EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS 1
Graduate School of Frontier Biosciences, 2 and Department of Pathology, Medical 6 School, Research Institute for Microbial Diseases, Osaka University, 2‐2 Yamada‐oka, 3 Suita, Osaka 565‐0871, Japan; Laboratory 4 of Molecular Embryology, Laboratory of Stem Cell Biology, Department of Bioscienc‐ es, Kitasato University School of Science, 1‐ 15‐1, Kitasato, Minami‐ku, Sagamihara, 5 Kanagawa 252‐0373, Japan; Department of 9 Anatomy and Cell Biology, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida‐kanoe‐ cho, Sakyo‐ku, Kyoto 606‐8501, Japan; 7 Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Sakaecho 35‐2, Itabashi‐ku, Tokyo 173‐0015, Japan.; 8 Precursory Research for Embryonic Science 10 and Technology (PRESTO), Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), Yoshida‐kanoe‐cho, Sakyo‐ku, 11 Kyoto 606‐8501, Japan; Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara‐cho, Shogoin Yoshida, Sakyo‐ 12 ku, Kyoto 606‐8507, Japan.; Institute for Integrated Cell‐Material Sciences, Kyoto University, Yoshida‐Ushinomiya‐cho, Sakyo‐ 13 ku, Kyoto 606‐8501, Japan.; Core Research for Evolutional Science and Technology (CREST), JST, 7 Gobancho, Chiyoda‐ku, To‐ kyo 102‐0075, Japan. Correspondence: Tohru KIMURA, PhD, Laboratory of Molecular Embryology, La‐ boratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1‐15‐1, Kitasato, Minami‐ku, Sagamihara, Kanagawa 252‐0373, Japan, Tel: +81‐042‐778‐8864; Fax: +81‐042‐778‐ 8864, E‐mail: tkimura@ kitasato‐u.ac.jp; Grant support: Supported in part by the Ministry of Education, Science, Sports and Culture, and by PRESTO, ERATO, CREST of JST.; The English in this document has been checked by at least two professional edi‐ tors, both native speakers of English.
Induction of Primordial Germ Cell‐Like Cells From Mouse Embryonic Stem Cells by ERK Sig‐ nal Inhibition TOHRU KIMURA1,2,3,4, YOSHIAKI KAGA1, HIROSHI OHTA5, MIKA ODAMOTO3,4, YOICHI SEKITA2, KUNPENG LI1, NORIKO YAMANO1, KEITA FUJIKAWA3,4, AYAKO ISOTANI6, NORIHIKO SASAKI7, MASASHI TOYODA7, KATSUHIKO HAYASHI5,8, MASARU OKABE6, TAKASHI SHINOHARA9, MITINORI SAITOU5,10,11,12, AND TORU NAKANO1,2,13 Key Words. Primordial Germ Cells • In Vitro Differentiation • Embryonic Stem cells • Mesoderm • ERK Signal
ABSTRACT Primordial germ cells (PGCs) are embryonic germ cell precursors. Specifica‐ tion of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces meso‐ dermal differentiation in vitro. Using this mesodermal differentiation sys‐ tem, we demonstrated that PGC‐like cells were efficiently induced from mouse ESCs by ERK signaling inhibition. Inhibition of ERK signaling by a MEK inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC‐like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK sig‐ naling suppresses mesodermal differentiation but activates germline dif‐ ferentiation program in this mesodermal differentiation system. Our find‐ ings provide a new insight into the signaling networks regulating PGC spec‐ ification. STEM CELLS 2014; 00:000–000
Received August 31, 2013; accepted for publication June 06, 2014 ©AlphaMed Press 1066‐5099/2014/$30.00/0 This article has been accepted for publica‐ tion and undergone full peer review but has not been through the copyediting, typeset‐ ting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.1781
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Induction of PGC‐like cells by ERK inhibition
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INTRODUCTION Gametes are derived from a founder population of pri‐ mordial germ cells (PGCs) [1], and the specification of PGCs occurs under the influence of mesoderm‐inducing signals. On embryonic day (E) 7.0 in mice, PGCs emerge from a subset of epiblast cells that migrate to the extraembryonic region together with future extraembryonic mesoderm cells during gastrulation. PGCs return to the embryonic region on E7.75 and col‐ onize the genital ridges by E11.5. After settlement into the genital ridges, germ cells undergo spermatogenesis and oogenesis to produce sperm and oocytes, respec‐ tively. During specification to germ lineage cells, the gene expression profiles and epigenetic status of the cells are drastically altered by Prdm1 (Blimp1), Prdm14, and Tcfap2c (AP2 ), transcription factors essential for PGC specification [2‐6]. Germ cell‐specific genes, such as Dppa3 and Dnd1, are induced in the nascent PGCs, i.e., the PGCs that are just emerging in the extraembryonic region on E7.0 [7]. In contrast, mesoderm‐related genes are repressed in the nascent PGCs, although they are transiently induced by the mesoderm‐inducing signals [7, 8]. PGCs undergo epigenetic reprogramming, which involves genome‐wide alterations of repressive histone modification and DNA methylation patterns [9]. All the‐ se changes are presumed crucial for the determination of cell fate toward PGCs. Two signaling pathways, bone morphogenetic pro‐ tein (BMP) and Wnt play important roles in PGC specifi‐ cation. BMP4 secreted from extraembryonic ectoderm is critical for PGC formation in vivo [10]. In addition, BMP4 is sufficient to induce Prdm1 and Prdm14 in the epiblast and to promote the