Stem Cell Research (2014) 12, 517–530

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Generation of male germ cells from mouse induced pluripotent stem cells in vitro☆ Yangfang Li a,b , Xiuxia Wang a , Xue Feng a , Shangying Liao a , Daoqin Zhang a , Xiuhong Cui a , Fei Gao a , Chunsheng Han a,⁎ a b

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China Department of Neurophamacology, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China

Received 8 September 2012; received in revised form 6 December 2013; accepted 17 December 2013 Available online 26 December 2013

Abstract Germ cells are the only cell type that passes genetic information to the next generation. In most metazoan species, primordial germ cells (PGCs) were induced from epiblasts by signals from the neighboring tissues. In vitro derivation of germ cells from the pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) are of great values for the treatment of infertility, for animal breeding, and for studying the mechanism of germ cell development. Although the derivations of male germ cells from PSCs have been previously reported, most of the studies failed to conduct the induction in a well-controlled and highly efficient manner. Here, we report the derivation of induced PGC-like cells (iPGCLCs) from mouse iPSCs via induced epiblast-like cells (iEpiLCs) as being monitored by the expression of enhanced green fluorescent protein gene under the control of the promoter of stimulated by retinoic acid 8 (Stra8-EGFP). The identity of iPGCLCs was characterized by examining the expression of multiple marker genes as well as by the recovery of spermatogenesis after they were transplanted to the testis of infertile W/Wv mice. Furthermore, iPGCLCs were either induced to germline stem cell-like cells (iGSCLCs) or reverted back to embryonic germ cell-like cells (iEGCLCs). In conclusion, we have established an efficient procedure for inducing iPSCs into iPGCLCs that can be further expanded and induced to more developed germ cells. This work indicates that the technology of in vitro germ cell induction is becoming more sophisticated and can be further improved. © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Introduction Germ cells in sexually reproducing organisms are the only cell type that is able to transmit genetic information ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1-5 Beichen West Road, Chaoyang District, Beijing 100101, China. Fax: + 86 10 64807105. E-mail address: [email protected] (C. Han).

to the next generation. Errors in germ cell development result in infertility, and mutations carried by germ cells lead to disorders in the offspring. In vitro derivation of germ cells may open an avenue for infertility treatment and genetic defect correction. In mammals, primordial germ cells (PGCs) are induced from a small group of epiblast cells by bone morphogenetic proteins (BMPs) such as BMP4 and BMP8b, which are produced by the extraembryonic ectoderm (Lawson and Hage, 1994; Ying et al., 2000). PGC specification occurs at around E6.25 in mice, marked by the sequential expression of two transcription factors Blimp1/Prdm1 and Prdm14 in response to BMPs (Saitou, 2009). These two transcription factors play key roles for PGC specification and their early stage development by regulating the expression of

1873-5061/$ - see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scr.2013.12.007

518 a large number of genes (Ohinata et al., 2005; Vincent et al., 2005; Yabuta et al., 2006; Kurimoto et al., 2008; Yamaji et al., 2008). Their expression distinguishes PGC precursors from surrounding cells in the epiblast, which have acquired a somatic fate (Saitou et al., 2012). The specification of PGCs can be viewed as the reprogramming of a somatic fate back to the pluripotent state as the expression of some pluripotent marker genes such as Nanog, Sox2 decrease in early Blimp1-positive cells but increase later in PGCs while the expression of many somatic genes are initially high in Blimp1-positive cells but decrease later in PGCs (Yabuta et al., 2006; Kurimoto et al., 2008). The full-grown PGCs are regarded as a group of alkaline phosphatase-positive cells at E7.25 in mice (Ginsburg et al., 1990), which are also marked by the re-expression of Stella, a maternal effect gene that is also expressed zygotically until blastocyst stage (Payer et al., 2003). During their migration to the developing gonad at the genital ridge, PGCs undergo further epigenetic reprogramming and active proliferation, in which the interaction between stem cell factor (SCF) and its receptor c-Kit plays an essential role (Besmer et al., 1993). SCF also promotes the survival and proliferation of cultured PGCs (Dolci et al., 1991). Post-migration PGCs, marked by the expression of several RNA binding proteins such as MVH, DAZL, NANOS3, undergo sex dimorphic development—while female germ cells quickly initiate meiosis and arrest at the meiosis I stage, the male ones mitotically divide for several rounds and also enter a quiescent stage when they are known as gonocytes (McLaren, 2003). After birth, gonocytes resume mitosis and mature into spermatogonial stem cells (SSCs), which undergo both self-renewal and differentiation to power the almost life-long spermatogenesis (Phillips et al., 2010). SSCs, marked by the expression of genes such as GFRα1, NGN3, PLZF, can be cultured as germline stem cells (GSCs) for a long term (Kanatsu-Shinohara et al., 2003; Kubota et al., 2004). The initiation of meiosis is believed to be stimulated by retinoic acid (RA) and to be mediated by a downstream gene named Stra8 (stimulated by retinoic acid 8) (Bowles et al., 2006; Koubova et al., 2006). The induced differentiation of pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into germ cells has been reported previously (Hubner et al., 2003; Toyooka et al., 2003; Clark et al., 2004; Geijsen et al., 2004; Nayernia et al., 2004, 2006; Kee et al., 2006, 2009; Wei et al., 2008; Panula et al., 2010). Despite that the inducing methods used in these studies were different in terms of the culture methods, the reporter constructs, and the inducing agents, one prominent common feature of these studies was that the induction efficiencies were very low because of the uncontrolled inducing processes. In 2011, Hayashi et al. reported the high efficiency induction of PGC-like cells (PGCLCs) from mouse ESCs and iPSCs by using a procedure that closely followed the developmental pathway of germ cells in vivo. In this procedure, ESCs and iPSCs were first induced into epiblast like cells (EpiLCs), which were subsequently induced to PGCLCs (Hayashi et al., 2011). In the present study, we reported the induction of mouse iPSCs derived from mouse embryonic fibroblasts (MEFs) and adult tail-tip fibroblasts (TTFs) into iPGCLCs via iEpiLCs using a two-step inducing procedure similar to what was reported by Hayashi et al. (2011). iPGCLCs and iEpiLCs were named so that their fully induced identities from somatic cells were

