Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis

ORIGINAL ARTICLE Embryology

Inhibition of glycogen synthase kinase-3promotes efficient derivation ofpluripotent stem cells from neonatalmouse testisSeyedeh-Faezeh Moraveji1,2,†, Farnoosh Attari3,†,Abdolhossein Shahverdi1,4, Houri Sepehri3, Ali Farrokhi1,Seyedeh-Nafiseh Hassani1, Hananeh Fonoudi1, Nasser Aghdami1,and Hossein Baharvand1,2,*1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology,ACECR, P.O. Box 19395-4644, Tehran, Iran 2Department of Developmental Biology, University of Science and Culture, ACECR, Tehran,Iran 3Department of Animal Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran 4Department of Embryology,Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

*Correspondence address. Tel: +98-21-22306485; Fax: +98-21-23562507; E-mail: [email protected]

Submitted on January 23, 2012; resubmitted on April 27, 2012; accepted on May 14, 2012

background: Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newbornand adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better androbust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germ-line-derived pluripotent stem cells (gPSCs).

methods: We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stemcells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence orpresence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches.

results: The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogensynthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotencyof the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formationof chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chem-ically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3.

conclusions: To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely byinhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for geneticmodification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naı̈vegPSCs lines in other mammals.

Key words: Mouse testis / germline-derived pluripotent stem cells / CHIR99021 / glycogen synthase kinase-3 / extracellular signal-regu-lated kinases

† These authors contributed equally to this work.

& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]

Human Reproduction, Vol.0, No.0 pp. 1–13, 2012

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IntroductionEmbryonic and induced pluripotent stem cells (ESCs and iPSCs) haveextensive capacity for proliferation, self-renewal and differentiation.Therefore, they provide a pluripotent source for a variety of applica-tions including developmental biology, drug screening, toxicology,disease research and cell-based therapy. However, standardmethods for derivation of ESCs, or patient- or disease-specific iPSClines, result in limitations in their possible biomedical applicationsbecause of ethical and immunological concerns. Some key advanceshave been achieved for the production of safe iPSCs including theuse of non-integrating viruses (such as adenovirus or episomalplasmid transfection), which are known to be stable in mammaliancells for extended periods without integrating into the genome (Stadt-feld et al., 2008; Zhou and Freed, 2009; Nishimura et al., 2010), treat-ment of cells with cell-penetrating recombinant reprogramming factorproteins (Kim et al., 2009), transposon-based systems (Woltjen et al.,2009) and the delivery of reprogramming factors in plasmids (Okitaet al., 2008; Yu et al., 2009; Si-Tayeb et al., 2010), repeated transfec-tion with modified mRNA encoding reprogramming factors (Warrenet al., 2010) and micro RNAs (Lin et al., 2011; Tian et al., 2011).However problems exist, such as low reprogramming efficiency,genomic alterations and immunogenicity (for review see Seifinejadet al., 2010; Stadtfeld and Hochedlinger, 2010; Gonzalez et al.,2011). Recently, it was reported that iPSCs could elicit an immune re-action in mice (Zhao et al., 2011). It has been demonstrated that thetransplantation of ESCs led to teratomas, whereas most of the iPSCswere not able to form teratomas, or produced teratomas that wereattacked or rejected by the immune systems of the host syngeneicmice. This immunogenicity of iPSC derivatives may relate to epigeneticdifferences between iPSCs and ESCs, and/or mutations in the codingsequences of iPSCs (Marchetto et al., 2009; Ji et al., 2010; Kim et al.,2010; Polo et al., 2010; Gore et al., 2011; Lister et al., 2011) could giverise to the ectopic expression of minor antigens.

In order to reach the final goal of clinical applications, it is necessaryto develop technologies to overcome iPSC limitations or search forother sources of PSCs that have minimal ethical and immunologicalconcerns.

Although recent reports (Conrad et al., 2008; Golestaneh et al.,2009; Kossack et al., 2009; Mizrak et al., 2010) claimed the derivationof multipotent or ESC-like cells from adult human testicular testis byexposing human testicular cells to specific ESC conditions in vitro,the pluripotency of these cells has been questioned (Ko et al., 2010;Tapia et al., 2011).

Studies have demonstrated that both newborn and adult male murinetesticular cells undergo a ‘self-reprogramming’ or spontaneousconversion process into multipotent stem cells or pluripotent embryon-ic stem (ES)-like cells in the absence of genetic manipulation when thesecells are cultured in vitro for an extended time (Kanatsu-Shinohara et al.,2004; Guan et al., 2006; Seandel et al., 2007; Izadyar et al., 2008;Kanatsu-Shinohara et al., 2008; Ko et al., 2009). These mice ESC-likespermatogonia-derived stem cells were termed multipotent germlinestem cells, multipotent adult germline stem cells, multipotent adultspermatogonia-derived stem cells or germline-derived pluripotentstem cells (gPSCs) according to different reports. The gPSCs resemblednaı̈ve mouse ESCs (mESCs) in terms of morphology, continual passagingwithout a decline in colony-forming ability or change in karyotype

