated the ability of the Pro-iPSCs to form multipletissue types by EB and teratoma assays. However, the ability toachieve controlled differentiation towards a specific lineagewould further strengthen the scope of potential applications forthese cells. It is inherent for the stem cells to differentiate tocells of multiple phenotypes, however, guiding the stem cellsto differentiate to a specific lineage following developmentalpathways is challenging. We utilized an existing 2-step differ-entiation strategy and demonstrated that the Pro-iPSCs canpotentially differentiate into neural lineage cells. The Pro-iPSCswere first differentiated into neurospheres, followed by termi-nal differentiation into neurons in the presence of retinoic acidon poly-lysine and laminin coated dishes. Light microscopic ob-servations suggested typical morphology of neural cells with longfilamentous cellular extensions (Fig. S7). Immunostaining of thedifferentiated colonies with nestin and beta III tubulin antibodiessuggested the expression of these proteins in the differentiatedcells (Fig. 5).

wn on ultra low attachment plates as described in the methods section for 20 days. Thehe cells were immunostained with antibodies against nestin (i) and beta III tubulin (ii)..

M. Khan et al. / Biomaterials 34 (2013) 5336e5343 5343

4. Conclusion

Proteins, especially transcription factors, can modulate the geneexpression of the host cells leading to complete transformation ofthe parental phenotype. Delivery of protein into cells has immenseapplication in various fields of research and therapeutics. Here wehave demonstrated that a simple cationic bolaamphiphile derivedfrom 1,12-diaminododecane can deliver proteins of different char-acteristics into human fibroblasts. We characterized and optimizedthe protein delivery system reported in this manuscript. The nu-clear localization of the transduced proteins indicates that theproteins dissociate by a mechanism which has yet to be identified.We have shown in this manuscript a single application of gener-ating human iPSCs via delivery of 4 transcription factors, using acationic bolaamphiphile as a non-viral based carrier. The iPSCsgenerated by this methodology were shown to perform similarly tohuman embryonic stem cells or iPSCs generated by other methods.The morphological appearance, colony formation, proliferation, theactivation of endogenous pluripotency genes, in vitro differentia-tion potential and the ability to form teratomas are some of thebasic criteria required for a cell line to be considered as iPSCs. Wehave shown all these assays to be positive for the reprogrammedcells reported in this manuscript.

Acknowledgments

We would like to thank members of A.C.A.W. and Y.Y.Y. labs fortheir technical assistance and helpful discussions. Authors wouldlike to thank Dr Steve Oh (Bioprocessing Technology Institute,Singapore) for the generous gift of iPSCs developed using viralvectors. Funding was provided by the Institute of Bioengineeringand Nanotechnology (Biomedical Research Council, Agency forScience, Technology and Research, Singapore).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2013.03.072.

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