Y. Li et al. emphasized. iPGCLCs were transplanted into the testes of the W/Wv mice which were sterile due to the lack of germ cells, and partial recovery of spermatogenesis was observed. We found that these iPGCLCs were able to either differentiate to germline stem cell-like cells (iGSCLCs) which were similar to spermatogonial stem cells (SSCs) or de-differentiate back to pluripotent embryonic germ cell-like cells (iEGCLCs). These results indicated that the two-step inducing procedure was a highly efficient technique, and further inductions could be conducted to derive more differentiated germ cells.

Materials and methods Animals and antibodies The F1 progenies of C57BL6 and DBA/2 mice were used for the isolation of TTFs, and ICR mice for MEFs. Mice of these inbred strains were purchased from Vital River Co., Ltd. The W/Wv mice were generated by crossing C57BL/6J-KitW-v/J and WB/ReJ KitW/J, and were used as recipient mice for germ cell transplantation. These two strains were purchased from Jackson Laboratory. The antibodies used in the present study were listed in Supplementary Table 1.

Cell culture To isolate TTFs, a fragment of the tail from a 6-week-old mouse was peeled and cut into 1-cm pieces. Two pieces were placed on a well of a 6-well plate which was pre-coated with gelatin. The pieces were cultured in 2 ml DMEM (Gibco) containing 10% FBS (Hyclone). Fibroblasts migrating out of the tail pieces were passaged and the passage 4 cells were used for iPSC induction. MEFs were isolated from the E13.5 embryos. The heads, tails, limbs, visceral tissues and gonads of the embryos were removed. The remaining bodies were minced and digested with 0.25% trypsin at 37 °C for 15 min. Isolated cells were transferred onto a 100-mm culture dish in 10 ml DMEM containing 10% FBS. MEFs were used at passage 3 for iPSC induction.

Retroviral production and iPSC induction 293FT cells (3 × 105 cells per 6 cm2 dish) were transfected with packaging vector pCL-10A1 (Imgenex plasmid 10047p; Imgenex, San Diego, CA, USA) and each of the retroviral vectors pMXs-mOct4, pMXs-mSox2, pMXs-mKlf4, pMXs-mc-Myc (Addgene plasmids 13366, 13367, 13370, and 13375; Addgene, Cambridge, MA, USA), respectively. Culture Medium was collected from four 6 cm2-dishes at 48 h and 72 h after transfection, pooled, and filtered through a 0.45 μm filter as viral supernatant used for infection. MEFs and TTFs (2 × 105 cells per 10 cm2 dish) were infected with the viral supernatant supplemented with 4 μg/ml polybrene. 4 days after infection, the cells were reseeded on mitomycin C treated MEFs and maintained in ESC medium (DMEM, 15% FBS, 1000 U/ml LIF for MEF derived iPSCs, or DMEM/F12, 20% SR, 1000 U/ml LIF for TTF derived iPSCs), as reported previously (Takahashi et al., 2007; Zhao et al., 2009). iPSC colonies were picked 17–21 days after being cultured on MEFs.

Generation of male germ cells from mouse induced pluripotent stem cells in vitro

iPSC culture Mouse iPSCs were maintained on mitomycin C treated MEF feeder in DMEM culture medium supplemented with 15% FBS, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, 50 U/ml penicillin, 50 μg/ml streptomycin (all Gibco) and 1000 U/ml LIF (Millipore, Billerica, MA, USA). Cells were passaged every 3 days with trypsin replacement enzyme TypLE (Gibco). Before the initiation of epiblast differentiation, iPSCs were planted on the gelatin-coated dish for 1–2 passages in order to remove MEF feeder cells in the feeder-free culture medium (GMEM, 1% FBS, 10% SR, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, 50 U/ml penicillin, 50 μg/ml streptomycin, 1000 U/ml LIF and 0.8 μM PD0325901 (Calbiochem, Darmstadt, Germany) and 3 μM CHIR99021 (Biovision, Milpitas, CA, USA).

Teratoma formation Mouse iPSCs were injected subcutaneously into the dorsal flank of 8-week-old nude mice (1 × 106 cells per mouse). 4 week after injection, teratomas were formed and removed to be fixed in 4% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.

Establishment of Stra8-EGFP transfected iPSC lines A 1.4-Kbp genomic region starting from − 1400 bp to + 7 bp relative to the transcription start site of Stra8 was PCR cloned and sequencing confirmed. It was subcloned to the pEGFP-N1 plasmid containing the EGFP coding region and the neomycin resistant gene as was previously described (Nayernia et al., 2004). Linearized plasmid DNA was introduced into iPSCs by electroporation, and the transduced cells were plated on MEF feeder cells in ESC medium. The selection with 300 μg/ml G418 was started the next day for 7 days. iPSC colonies resistant to G418 were picked and transferred into 96-well culture plate and expanded subsequently. iPSC lines with stra8-EGFP cassette integrated into genome were identified by PCR genotyping.