(de Rooij and Mizrak, 2008), spontaneous differentiation into derivativesof the three germ layers, their contribution to chimeras after injectioninto blastocysts (Kanatsu-Shinohara et al., 2004; Guan et al., 2006;Seandel et al., 2007) or germ-line transmission (Ko et al., 2009).gPSCs can be propagated in the medium that contains serum and leuke-mia inhibitory factor (LIF), and have been induced to directly differenti-ate into functional hepatocytes, cardiomyocytes, neurons and glial cellsin vitro (Baba et al., 2007; Guan et al., 2007; Streckfuss-Bomeke et al.,2009; Fagoonee et al., 2010). Thus, gPSCs could differentiate into therequired cell type and be transplanted back to the autologous patientwithout ethical or immunological problems, or they can be geneticallycorrected for using in regenerative medical therapies (Tapia et al., 2011).

However, gPSCs have been generated with very low efficiency andafter several weeks following testicular culture. Small molecules (SMs)may offer one possible solution to this challenge (for review see Fenget al., 2009; Li and Ding, 2010; Efe and Ding, 2011; Lyssiotis et al.,2011; Yuan et al., 2011). SMs can reversibly perturb specific functionsof a single protein (or multiple proteins) with exquisite temporalcontrol in the absence of genetic modification(s). Recently, severalreports have demonstrated the efficient and reproducible generationof ESCs from mouse strains previously considered to be refractoryand non-permissive to ESC establishment, such as BALB/c, C57BL/6,DBA/2, NMRI, FVB/N, NOD (Buehr and Smith, 2003; Umeharaet al., 2007; Hanna et al., 2009; Nichols et al., 2009a; Sato et al.,2009; Gertsenstein et al., 2010; Kiyonari et al., 2010; Wray et al.,2010; Hassani et al., 2011) and from rat (Buehr et al., 2008; Li et al.,2008; Kawamata and Ochiya, 2010; Leitch et al., 2010) and iPSCs pro-duction (Feng et al., 2009; Nichols et al., 2009b; Li and Ding, 2010; Wrayet al., 2010; Efe and Ding, 2011; Hassani et al., 2011; Lyssiotis et al.,2011; Yuan et al., 2011) by treatment with SM(s).

In this study, we introduce a simple and efficient protocol for thegeneration of gPSCs from neonatal mouse testis in conventionalmESC medium supplemented with SMs and without genetic manipu-lation. Thus far, no report has shown the reprogramming of somaticcells using just one SM. Additionally, we have demonstrated thatgPSCs could propagate and maintain their pluripotency under feeder-free defined conditions in a chemically defined N2B27 supplementedmedium, as previously reported for mESCs (Ying et al., 2008). Ourmethod helps to overcome the problems of gPSC establishment andclarifies a new signaling pathway involved in the generation of gPSCsfrom different mouse strains.

Materials and Methods

Isolation of testicular cells and primaryculture conditionAll animal care was in accordance with the approval of the Royan Institu-tional Review Board and Institutional Ethical Committee. Testicular cellswere isolated from neonatal male mice (3 day old, NMRI and C57BL/6strains, Pasteur Institute, Tehran, Iran). Embryonic day (E) 12.5–13.5fetuses (NMRI strain) were used to produce mouse embryonic fibroblasts(MEFs) as feeder cells.

Isolation of testicular cells was performed as previously described(Izadyar et al., 2008) with slight modification. Briefly, the bilateral testesof 5–10 neonatal mice were collected in phosphate-buffered saline(PBS), placed on ice and then transferred to the laboratory within15 min of decapsulation. Seminiferous tubules were placed in a dish that

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contained knockout Dulbecco’s modified Eagle’s medium (Ko-DMEM,Invitrogen) and separated from one another by pipetting for 1 min. Theseparated tubules were collected by centrifugation at 500g for 2 min,then suspended in Ko-DMEM supplemented with collagenase, dispaseand hyaluronidase (each at 1 mg/ml; all enzymes purchased fromSigma-Aldrich) for 10 min with incubation at 378C (first enzyme digestionstep). After a second centrifugation (500g for 2 min), in which the majorityof interstitial cells and blood vessels were removed, a second digestionstep was performed in Ko-DMEM by adding fresh enzymes to the semin-iferous cord fragments. After filtration through a 70-mm nylon filter, iso-lated cells were replated (2.5 × 105 cells/per gelatin-coated center-wellplate, 2 cm2, Falcon) at 378C in an atmosphere of 5% CO2 in air inKo-DMEM supplemented with L-glutamine (2 mM, Invitrogen), non-essential amino acids (NEAAs, 1%, Invitrogen), penicillin (100 U/ml, Invi-trogen), streptomycin (100 mg/ml, Invitrogen), b-mercaptoethanol(0.1 mM, Sigma-Aldrich) and 2% fetal calf serum (FCS, HyClone).

One day after plating, most testicular cells were attached to the gelatin-coated surface. The medium was replaced with the same mediumsupplemented with 1% FCS, glial-derived neurotrophic factor (GDNF,20 ng/ml, Sigma-Aldrich), mouse epidermal growth factor (EGF, 20 ng/ml, Sigma-Aldrich), human basic fibroblast growth factor (bFGF, 10 ng/ml;Royan Institute) and mouse LIF (1000 U/ml; Millipore) in the absence(control) or presence of SM. The culture medium was changed everyother day.

We applied a list of selected SMs based on previous reports that haveshown enhancement of generation of ESCs or iPSCs (Feng et al., 2009;Nichols et al., 2009b; Li and Ding, 2010; Wray et al., 2010; Efe andDing, 2011; Hassani et al., 2011; Lyssiotis et al., 2011; Yuan et al.,2011), including PD0325901 [PD, 1 mM, extracellular signal-regulatedkinases (Erk1/2) Inhibitor], pifithrin a (10 mM, P53 inhibitor), pluripotin(0.3 mM, Ras/ERK1/2 inhibitor and phosphoinositide-3-kinase activator),CHIR99021 (CHIR, 3 mM, glycogen synthase kinase-3 (GSK-3) inhibitor),tranylcypromin (2 mM, lysine-specific demethylase 1 inhibitor), reversine[5 mM; Sigma-Aldrich, mitogen/extracellular-regulated kinase inhibitorand PI3K activator], PD (1 mM) + SB431542 [SB, 10 mM; Sigma-Aldrich,transforming growth factor-b (TGF-b) inhibitor] and bisperoxovanadium[bpV, 2 mM, Axxora, phosphatase and tensin homolog deleted onchromosome 10 (PTEN) inhibitor]. All other materials were purchasedfrom Stemgent.

After 5 days, we observed sharp-edged, round- or domed-shaped col-onies that morphologically resembled mESC colonies on the top of themonolayer of testicular cells. At this step, to expand ES-like cells, theESC-like colonies were isolated mechanically using a thin Pasteur pipetteand replated on a 24-well dish that included fresh mitotically arrestedMEF in the mESC medium in the absence or presence of SMs. MouseESC medium contained the same components in addition to 15% FCSand 1000 U/ml LIF. After 2 days, the colonies were dissociated withtrypsin/EDTA (0.05% w/v, Invitrogen) and plated into 12-well disheswhich included fresh mitotically arrested MEF, and cultured for six add-itional days in the mESC medium. Afterwards, SM(s) were omitted. Celllines were subsequently passaged every 2–3 days. The mESC mediumwas renewed daily.

Alkaline phosphatase andimmunofluorescence stainingAlkaline phosphatase (AP) staining was performed using a kit(Sigma-Aldrich) according to the manufacturer’s recommendations.Mouse ESCs were used as a positive control. For immunofluorescence stain-ing, undifferentiated germ line-derived ESC-like colonies were fixed in 4%paraformaldehyde in PBS, pH 7.4 (Invitrogen), for 20 min. Cells werewashed twice with 0.1% Tween-20 in PBS to remove residual fixative and

permeabilized with 0.2% Triton X-100 in PBS for 20 min prior to blockingin 10% normal goat serum in PBS for 60 min followed by incubation withprimary antibody solution overnight at 48C, or for 1 h at 378C. Theprimary antibodies used in this study were Oct-4 (1:50; Santa Cruz Biotech-nology, SC-5279), Nanog (1:100; Santa Cruz Biotechnology, SC-30329) andstage-specific embryonic antigen-1 (SSEA-1; 1:50, R&D, MAB2155) for de-termination of undifferentiated state, and Gata4 (1:200; Santa Cruz,SC-1237), Mef2c (1:200; Abcam, ab64644), a-Mhc (1:200; Abcam,Ab15), Tuj1 (1:500; Sigma-Aldrich, T-8660), Map2 (1:200; Sigma-Aldrich)Tbx5 (1:200; Santa cruz, SC-48782) and FoxA2 (1:100; Abcam, ab60721)for differentiated cells. Then, cells were washed twice with 0.1%Tween-20 in PBS for 5 min and incubated with the appropriate secondaryantibody in PBS. Fluorescence-conjugated secondary antibodies, goat anti-mouse immunoglobulin (Ig)G fluorescein isothiocyanate (FITC, 1:200;Sigma, F9006), mouse anti-goat IgG phycoerythrin (1:200, Santa Cruz,SC-3725), rabbit anti-mouse IgG Texas red (1:400; Jakson Lab,315-075-003), rabbit anti-Goat IgG FITC (1:200; Sigma-Aldrich, F7367)and goat anti-rabbit IgG FITC (1:200; Sigma-Aldrich, F1262) were used, asappropriate, for 1 h at 378C. After two washes with PBS + 0.1% Tween20 for 5 min, cells were counterstained with 4′,6-diamidino-2-phenylindole(DAPI; Sigma-Aldrich) and analyzed with a fluorescent microscope(Olympus). Immunostaining without primary antibodies was also used as anegative control for the cells (Supplementary data, Figs S1–S3).