Establishment of Stra8-EGFP transgenic mice and detection of the specificity of Stra8 promoter Transgenic lines were generated by pronuclear microinjection of ICR mice with Stra8-EGFP plasmid according to standard procedures. Briefly, the Stra8-EGFP cassette was excised from the plasmids with ScaI to remove vector sequences and was purified by gel electrophoresis. The DNA was quantified and diluted to a concentration of 3 mg/ml in injection buffer. The DNAs were injected into the male pronuclei of fertilized eggs. The injected embryos were transferred into the uteri of ICR pseudopregnant hosts. Transgenic mice harboring the Stra8-EGFP sequences were genotyped by PCRs using Stra8-EGFP sequence specific primers. The founder transgenic mice were bred for several generations to increase the number of transgenic mice. To detect the specificity of the Stra8 promoter, the embryos of the transgenic mice were collected at 14.5 and 15.5 dpc, and were fixed with 4% formaldehyde. 6-μm serial sections were

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processed, and were immunostained with anti-rabbit MVH, anti-rabbit STELLA and anti-rabbit GFP antibodies, respectively.

In vitro differentiation For iEpiLC induction, 5 × 105 iPSCs were plated on wells of gelatin-coated 6-well plates and treated with 20 ng/ml Activin A and 5 ng/ml bFGF (Peprotech, Rocky Hill, NJ, USA) combined with 1% SR in differentiation medium (GMEM with N2, B27, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine [all from Gibco]). For iPGCLC differentiation, iEpiLCs were induced with 50 ng/ml BMP4, 50 ng/ml BMP8b, and 50 ng/ml SCF (all from R&D Systems) in differentiation medium containing 10% SR for 1–6 days. FACS sorted EGFP + iPGCLCs were reseeded on MEF feeder layer (4 × 105 cells per well of a 6-well plate) and were incubated in ESC medium supplemented with 0.8 μM PD0325901, 3 μM CHIR99021 and 10 ng/ml bFGF for induction of iEGCLCs, or in differentiation medium supplemented with 20 ng/ml BMP4 and 1 μM RA for the induction of iGSCLCs.

FACS analysis and cell sorting iPGCLCs were dissociated with TypLE (Gibco), neutralized with DMEM containing 10% FBS, washed twice and then resuspended with PBS containing 0.5% BSA. Single cell suspension (1 × 106 cells/ml) was used for sorting. The flow cytometry was performed on a FACSAriaII cell sorter (BD Biosciences, San Diego, CA, USA). iGSCLCs and iEGCLCs were collected by digestion with TypLE at day 14 of induction, and were fixed with 4% paraformaldehyde for 20 min at RT followed by two washes with PBS/BSA. For MVH staining, the fixed cells were permeabilized with 0.3% Triton X-100 in PBS on ice for 10 min and washed with PBS/BSA again. The permeabilization step was omitted for staining cell surface markers such as SSEA-1. The cells were blocked with 0.5 μl mouse CD16/CD32 antibody for 30 min at RT, and incubated with the rabbit MHV antibody or the rat SSEA-1 antibody diluted 100 times for 3 h at RT. After two washes, the cells were incubated with PE-conjugated secondary antibody for 1 h at RT. The cells were resuspended in PBS and analyzed by using FACSCalibur (BD Biosciences) after two washes. The specificity of staining was determined using matched isotype control antibodies.

Immunocytochemistry and AP staining After the cells in culture were fixed with 4% formaldehyde for 20 min and rinsed with PBS twice, the fixed cells were permeabilized with 0.3% Triton X-100 in PBS for 20 min and blocked with 5 mg/ml BSA in PBS. The cells were subjected to specific immunostaining overnight by using the following primary antibodies at 4 °C: anti-NANOG, anti-SSEA-1, anti-MVH, anti-STRA8, anti-GFP, anti-DAZL, anti-STELLA, anti-GFRα1, anti-NGN3, anti-γH2AX, and anti-SCP3. The dilution for most of the primary antibodies was 1:200 except for being 1:400 for anti-SCP3. For immunofluorescence assays, TRITC-conjugated secondary antibody and FITC-conjugated secondary antibody were used at 1:200

520 dilution for 1 h, and nuclei were stained with 1 μg/ml DAPI for 10 min at RT. Secondary antibodies were purchased from Santa Cruz Company. Alkaline phosphatase activity was analyzed using the alkaline phosphatase detection kit (Chemicon, Chandlers Ford, UK) according to the manufacturer's instructions. Microscopy was performed using a Zeiss LSM 710 Meta confocal microscope (Carl Zeiss).

Transplantation and immunohistochemistry The sorted EGFP+ iPGCLCs were suspended with PBS, and microinjected into the seminiferous tubules of 6-week W/Wv mice through the efferent duct at a density of approximately 1 × 105 cells per testis. After two months of transplantation, the recipient testes were harvested and fixed with 4% formaldehyde overnight and frozen in Tissue-Tek OCT compound (Cryochrome; Shandon, Pittsburgh, PA, USA). 6-μm sections were processed for immunostainings. The following primary antibodies were used: anti-mouse DAZL, anti-rabbit MVH, anti-mouse γH2AX and anti-rabbit SCP3. The images were recorded with Zeiss LSM 710 Meta confocal microscope (Carl Zeiss).

Isolation of mouse E11.5 PGCs and SSCs Gonads containing PGCs were isolated from the E11.5 embryos with the sharp tweezers under the light microscope, and then were washed three times with PBS. The gonads were dissociated into cell suspension by incubation with 0.25% trypsin for 5 min. After removed tissue clumping with 0.45 μm filter, the cells were collected by centrifugalization and were used for examining the expression of PGC marker genes (Horii et al., 2003). The isolation of SSCs followed our previously described protocol with minor modifications (Zhang et al., 2012). Briefly, the testes were harvested from the 5–6-dpp pup mice; the tunica albuginea was removed with fine forceps. The seminiferous tubules were digested with 1 mg/ml type IV collagenase (Sigma) and 500 μg/ml DNase I (Sigma) for 5–10 min at 37 °C until the surface of the tubules became loosen. The tubules were dissociated into small fragments by gentle pipetting, which were placed onto the gelatin-coated dish with culture DMEM medium supplemented with 10% FBS. After overnight incubation, germ cells on top of somatic layer were collected by gentle pipetting, and were transferred to a second dish with SSC culture medium. Within 1 week, SSC colonies appeared on top of the flat somatic cell layer and were passaged and maintained as previously described (Zhang et al., 2012).