RNA isolation, reverse transcriptionand quantitative RT–PCRTotal RNA was isolated using TRIzolw reagent (Invitrogen) according tothe manufacturer’s protocol. To remove genomic DNA contamination,all RNA samples were subjected to DNase I (EN0521, Fermentas)treatment.

cDNA synthesis was performed using the RevertAidTM H Minus FirstStrand cDNA Synthesis Kit (K1632, Fermentas), 0.2 mg randomhexamer primer and 1 mg total RNA per reaction, according to the man-ufacturer’s instructions. For every reaction set, one RNA sample wasincluded without the addition of reverse transcriptase to provide a noRT control (RT2 reaction) as a negative control in the subsequent PCR.

Gene expression was assessed by quantitative RT–PCR for candidategenes in a Rotor-Gene 6000 (Corbett Life Science) using the followingprogram, stage 1: 958C for 10 min, stage 2: 958C for 10 s, 608C for20 s and 728C for 30 s, for 40 cycles. At the end of the run, a meltingprofile was determined to demonstrate the synthesis of a single PCRproduct. The primers were designed using the primer design software,Perlprimer (Marshall, 2004). Primer sequences, expected fragment sizeand Gene Bank accession numbers are listed in Supplementary data,Table S1.

The PCR mix in each well included 10 ml of SYBRwPremix Ex TaqTM II(RR081Q, Takara Bio., Inc.), 6 ml dH2O, 1 ml each of the forward andreverse primers (5 pmol/ml), 2 ml of single-strand cDNA (16 ng/ml) in afinal reaction volume of 20 ml. Each experiment included at least three bio-logical replicates and each replicate was analyzed in duplicate.

For relative quantification, a standard curve was generated in every in-dividual run using a serial dilution of the pool of cDNA samples (50, 10, 2,0.4 and 0.08 ng) with high expression values.

Data were analyzed using the relative standard curve method. Theoutput data from Rotor-Gene 6000 Analysis software (version 1.7;Corbett Life Science) were transferred to Microsoft Excel for analysis.For each unknown sample, the relative amount was calculated by normal-ization of each target gene to the geometric mean of two reference genes,B2m and beta Tubulin, and then calibrated against a control group (day 0testicular cells).

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Spontaneous and direct differentiationof gPSCs into cardiomyocytes, endodermaland neuronal cells in vitroFor demonstrating the pluripotency of ESC-like lines, spontaneous differ-entiation was done by embryoid body (EB) formation in low attachmentbacterial plates (Grainer, Germany; 628–102) for 6 days; EBs werefurther plated for an additional 5 days on gelatin-coated tissue cultureplates in the mESC medium without LIF.

For direct cardiomyocyte differentiation (Moghadasali et al., 2007; Far-okhpour et al., 2009), 800 gPSCs were cultured for 2 days in 20 ml of ESCmedium that contained 1024 M ascorbic acid (vitamin C; Sigma-Aldrich,A4403) in the absence of LIF in hanging drops to produce EBs. Subse-quently, EBs were cultured as suspensions in bacterial dishes for 5 add-itional days. On Day 7, EBs were plated separately in 1% gelatin-coatedwells of a 24-well tissue culture plate for an additional 14 days to allowadherence and development of beating cardiomyocytes. In order toconfirm the functionality of gPSCs-derived cardiomyocytes, the effect oftreatment with diltiazem, a calcium channel blocker, on cells was evalu-ated. Diltiazem (1025 M; Sigma-Aldrich, D2521) was added to beatingEBs for 2–3 min. Subsequently, the number of beats per minute wascounted and compared with the number of beats per minute beforetreatment.

In order to promote directed differentiation of gPSCs into a neural fate(Moghadasali et al., 2007; Farokhpour et al., 2009), EBs were produced byculturing 105 gPSCs in a 3 ml medium in non-adhesive bacterial dishes thatcontained ESC medium without LIF. Subsequently, EBs were cultured insuspension in the presence of retinoic acid (1 mM; Sigma-Aldrich,R2625) in the same medium. At Day 6, the EBs were plated ontopoly-L-lysine-coated dishes in ESC medium supplemented with 5% FCSfor 5 days, for further differentiation of precursor cells into matureneurons.

To induce endodermal cell differentiation, 4 day EBs were plated ingelatin-coated dishes and were treated with Activin A (50 ng/ml; R&D,338-AC) in the mESC medium without LIF and serum supplementedwith 1% N2 supplement (Invitrogen) and 2% B27 supplement (Invitrogen)for a further 6 days. The medium was changed every 2 days.

Production of chimeric miceTo examine the differentiation potential of ES-like cells in vivo, chimeraswere generated. Briefly, 3.5 day post coitus blastocysts were collectedfrom superovulated female BALB/c mice and placed in the M2 medium.Then, 10–15 single-cell ESC-like colonies were injected into each blasto-cyst. Approximately 10 injected blastocysts were transferred into theuterine horns of pseudo-pregnant BALB/c × C57BL/6 F1 mice. Chimeraswere identified by coat color.

Culture of gPSCs under defined conditionsFor expansion of gPSCs under defined conditions, MEF was replaced bygelatin (0.1%; Sigma-Aldrich), and N2B27-supplemented medium wasused instead of conventional mESC medium. Serum-free N2B27-supple-mented medium (100 ml) contained the following: 45 ml DMEM/Ham’sF12 (DMEM/F12, Invitrogen), 45 ml neurobasal (Invitrogen), 1 ml N2 sup-plement, 2 ml B27 supplement, 2 mM L-glutamine, 1% NEAAs, penicillin(100 U/ml), streptomycin (100 mg/ml), 0.1 mM b-mercaptoethanol,1000 U/ml mouse LIF, 5 mg/ml bovine serum albumin (Sigma-Aldrich),an inhibitor of the Erk1/2 cascade (Ying et al., 2008), PD0325901 (PD,1 mM; Stemgent) and CHIR (3 mM).

Statistical analysisThe experiment of gPSC generation was repeated more than 10 times. Allother experiments were conducted in at least three independent cultures.Real-time data were expressed as mean+ SD and analyzed with one-wayanalysis of variance followed by the post hoc Tukey honest significant differ-ence test for multiple comparisons. P-values , 0.05 were considered sig-nificant. The data for the number of ESC-like colonies at Day 6 andtreatment with diltiazem were analyzed by Student’s t-test.

Results

Screening of SMs to generate gPSCs fromtesticular cellsIn initial attempts to derive gPSCs, we obtained testes from neonatalNMRI mice and produced cell suspensions by enzymatic digestion. Wethen plated isolated cells on gelatin-coated dishes in the Ko-DMEMmedium supplemented with 2% FCS for 1 day. The experimentalscheme for establishment of gPSCs used in the current study isillustrated in Fig. 1A.

Then, we explored SM to induce the derivation of mESC-like col-onies in Ko-DMEM medium supplemented with GDNF, EGF, bFGF,LIF, FCS (1%) and in the absence (control) or presence of SMs for5 days. At Day 6 post-digestion mESC-like colonies appeared in theCHIR group, with a packed spindle- to round-shaped morphologywith smooth borders. In the experiments including the CHIR group,all colonies were mESC like. After plating 250 000 testicular cells,164.1+29.9 ESC-like colonies were observed at Day 6. Therefore,the percentage of ESC-like colony formation in the CHIR group was0.065%. The remaining SMs or their combinations did not result inthe formation of mESC-like colonies after 6 days or later underthese conditions but produced cell aggregates which were notexpanded (Fig. 1B and Supplementary data, Fig. S4).

Subsequently, the mESC-like colonies were transferred onto freshfeeder layers of mitotically arrested MEF in the mESC medium inthe presence of CHIR (Fig. 1B). Then, some colonies were pickedup from the plate by a thin Pasteur pipette under stereomicroscopeand replated on fresh MEF in the mESC culture medium for 2 addition-al days (Day 8); growth factors were eliminated. We found this pro-cedure influenced the generation of gPSCs in comparison withtrypsinization of whole ESC-like colonies at Day 6. At Day 8, cellswere dissociated into single cells with trypsin/EDTA and replatedinto 12-well plates, including MEF. Following an additional 6 days ofculture, typical mESC-like colonies could be identified, which we con-sidered them to be passage one. The mESC-like colonies expanded asdome shaped and compact colonies, with high nuclear–cytoplasmicratios. Under the phase contrast microscope it was difficult to distin-guish the individual cells in the mESC-like colonies, although nucleicould be recognized in some cells and contained one to three darkprominent nucleoli (Fig. 1B). Daily observation showed that the col-onies continued to increase in number and size by proliferationwithout differentiation. After this step, CHIR was omitted from theESC medium. Cell lines were then propagated by enzymatic subcultureevery 2–3 days with a doubling rate comparable to mESCs. The mESCmedium was changed daily. We designated these to be putative gPSCcolonies.

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Figure 1 The process of establishing mouse gPSCs. (A) Schematic of the derivation of gPSCs. Three-day derived testicular cells were plated ongelatin-coated plates in the Ko-DMEM medium supplemented with 2% FCS. The next day, the medium was replaced by a cocktail of growthfactors GEFL ( = GDNF + EGF + bFGF + LIF) plus FCS (1%), in the absence (control) or presence of SM. At Day 6, the ESC-like colonies were isolatedmechanically and replated on a mitotically arrested MEFs feeder layer in the mESC medium. After 2 days, colonies were passaged by trypsin/EDTAtreatment and plated on a fresh MEF feeder layer in the same conditions. Typical mESC-like colonies were observed after 4–6 days. These colonieswere passaged on Day 14 in the absence of SM (passage 1, P1). (B) Phase contrast microscopy of cultured cells during generation and passaging ofgPSCs. In the absence of SM (control) we only observed fibroblast-like aggregates which did not produce gPSCs after passaging in these conditions.However, in the presence of CHIR99021 (CHIR), a GSK-3 inhibitor, mESC-like colonies, with packed spindle to round morphology and smoothborders, appeared at Day 6. After passaging, typical mESC-like colonies were observed at Day 14. These colonies could be passaged every 2–3days. The results were also similar regardless of genetic background in both outbred NMRI and inbred C57BL/6 strains. The lines were referredto as RNn/gPSC and RBn/gPSC, which were derived from neonatal male NMRI and C57BL/6 strains, respectively (R: ‘Royan’ which means‘embryo’ in Persian; ‘N and ‘B’, generated from NMRI and C57BL/6 strains; and ‘n’ for ‘neonatal’).