Real-time RT-PCR analysis Total RNA was isolated using the QIAGEN RNeasy mini RNA kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. About 1 μg of total RNA from each sample

Y. Li et al. was used to synthesize the first-strand cDNA with random primers using the Superscript III kit (Promega). Real-time PCR was set up with 1 μl of the RT reaction, 10 μl SYBR Green PCR Master Mix (Qiagen), 8 μl sterile water and 1 μl forward and reverse primers. The reactions were carried out on the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). A list of primers was provided in Supplementary Table 2. Expression of interested genes was determined with GAPDH as the internal control using the ΔCt method.

Statistical analysis All data were shown as mean ± SD from at last three independent experiments. Differences between groups were examined statistically using 2-tailed Student's t test. Values of P b 0.05 were considered significant.

Results Generation of iPSCs from MEFs and TTFs We reprogrammed MEFs and TTFs to iPSCs through retroviral transduction with the four “Yamanaka transcription factors”—Oct4, Sox2, Klf4 and c-Myc (Takahashi and Yamanaka, 2006). iPSC colonies were picked up 17 days after transduction based on their ESC-like morphology. Two lines, one from the MEFs (mMiPS14) and the other from the TTFs (mTiPS3), were expanded and further characterized. These iPSC lines expressed pluripotent markers NANOG and SSEA-1, and were alkaline phosphatase positive (Supplementary Fig. 1A). Once transplanted into the nude mice, these iPSCs formed teratomas that consisted of various tissues from the three germ layers including osteoblast, muscle, neural tissue, epithelium and glandular tissue (Supplementary Figs. 1B–G).

In vitro differentiation of iPSCs into iEpiLCs and iPGCLCs We used a 2-step induction procedure to derive iPGCLCs from iPSCs via iEpiLCs (Fig. 1A). To monitor the induction, we constructed a reporter plasmid with the coding region of the enhanced green fluorescent protein (EGFP) being under the control of a 1.4-Kbp promoter of the mouse Stra8. Stra8 is expressed in female germ cells from E12.5 to E16.5 while in male germ cells from 2 dpp and onward (Menke et al., 2003; Zhou et al., 2008; Snyder et al., 2010). The 1.4-Kbp promoter was reported to drive the expression of EGFP specifically in a subpopulation of spermatogonia (Nayernia et al., 2004). To confirm this, we produced Stra8-EGFP transgenic mice and examined the expression of EGFP on sections of ovaries and testes. The expression of EGFP was

Figure 1 Induction of iEpiLCs from iPSCs in vitro.A: The schema of induction of iPGCLCs from iPSCs stably transfected with the Stra8-EGFP construct via iEpiLCs. B: Morphology and fluorescence of the induced cells at days 1, 2, and 3 of induction. Note that the cells were EGFP-negative. C: Time course of expression of genes representing epiblasts (Cer1, Fgf5, Dnmt3a, Dnmt3b), PSCs (Nanog, Rex1, Prdm14, Stella) during the induction. The expression of genes was determined by real-time PCRs with GAPDH as the internal control, and was normalized by the expression at day 0 of induction. For each time point, the average values from three independent experiments with standard deviation (SD) are shown.

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522 detected specifically in female germ cells of E14.5 and E15.5 embryos and in male germ cells at 6 days postpartum (dpp) (Supplementary Fig. 2). These observations were consistent with the results of a previous study (Nayernia et al., 2004). The plasmid was introduced into the cells of both mMiPS14 and mTiPS3 lines by electroporation. The integration of Stra8-EGFP in the genome was confirmed by PCRs for G418 resistant clones. One such clone from mMiPS14 cells was chosen for the subsequent experiments while all mTiPS3 clones were pooled together for further study. During the first step induction, iPSCs grew rapidly for the first 2 days after Activin A and bFGF were added and then some cells began to die at day 3 (Fig. 1B). These cells were not fluorescently green at this stage. The expression of Nanog, Rex1, Prdm14 and Stella, whose expression is detected in pluripotent cells (Chambers et al., 2003; Geijsen et al., 2004; Mise et al., 2008; Yamaji et al., 2008), was decreased during the 3-day period while the expression of genes that are highly expressed in epiblast such as Cer1, Fgf5, Dmnt3a and Dmnt3b (Bao et al., 2009; Hayashi et al., 2011) was increased (Fig. 1C). The change patterns of these genes were the same for both mMiPS14 and mTiPS3 cells. These results indicated that iEpiLCs were induced in vitro. Treatment of the days 1, 2, and 3 iEpiLCs with BMP4 for two more days resulted in green fluorescent cells, and the highest number of green cells was derived from the day 2 iEpiLCs (Fig. 2A). In contrast, no green cells were observed from similarly treated iPSCs or iEpiLCs without growth factors treatment. Moreover, the combination of BMP4, BMP8b, and SCF was the most potent in the induction of iPGCLCs as shown by the significantly higher expression of Mvh although the proportion of green cells were not significantly higher compared with the treatment with BMP4 only (Fig. 2B). Subsequently, the day 2 iEpiLCs were treated with BMP4, BMP8b and SCF for different days to determine the optimal time period for iPGCLC induction. As shown by Fig. 2C, treatment for 3–4 days resulted in higher proportions of green cells (44%–41%) than the other periods (less than 21%). Treatment longer than 4 days leads to large scale cell death. We next used FACS to isolate green fluorescent cells at days 1, 3 and 5 of treatment and examined the expression of three panels of marker genes for PSCs, epiblast cells, and PGCs. The untreated iEpiLCs and PGCs isolated from E11.5 embryos were included as controls. As shown by Fig. 2D, the expression of PGC marker genes such as Blimp1, Mvh, Nanos3, Stella, Dnd1, Prdm14 was up-regulated from day 1 to day 3 of treatment while kept relatively constant from day 3 to day 5 in cells derived from both lines. The expression of pluripotent stem cell marker genes Nanog and Eras only increased slight during the treatment while that of epiblast marker genes Dnmt3b and Fgf5 decreased slightly. The expressions of these genes from day 3 to day 5 of treatment were close to their expression in isolated PGCs. Considering the percentages of green cells, the proportions of dead cells and the expression of marker genes altogether, we decided that a 4-day treatment was the optimal time period for iPGCLC induction. Therefore, the 2-step induction procedure was finalized as a 2 day iEpiLC induction with Activin A and bFGF followed by a 4 day iPGCLC induction with BMP4, BMP8b, and SCF (Fig. 1A).