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The same protocol was used to evaluate the universality of inhib-ition by GSK3 of the production of gPSC lines from another mousestrain, C57BL/6. Results were similar to those of the NMRI strain

(Fig. 1B). These results clearly demonstrated that mouse gPSC gener-ation was promoted by the inhibition of GSK-3 only, in two strains ofmice with different genetic backgrounds.

Figure 2 Characterization of established, undifferentiated gPSCs. (A) The expression of mESC-specific markers, AP and immunofluorescence stain-ing for Oct4, Nanog and SSEA1 in the gPSC lines established by CHIR after at least 20 passages. (B) Real-time RT–PCR analysis of pluripotencymarkers (Oct4 and Nanog) and the germ cell-specific marker (Dazl) in the gPSCs and mESC lines. Testicular cells (d0) and MEF were used as controls.The number of biological replicates was at least three. Data are mean+ SD. One-way ANOVA and Tukey test were used. aP , 0.05.

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The established lines were named RNn/gPSC and RBn/gPSC (R:‘Royan’ which means ‘embryo’ in Persian; ‘N and ‘B’, derived fromNMRI and C57BL/6 strains; and ‘n’ for ‘neonatal’).

The established cell lines were successfully cryopreserved andthawed with no loss in proliferation or differentiation capacities.

Characterization of undifferentiated gPSCsThe gPSC lines were passaged 1:3–1:6 for more than 20 times follow-ing trypsin digestion, with an estimated doubling time of 48 h. Thesecells maintained their undifferentiated state in multiple passages andshowed clonal growth from single cells. To examine the pluripotencyof generated gPSC lines, they were evaluated for mESC markers(Fig. 2). AP was highly expressed in gPSCs and the cells were stronglypositive for pluripotency markers Oct4, Nanog and SSEA-l (Fig. 2A).The expression of pluripotent markers, Oct4, Nanog and AP wasnot detected by immunofluorescence staining in non-SM-treatedcontrol cells (fibroblast-like aggregates; Supplementary data, Fig. S5).

We further examined the expression of Nanog and Oct4, pluripo-tency markers, and Dazl, a germ-line stem cell marker in establishedgPSCs by quantitative RT–PCR. We found that gPSCs expressedthe pluripotency genes Oct4, Nanog and the germ-cell specificmarker Dazl, all at a level similar to that in mESCs (Fig. 2B).

Similar to mESCs, gPSC lines (RN/ngPSC and RB/ngPSC) showedthe ability to differentiate spontaneously into derivatives of all threegerm layers by EB formation, as indicated by expression of the lineage-specific marker genes for ectoderm (Nestin, Map2, Mash1 and Tuj1),mesoderm (Gata4 and Brachyury) and endoderm (Afp, Sox17 andFoxA2), and after plating (Fig. 3A and B).

To further confirm the pluripotency of our gPSCs, we tested thecapacity of C57BL/6-derived gPSCs to contribute to chimeric mice.RBn/gPSCs were injected into host 3.5 day old blastocysts of theBALB/c strain. We transferred 155 injected blastocysts into theuteri of 19 pseudo-pregnant female mice. From 10 pregnant fostermothers, we obtained 32 live pups. The pups were of a similar sizeto a normal birth pup, with no abnormalities. Nine out of 32 pupswere chimeric and four out of nine chimeric pups were male andhighly chimeric but they did not show germ-line transmission (Fig. 3C).

Next, we investigated directed differentiation of the generatedRNn/gPSC line into cardiomyocytes and neuronal cells (Fig. 4A–C).The cells that differentiated into cardiomyocytes demonstratedbeating (Fig. 4A). Furthermore, negative chronotropic effects on cardi-omyocytes were observed after diltiazem treatment (Fig. 4B). Im-munofluorescent staining revealed the expression of muscle markersthat included a-Mhc, Mef2c, Gata4 and Tbx5 (Fig. 4C). RT–PCR ofdifferentiating cardiomyocytes showed the expression of cardiac-specific genes, including Mlc2a, Nkx2.5, Mlc-2v, cTNT and a-Actinin(data not shown). Endodermal cells expressed FoxA2 after activin Atreatment (Fig. 4D). Neurons were also generated by directed differ-entiation of gPSCs and were positive for Tuj1 and Map2 (Fig. 4E).Therefore, with the above in vitro and in vivo differentiation assays,our results have revealed that the gPSC lines produced by treatmentwith CHIR are pluripotent.