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Characterization of iPGCLCs EGFP+ and EGFP− cells were sorted by FACS and examined for protein and mRNA expression of PGC marker genes after a short period of culture in differentiation medium containing 10% SR for 12 h (Fig. 3). As shown by the co-immunostaining of STRA8 and EGFP, almost all the EGFP+ cells were STRA8+ (99.6%), although the signal intensities of these two proteins varied in different cells, while all the EGFP− cells were STRA8− (Supplementary Table 3). Moreover, the proteins DAZL, MVH, and STELLA were all detected immunocytochemically in EGFP+ cells but were absent in EGFP− cells except for STELLA, which was weakly present in EGFP− cells. Therefore, all the EGFP+ cells were iPGCLCs. The mRNA expression of PGC marker genes Blimp1, Prdm14, Mvh, Stra8 and Nanos3 was also significantly higher in EGFP+ cells than in EGFP− cells while the expression of pluripotent stem cell marker genes Rex1, Eras was significantly lower in the former than in the later. As it has been known that Stella, Nanog and Oct4 are expressed in both PSCs and PGCs, their expression in EGFP+ and EGFP− cells was not significantly different except for that Oct4 was expressed at a higher level in EGFP+ cells than in the EGFP− cells originated from the mMiPS14 line. These results further demonstrated that the stra8-EGFP+ iPGCLCs were similar to natural PGCs in terms of gene expression. Based on these results and the FACS analysis of EGFP+ cells, the efficiency of iPGCLC induction from iEpiLC was estimated to be approximately 41%. We next assessed whether these iPGCLCs were able to establish spermatogenesis upon being transplanted into the testes of W/Wv mice, which contain few undifferentiated spermatogonia (Geissler et al., 1988). Two months after transplantation, the recipient mice were sacrificed and sections of testis were immunostained to detect differentiated germ cells. As shown by Fig. 4, the seminiferous tubules of W/Wv mice without transplantation contain almost no germ cells as indicated by the DAZL staining while 50–70% tubules of the transplanted testes contained large numbers of DAZL positive or MVH positive germ cells. In some tubules, spermatocytes were identified by the positive staining of SCP3 and γH2AX, and such tubules were observed in about 50% of the transplanted testes. Moreover, teratomas were not seen in any of the transplanted testes. These results indicated that our induced iPGCLC had acquired spermatogenic capability, and they could develop to more mature germ cells when the spermatogenic niche was available.

Bi-directional differentiation of iPGCLCs in vitro We next examined whether iPGCLCs could further differentiate into GSCs and/or de-differentiation to EGCs in vitro. EGFP+ iPGCLCs and EGFP− cells were treated with RA and BMP4 for 14 days. The EGFP− cells were still EGFP negative after the treatment, and underwent massive cell death. The green fluorescent signal of the EGFP+ iPGCLCs became brighter after the treatment. The colonies of these cells had the typical morphology of GSCs, and these cells were termed as iGSCLCs. When the iPGCLCs were cultured using the ESC medium containing bFGF, LIF, and 2 inhibitors (MAPK inhibitor PD0325901 and GSK3 inhibitor CHIR99021),

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Figure 2 Induction of iPGCLCs from iEpiLCs. A: The effects of BMP4 and the combination of BMP4, BMP8b and SCF on the induction of Stra8-EGFP+ cells from mMiPS14 cells and iEpiLCs induced by Activin A and bFGF for 1, 2 and 3 days. The induction of iPGCLCs from different cells continued for 2 days, and the appearance of Stra8-EGFP+ cells were checked by fluorescent microscopy. Scale bar = 100 μm. B: Comparison of the effects of BMP4 and BMP4 + BMP8b + SCF on the induction of Stra8-EGFP+ cells and the expression of Blimp1, Stella and Mvh. The day 2 iEpiLCs were treated with BMP4 or the triple factors for 2 days. The proportion of the EGFP + cells was determined by FACS, and the expression of the marker genes was quantified by Q-PCRs. Error bars indicate SD of three independent experiments. Comparisons were conducted using t-test. * denotes P b 0.05. C: Morphology, green fluorescence, and FACS result of iPGCLCs induced from day 2 iEpiLCs by BMP4 + BMP8b + SCF for different days (day 1 to day 6). Undifferentiated mMiPS14 cells were included as negative controls. D: Time courses of expression of genes representing germ cells (Nanos3, Mvh, Stella, Prdm14, Blimp1, Dnd1), PSCs (Eras, Nanog) and epiblasts (Dnmt3b, Fgf5) during the iPGCLC induction. The expression of genes in E11.5 PGCs was as references. The expression of genes was determined by real-time PCRs with GAPDH as the internal control, and was normalized by the expression at day 0 of PGCLC induction. For each time point, the average values from three independent experiments with SDs are shown.

the EGFP fluorescence became lost gradually and undetectable 7 days after treatment. These cells eventually adopted the tight round colony morphology typical of PSCs about 14 days in culture and were termed iEGCLCs (Fig. 5A). As also indicated by the FACS results, the un-induced mMiPS14 cells were basically EGFP negative, while about 41% of the iPGCLCs

were of low or high level of EGFP fluorescence, and the cells of high EGFP signal were increased to 57.2% in the iGSCLC population compared with iPGCLCs, suggesting the efficiency of iGSCLC induction was about 57% (Fig. 5B). As shown in Fig. 5C, the iGSCLCs were immunostained positive for germ cell marker proteins DAZL and MVH as well

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Figure 3 Characterization of Stra8-EGFP+ iPGCLCs by immunostaining and quantitative PCRs. A–D: Co-localization of EGFP with STRA8 (A), MVH (B), STELLA (C) and of DAZL and STRA8 (D) in sorted Stra8-EGFP+ and Stra8-EGFP− cells at day 4 of iPGCLC induction from mMiPS14 and mTiPS3-derived iEpiLCs, which were treated with BMP4, BMP8, and SCF for 4 days. Cell nuclei were stained with DAPI (blue). Scale bar = 50 μm. D: Gene expression analysis of Stra8-EGFP+ and Stra8-EGFP− cells. The expression was determined by real-time PCRs with GAPDH as the internal control, and was normalized by the expression of Stra8-EGFP− cells of the mTiPS3. The average values from three independent experiments with SDs are shown. * indicates P b 0.05. Statistical analysis was performed using t-test.

as SSC marker proteins NGN3 and GFRα1. Some of them were also immunostained positive for spermatocyte marker

proteins SCP3 and γH2AX in their nuclei (Yuan et al., 2000; Mahadevaiah et al., 2001). The green fluorescence of these

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Figure 4 Recovery of spermatogenesis by transplanted iPGCLCs in the testes of WWv mice. A: A testis section of the W/Wv mouse was immunostained by the antibody for DAZL. Note that the germ cells were negatively stained while non-specific staining was seen around the seminiferous tubules probably as a result of the use of the secondary antibodies against the mouse primary antibodies. B: Some tubules of the transplanted testes were filled with DAZL positive cells (open arrow). C: SCP3 staining was seen in DAZL-positive cells in the section of a tubule (open arrow head). D: Some tubules of another transplanted testes were filled with MVH-positive cells (filled arrow head). E–F: Some cells were positively stained for both MVH and γH2AX (white arrows). F is an amplified version of the boxed area in E. Nuclei were stained blue with DAPI. Three batches of mMiPS14 derived iPGCLCs were transplanted into 12 testes, of which 6 were observed to partially recover spermatogenesis 2 months after transplantation. G: HE staining on the testis section of the W/Wv mouse. H: After two months of transplantation, the testis section was stained with HE. Scale bar = 50 μm.

cells was weak or disappeared, consistent with that Stra8 promoter was activated in premeiotic germ cells. iGSCLCs were negative for both NANOG and SSEA-1, while iPGCLCs were positive for NANOG and SSEA-1 but negative for NGN3 and GFRα1. The iEGCLCs were positive for NANOG (Yamaguchi et al., 2005) and SSEA-1 but were negative for DAZL and MVH. These results demonstrated that iPGCLCs, iGSCLCs, and iEGCLCs were different cell types although they shared some marker proteins.

Quantitative analysis of the induction of iGSCLCs and iEGCLCs from iPGCLCs We first used FACS to conduct quantitative analysis of the induction of iGSCLCs and iEGCLCs from iPGCLCs based on the expression of MVH, SSEA-1 and the EGFP fluorescence. As shown by 6A, the expression of MVH and SSEA-1 correlated with EGFP fluorescence positively and negatively, respectively, under different inducing conditions. While BMP4, RA, and their combination all induced MVH+STRA8-EGFP+ iGSCLCs, the combination of the two factors was the most potent. The percentages of MVH+EGFP+ cells were 35%, 48%, and 66% for the three treatments, respectively. In contrast, this type of cells were only 0.1% with the treatment of 2 inhibitors, LIF, and bFGF (2i + LIF + bFGF) while 98.5% of the cells were MVH− EGFP−. In parallel, BMP4, RA, and BMP4 + RA treatments of iPGCLCs resulted in 48%, 78%, and 88% SSEA-1−EGFP+ iGSCLCs. With the 2i + LIF + bFGF treatment, this type of cells constituted only 0.4% of the whole population, of which 70% were SSEA-1+STRA8-EGFP− cells. As a control for iGSCLCs and iEGCLC, iPSCs hardly expressed EGFP and MVH, while 97.1% of

them were positive to SSEA-1. These data indicated that iGSCLCs were MVH+SSEA-1−EGFP+ while iEGCLCs were MVH− SSEA-1+EGFP−, consistent with the immunostaining results (Fig. 5). We next used quantitative RT-PCR to examine the expression of a panel of marker genes during the induction of iGSCLCs and iEGCLCs at day 7 and day 14 of treatment (Fig. 6B). The original iPSC line mMiPS14, the iPGCLCs from this line, and isolated SSCs were used as controls. The expression changes of the premeiotic germ cell markers Dazl, Mvh, the undifferentiated spermatogonial marker Plzf, the differentiating spermatogonial marker c-Kit, as well as the spermatocyte marker Scp3 were similar under different inducing conditions. They were low in mMiPS14 cells, slightly increased in iPGCLCs, and were further increased by the treatments of BMP4, RA, and their combination. Again, RA was more potent than BMP4, and their combination was the most potent. The expression also increased as the treatment time went from day 7 to day 14. In contrast, the expression of these genes in the 2i + LIF + bFGF treated cells was similar to and lower than the levels in mMiPS14 cells and iPGCLCs, respectively. The expression of pluripotent stem cell marker genes Nanog and Eras was similarly higher in mMiPS14 cells, iPGCLCs, and the 2i + LIF + bFGF-treated cells than in cells treated with BMP4, RA, or their combination. Comparing different cell types in terms of the expression of these genes, it was apparent that cells treated BMP4 + RA were most similar to SSCs while cells treated with 2i + LIF + bFGF were most similar to mMiPS14 cells. These results, again, suggested that iPGCLCs were a transition cell type, which were able to differentiate to iGSCLCs and dedifferentiate back to the pluripotent state.