Expansion of gPSCs in chemically definedconditions, supplemented with PD 1 CHIRTo examine the expansion of gPSCs in defined conditions, we culturedgPSCs under feeder-free conditions in a chemically definedN2B27-supplemented medium with inhibition of both GSK-3 andErk1/2 by CHIR and PD, respectively (Ying et al., 2008). gPSCswere propagated in these culture conditions with no feeder celllayer or serum for at least 20 passages. gPSCs were differentiated inthe presence of CHIR only. However, we found that PD + CHIRalong with LIF replaced the requirement for feeder and serum, andsupported robust long-term gPSC propagation. The cells showed amorphology which was typical of mESCs, expression of pluripotencymarkers (Fig. 5) and differentiation by EB formation (Supplementarydata, Fig. S6).

DiscussionHere, we report that testicular cells isolated from neonatal mice canbe efficiently reprogrammed into PSCs under relatively simple mESCculture conditions. The rate of conversion of testicular cells into

Figure 3 Spontaneous differentiation of gPSCs in vitro and chimeraformation. (A) Spontaneous differentiation of RNn/gPSCs which pro-liferated on MEF by EB formation. (B) RT–PCR analyses of variousdifferentiation markers for the three germ layers (ectoderm, meso-derm and endoderm) during EB-mediated differentiation [6-day EBand 5-day post-plating (10d)]. The gPSCs were expanded in the pres-ence or absence of MEF, under defined conditions. B2M, internalcontrol; RT2, no reverse transcriptase controls. (C) Chimeras wereproduced from the RBn/gPSC line.

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Figure 4 Directed differentiation of gPSCs in vitro. (A) Directed differentiation of RNn/gPSCs into beating cardiomyocytes. (B) The differentiatedcardiomyocytes responded to diltiazem, a negative chronotropic drug. Data are mean+ SD. The Students t-test was used. aP , 0.05. (C) The differ-entiated cardiomyocytes expressed aMhc, Mef2c, Gata4 and Tbx5 as detected by immunofluorescence staining. Nuclei were stained with DAPI(blue). (D) Differentiated cells expressing FoxA2 after treatment with Activin A. Nuclei were stained with DAPI (blue). (E) Neuronal cells as detectedby phase contrast microscopy and immunofluorescence staining for Tuj1 and Map2. Nuclei were stained with DAPI (blue).

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gPSCs is 0.065% of the initially plated testicular cells (an average 164colonies per 250 000 seeded testicular cells).

These cells were reprogrammed without genetic modification andwithout enrichment for spermatogonial stem cells in culture. Specific-ally, application of CHIR reduced the time required to reprogram tes-ticular cells and increased the fraction of cells that became gPSCs. Theresultant cells shared many features with ESCs. All cell lines were

passaged at least 20 times and maintained ESC morphology, as indi-cated by light microscopy. All cell lines showed strong positive stainingfor AP activity. Additionally, they expressed SSEA1 and the pluripo-tency markers Oct4 and Nanog. The germ cell-specific gene Dazlshowed down-regulation. The cell lines produced EB-like structuresin suspension with the apparent potential to differentiate into deriva-tives of the three germ layers in vitro. Additionally, they could be

Figure 5 Proliferation and characterization of gPSCs in chemically defined conditions. gPSCs were expanded under a chemically definedN2B27-supplemented medium by the inhibition of both the mitogen-activated protein kinase (Erk1/2) and GSK-3 (PD0325901 + CHIR) in the pres-ence of LIF. gPSCs expressed the pluripotency markers Oct4, Nanog and SSEA1, and differentiated in vitro by EB formation (Fig. 3B and Supplementarydata, Fig. S6).

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stimulated to differentiate into cardiomyocytes and neurons, and con-tributed to chimera development. The new cell lines therefore appearto be pluripotent. However, their potential for teratoma formation invivo and their contribution to forming tissues of all three germ layers inthe chimeric mice remains to be demonstrated.

In previous studies (Kanatsu-Shinohara et al., 2004; Guan et al.,2006; Seandel et al., 2007; Izadyar et al., 2008; Kanatsu-Shinoharaet al., 2008; Ko et al., 2009) the generation of gPSCs was a rareand stochastic process, and was uncontrollable. Reprogramming oftesticular cells in our study by CHIR occurred over 6–14 days; thisphenomenon has been shown consistently to occur within 2–4weeks of purified germline stem cell culture (Ko et al., 2010). Add-itionally, our gPSCs maintained expansion and pluripotency in feederand serum-free conditions in the presence of CHIR + PD in combin-ation with LIF for an extended period (at least 10 passages) and sharedtypical features of mESCs. It has been reported that CHIR + PD incombination with LIF could replace serum and allow cultivation ofmESCs in a chemically defined medium (Ying et al., 2008).