526 During the induction of iGSCLCs with BMP4, the cells expanded by 51-folds by day 20, equivalent to a doubling time of 3.4 days. However, the proliferation of cells was markedly reduced in the presence of RA as the cells expanded by 7.4-folds and 4-folds with the RA, and the RA + BMP4 treatments, equivalent to doubling times of 5.6 and 8.1 days, respectively. A number of these cells started to die at the beginning of induction, and then they grew rapidly from day 4 to day 16 when large scale cell death was observed. For the dedifferentiation into iEGCLC, a portion of the treated iPGCLCs also died at the beginning, but the cells grew up gradually and were expanded by 104-folds by day 20, with the doubling time being 3.0 days.

Discussion Only about a dozen of studies have been published on the induction of germ cells from PSCs since germ cell induction from ESCs was first reported in 2003 (Hubner et al., 2003). Such a low number of publications indicated that the derivation of germ cells from PSCs is still an immature technology. In the present study, we report the induced differentiation of mouse iPSCs into iPGCLCs using a procedure similar to what was previously reported by Hayashi et al. (2011). Our procedure, being different from theirs in several aspects (discussed later), represents a simpler yet equally efficient one. Our results indicated that this “two-step” inducing procedure was efficient and reproducible probably due to the sequential induction of EpiLCs and PGCLCs using well-defined key factors at each step. More importantly, we showed that our iPGCLCs could either be induced one more step further to iGSCLCs or be converted to iEGCLCs. These works indicate that different germ cells could be derived from iPSCs in an efficient way. One important component of our inducing system was the Stra8-EGFP reporter used to monitor the induction of germ cells. Different groups have used different promoters to drive the expression of reporter genes in their studies. These promoters are from genes such as Oct4 (Hubner et al., 2003), Mvh (Toyooka et al., 2003; Kee et al., 2009; Panula et al., 2010), Stra8 (Nayernia et al., 2004, 2006), Stella (Wei et al., 2008), Blimp1 and Prdm14 (Hayashi et al., 2011), which are expressed in germ cells at different stages of development but not in somatic cells. Oct4 is expressed in all cell types along the differentiation path from PSCs to SSCs. Consequently, some group used a truncated version of the Oct4 promoter, which is only active in PGCs but not in ESCs (Hubner et al., 2003). The expression of Blimp1 and Prdm14 starts in PGC precursors and ends in migrating PGCs and E13.5–14.5 postmigration PGCs, respectively (Ohinata et al.,

Y. Li et al. 2005; Yamaji et al., 2008). The expression of Stella starts in E7.25 PGCs and ends in E13.5 PGCs in female and E15.5 in male PGCs (Sato et al., 2002). Mvh is expressed in postmigration PGCs until round spermatids (Fujiwara et al., 1994). Stra8 was reported to be expressed in female germ cells from E12.5 to E16.5 while in male germ cells from 2 dpp and onward (Menke et al., 2003; Zhou et al., 2008; Snyder et al., 2010). The expression of EGFP in Stra8-EGFP transgenic mice generated in the present study, consistent with the results of a previous study (Nayernia et al., 2004), confirmed the activation stages of the Stra8 promoter. It is apparent that the expression of Stra8 in male germ cells starts at least as early as in undifferentiated spermatogonia although it is regarded as a premeiotic germ cell marker. More importantly, the male PGCs at E12.5 are already responsive to RA in terms of Stra8 expression if exogenous RA or RAR agonists or the CYP inhibitor were added to cultured embryonic male gonads (Koubova et al., 2006). Indeed, the activation of Stra8 promoter in germ cells is believed to be controlled by the availability of RA, RAR and cofactors. The level of RA is a balance between its production and degradation, which are catalyzed by retinaldehyde dehydrogenase (RALDHs) and CYP enzymes, respectively (Griswold et al., 2012). Several CYP enzymes, including some that may degrade RA, are expressed in the somatic cells of the embryonic testes but not the embryonic ovaries (Bowles et al., 2006). Therefore, the absence of Stra8 expression in male germ cells at the embryonic stage is because of the degradation of RA by CYP enzymes, of which the CYP26B1 has been known to be critical (Bowles et al., 2006). Based on these observations, it is not surprising that we could acquire a large number of Stra8-EGFP low expression PGCLCs in a short period of time because the CYP-expressing barrier is missing in the culture system and the B27 contains RA activity. These cells were more like the post-migration PGCs based on the expression of marker genes. Notably, the Stra8-EGFP expression was not activated in EpiLCs, suggesting that the activation of Stra8 promoter also needs other cofactors whose activities were induced by BMP4 and/or other factors. In line with this, the activation of Stra8 was not observed in the female gonads at E11.5 even in the absence of the Cyp26b1 expression, again suggesting that the germ cells have not been induced to a RA-responsive state (Koubova et al., 2006). These observations indicated that the activation of Stra8 promoter was induced but was not aberrantly caused by the integration of the reporter cassette to certain genomic locations. For the first time, we showed that iPGCLCs could be expanded and induced bi-directionally to either iGSCLCs or iEGCLCs in vitro. From the specification of PGCs to the generation of functional sperm, the development of male germ cells is a lengthy process encompassing many cell