Recently, it has been demonstrated that GSK-3 inhibitors (CHIRor BIO) have an impressive effect on mESC establishment usingstrains which were previously considered refractory and non-permissive (Buehr and Smith, 2003; Umehara et al., 2007; Hannaet al., 2009; Nichols et al., 2009a; Sato et al., 2009; Gertsensteinet al., 2010; Kiyonari et al., 2010; Wray et al., 2010; Hassaniet al., 2011). Additionally, it has been reported that CHIR, in com-bination with PD, A-83-01 and LIF, produced naı̈ve ESC-like ratiPSCs (Li et al., 2009a) or rat ESCs (Buehr et al., 2008; Li et al.,2008; Kawamata and Ochiya, 2010) capable of contributing to chi-merism. Moreover, it has been demonstrated that mouse and ratembryonic germ cells can be established with high efficiency usingCHIR in combination with PD0325901 and cytokine LIF by culturingprimordial germ cells from E8.5 to E12.5 mice and E10 rats (Leitchet al., 2010). It was also demonstrated that human pluripotent naivecells were derived from human ESCs by ectopic expression of OCT4and simultaneous treatment with LIF, inhibitors of GSK-3 and ERK1/2, and forskolin, a protein kinase A pathway agonist that can induceKLF2 and KLF4 expression (Hanna et al., 2010). Activation of theWNT pathway by a Wnt ligand or GSK-3 inhibitor (CHIR) hasalso been demonstrated to increase the induction of pluripotencyreprogramming of somatic cells (Lluis et al., 2008; Marson et al.,2008). CHIR can also replace SOX2 in the pluripotent reprogram-ming of both MEF and human neonatal keratinocytes which wereoverexpressed with OCT4 and KLF4 in combination with parnate,a lysine-specific histone demethylase 1 inhibitor (Li et al., 2009b).Activation of Wnt/b-catenin signaling can stimulate ESC self-renewaland support short-term pluripotency in humans and mice (Satoet al., 2004). However, these results are somewhat controversialbecause self-renewal of the ESCs in Wnt-3a-supplementedmedium has not been demonstrated over multiple passages(Dravid et al., 2005; Bakre et al., 2007). Additionally, although PDand CHIR are sufficient to generate mESCs from blastocysts,CHIR alone is not sufficient in this process (Hassani et al., 2011).

Although the exact mechanism(s) of GSK-3 inhibition in gPSC isola-tion remains unclear, it has been reported that b-catenin is theprimary GSK-3 substrate regulating the differentiation of mESCs.Stable b-catenin can interact with DNA-binding Tcf factors in thenucleus, where the complex activates transcription of target genes

(Lluis et al., 2011). Recently, the Doble group found that alternativeb-catenin-mediated signaling, through independent T-cell factor/lymphoid enhancer (Tcf/Lef) factors, can reinforce the pluripotentstatus of mESCs and impair their efficient differentiation (Kelly et al.,2011). These data are consistent with new findings (Wray et al.,2011) and can be explained by b-catenin-mediated repression ofTcf3 targets, including Oct4 and modulation of Oct4 target genes(Tam et al., 2008). The interaction between b-catenin and Oct4(Kelly et al., 2011) could reflect the recruitment of b-catenin byTcf3 to promoter sites co-occupied by Oct4 (Wray et al., 2011).Therefore, GSK-3 inhibition may act primarily through the stabilizationof intracellular b-catenin, and by converting Tcf3 complexes fromrepressors to activators or by displacing Tcf3 with other Tcf factorsthrough which b-catenin activates the pluripotency network (Wrayet al., 2011).

Taken together, we have shown for the first time that gPSCs can beestablished, with some efficiency, from neonatal mouse testes in vitroin the presence of CHIR, and gPSCs could be propagated underdefined conditions in the presence of chemicals. Notably, these cellsare not genetically modified, as required for the derivation of iPSCs,and there are no ethical concerns associated with ESC derivation.The ability to derive and expand gPSCs in vitro may facilitate the devel-opment of novel therapeutic strategies to produce immune-matcheddifferentiated cells for patient-specific treatment. Additionally, thesepluripotent cells offer a source of patient-specific stem cells appropri-ate for the study of genetic diseases in different cell lineages in vitro.Future studies will show whether CHIR or other inhibitors of GSK-3(Meijer et al., 2004) could affect the isolation of gPSCs lines withground-state properties—a basal proliferative state that is free of epi-genetic restriction and has minimal requirements for extrinsic stimuli—from other mammals, such as rats, and in biopsies of human testes.Furthermore, this signaling pathway, GSK-3 inhibition may help in dis-covering the mechanisms involved in germ cell-related testicular tera-toma formation and improve our understanding of human testicularcancer.

Supplementary dataSupplementary data are available at http://humrep.oxfordjournals.org/.

Authors’ rolesS.-F.M. and F.A. were involved in collection and/or assembly of data,data analysis and interpretation and manuscript writing; A.S., H.S. andN.A. were involved in data analysis and interpretation; A.F., S.-N.H.and H.F. were involved in collection and/or assembly of data, dataanalysis and interpretation; H.B. was involved in conception anddesign, financial support, data analysis and interpretation, manuscriptwriting, final approval of manuscript.

FundingThis study was funded by grants provided from Royan Institute and theIran National Science Foundation (INSF).

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Conflict of interestNone declared.

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