Figure 5 In vitro differentiation of iPGCLCs. A: Stra8-EGFP+ and Stra8-EGFP− cells were sorted using FACS and cultured under different conditions. Treated with RA and BMP4 for 14 days, the Stra8-EGFP+ cells (iPGCLCs) continued to proliferate to form iGSCLCs with increased EGFP intensity while the Stra8-EGFP− cells failed to proliferate actively and the EGFP signal was still not detected. Cultured with ESC medium supplied with inhibitors of GSK3 and MAPK, LIF, and bFGF, the Stra8-EGFP+ cells dedifferentiated to iEGCLCs, and the EGFP signal was lost. B: FACS analysis of the EGFP signals in iPGCLCs and iGSCLCs treated with BMP4 and RA in comparison with un-induced iPSC line mMiPS14. C: Immunostaining of iEGCLCs, iPGCLCs, and iGSCLCs for DAZL, MVH, GFRα1, NGN3, γH2AX, SCP3. Nuclei were stained with DAPI. Note that iEGCLCs were positive for PSC markers SSEA-1 and NANOG but negative for germ cell markers MVH and DAZL; iPGCLCs were positive for SSEA-1 and NANOG but negative for spermatogonial marker NGN3 and GFRa1; iGSCLCs were positive for DAZL, MVH, GFRα1, NGN3, γH2AX, SCP3 but negative for SSEA-1 and NANOG. Scale bar = 50 μm.

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Figure 6 Quantitative analysis of induction of iGSCLCs and iEGLCs from iPGCLCs. A: FACS analysis of Stra8-EGFP+ cells cultured under different conditions using signals of EGFP and MVH/SSEA-1 as two parameters, and undifferentiated iPSCs (mMiPS14) were as negative control. B: Comparisons of marker gene expression among different cell types including iPSCs (mMiPS14), iPGCLCs, iGSCLCs (BMP4, RA, BMP4 + RA), iEGCLCs (2i + LIF + bFGF), and SSCs. Error bars indicate SD of three independent experiments. * is for P b 0.05 using t-test. C: Proliferation curves of iPGCLCs under different induction conditions. Data were based on 3 independent experiments.

types. Most of the previous male germ cell induction studies failed to characterize their derived germ cells in details. For example, Toyooka et al. reported for the first time the induction of Mvh-GFP+/lacZ+ cells which differentiated to sperm in reconstructed testes (Toyooka et al., 2003). As pointed out earlier, Mvh is expressed in many types of male germ cells, the identity of the Mvh-GFP+/lacZ+ cells was unknown. Geijsen et al. derived both diploid and haploid male germ cells directly from EBs, and blastocysts could be derived when the haploid cells were injected to the recipient oocytes (Geijsen et al., 2004). However, again, such a spontaneous differentiation method precluded the characterization of germ cell types. Nayernia et al. reported the derivation of Stra8-EGFP+ cells, which further differentiated to sperm, either from teratocarcinoma cells or from ESCs induced by RA at concentration of 1 μM and 10 μM, respectively (Nayernia et al., 2004, 2006). Hayashi et al. first reported the 2-step PGCLC inducing procedure, and the detailed characterization using qPCRs, immunostaining, and

microarray analysis indicated that their germ cells were most similar to postmigration PGCs. However, they were unable to expand or further induce the isolated PGCLCs in culture. In our study, we were able to culture the sorted iPGCLCs for at least 16 days in the presence of BMP4, RA, or their combination. BMP4 and RA have been known to promote the in vitro proliferation of PGCs (Koshimizu et al., 1995; Pesce et al., 2002). We also found that BMP4 was much more potent than RA in promoting the expansion of iPGCLCs. More importantly, we found that the combination of these two factors stimulated not only the proliferation of iPGCLCs but also the further differentiation of these cells into iGSCLCs as indicated by the increased intensity of EGFP and the expression of SSC marker proteins such as GFRα1 and NGN3. Interestingly, some of the treated cells even expressed meiotic markers such as SCP3 and γH2AX, whose appearances were concurrent with the decrease of the Stra8-EGFP signal. This indicated that the combination of BMP4 and RA was potent inducer of more differentiated male

Generation of male germ cells from mouse induced pluripotent stem cells in vitro germ cells from iPGCLCs. The decreased proliferation of the treated cells beyond day 16 of treatment may be a result of the initiation of meiosis. Therefore, a condition that stabilizes the induced cells at the iGSCLC stage should be established so that larger amount of germ cells could be derived through the self-renewal of the stem cells. Another difference between our procedure and that of Hayashi et al. was that we induced iPGCLCs from EpiLCs on feeder-free gelatin-coated culture dish while they used the aggregates of EpiLCs for the induction. Although the proportion of EGFP+ cells derived using both methods were similarly high (44%), the amount of PGCLCs using our method could easily be amplified if larger dishes were used. Hayashi et al. claimed that ~ 105–106 PGCLCs could be derived using their method. We were able to acquire similar number of iPGCLCs in a 35-cm dish. Therefore, our procedure represents a simplified and equally efficient method for iPGCLC induction. The use of Stra8-EGFP contributed to the enrichment of iPGCLCs on the one hand but also imposed a safety concern when medical applications based on iPGCLCs was considered. Hayashi et al. reported that PGCLCs could be efficiently sorted based on the expression of SSEA1 and Integrin-b3. It will be important for us to examine whether our Stra8-EGFP+ iPGCLCs can also be selected based on similar cell surface markers in future experiments. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2013.12.007.

Acknowledgments This work was supported by grants from the Ministry of Science and Technology of China (2012CB966702 and 2013CB945001), National Natural Science Foundation of China (31271379, 31100984).

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