formation of PGC‐like cells from the epiblast [11, 12]. While BMP8b emitted from extraembryonic ectoderm is also important for PGC formation [13], its function is to restrict the inhibitory signals against BMP4 outside posterior and proximal epiblast cells [12]. In addition, BMP2 from visceral en‐ doderm appears to augment the role of BMP4 to ensure the generation of a sufficient number of PGCs [14]. Fi‐ nally, Wnt3 enables epiblast cells to respond to BMP4 to form PGCs [12]. In addition to the roles in PGC specification, these signals are involved in mesodermal differentiation. For example, Wnt3 is required for formation of the primi‐ tive streak, mesoderm, and node [15]. Mice deficient for BMP4 have little or no mesodermal tissues, truncat‐ ed or disorganized posterior structures, and a reduction in extraembryonic mesoderm [16]. Similar abnormali‐ ties in embryonic and extraembryonic mesoderm are also observed in mice lacking BMP2 or BMP8b [14, 17]. Thus, BMPs and Wnt3 regulate differentiation of both germline and mesodermal lineages in embryos from the egg cylinder to gastrulation stages. ERK signaling is a canonical mitogen‐activated pro‐ tein kinase (MAPK) signal [18]. This signaling pathway www.StemCells.com
initiates with activation of Ras GTPase by growth fac‐ tors. Ras then activates the kinase cascade, which se‐ quentially activates MAPKKK (Raf), MAPKK (MEK), and MAPK (ERK). Finally, ERK phosphorylated by MEK trans‐ locates into the nucleus and activates several transcrip‐ tion factors such as SRF and Elk‐1. The ERK signal axis is involved in various biological processes including prolif‐ eration, survival, and differentiation. In this study, we demonstrated that PGC‐like cells were induced from mouse embryonic stem cells (ESCs) in an in vitro mesodermal differentiation system using OP9 feeder cells. When seeded onto OP9 feeder cells without leukemia inhibitor factor (LIF), ESCs differenti‐ ate into a variety of mesodermal lineage cells [19‐24]. Using this mesodermal culture system, we demonstrat‐ ed that ERK signaling inhibition suppressed mesoderm differentiation but promoted induction of PGC‐like cells. A potential role of ERK signaling is discussed in the con‐ text of suppressing mesodermal programming during PGC specification.
MATERIALS AND METHODS Animals ICR mice were purchased from Nihon SLC (Shizuoka, Japan) and used for preparation of the PGCs. WBB6F1‐ W/Wv mice (Nihon SLC) were used for transplantation assay. Animal care was in accordance with the guide‐ lines of Osaka University and Kyoto University for ani‐ mal welfare.
Cell Lines The male Blimp1‐mVenus and stella‐ECFP (BVSC) ESCs were established from the embryo carrying Blimp1 (Prdm1)‐promoter driven Venus and Stella (Dppa3)‐ promoter driven CFP, which was backcrossed to C57/BL6 mice more than six times [11]. The OP9 feeder cells were used for mesodermal differentiation of the ESCs [20, 21]. The Sl/Sl‐m220 feeder cells, which ex‐ press a membrane bound form of stem cell factor (SCF), were used for derivation of embryonic germ cells (EGCs) and EGC‐like cells [25].
Differentiation Induction The BVSC ESCs were maintained with Glasgow Mini‐ mum Essential Medium (Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (JRH, Lenexa, KS, USA), 1% nonessential amino acids solution (NEAA) (Gibco, Gaithersburg, MD, USA), 1 mM sodium pyruvate (Gibco), 2.75 g/L sodium bicarbonate, 75 mg/L penicillin G, 50 mg/L streptomycin, 1000 U/mL LIF, 1 μM 2‐mercaptoethanol, and 2 inhibitors (2i; 1 μM of the MEK inhibitor PD0325901 and 3 μM of the GSK3 inhibitor CHIR99021) (Stemgent, Cambridge, MA, USA). The ESCs were cultured without 2i for 3 days before differentiation induction. The ESCs were cultured on gelatin‐coated dishes. OP9 stromal cells were cultured ©AlphaMed Press 2014
3 in Minimum Essential Medium‐alpha (Gibco) supple‐ mented with 20% FCS (JRH), 1% NEAA (Gibco), 2.2 g/L sodium bicarbonate, 2 mM L‐glutamine (Gibco), 75 mg/L penicillin G, and 50 mg/L streptomycin. The mes‐ odermal differentiation induction of ESCs on OP9 stro‐ mal cells was described previously [20, 21]. The cells were analyzed using a BD FACSAria system (BD Biosci‐ ences, Franklin Lakes, NJ, USA) and photographed under an Olympus IX70 and IX71 inverted microscope (Olym‐ pus, Tokyo, Japan).
Derivation of EGCs Derivation of EGCs was performed as described previ‐ ously [25, 26]. Briefly, gonadal cell suspension was pre‐ pared from E11.5 embryos. The cells induced from the BVSC ESCs were sorted using a BD FACSAria system. The cells were seeded onto mitomycin C‐treated Sl/Sl‐m220 feeder cells and cultured with Dulbecco's modified Ea‐ gle’s medium (Gibco) supplemented with 15% knockout serum replacement (Gibco), 1% NEAA, 1 mM sodium pyruvate, 1000 U/mL LIF, 20 ng/mL bFGF (R&D Systems, Minneapolis, MN, USA) and 2 μM retinoic acid (RA; Sigma‐Aldrich) for 5 days. The EGC colonies were visual‐ ized by staining with an Alkaline Phosphatase Staining Kit (Sigma‐Aldrich). The number of adherent PGCs at 8 h post‐seeding was defined as the number of seeded PGCs. Multilayered colonies with more than 20 cells were considered to be EGC colonies, as described pre‐ viously [26, 27].
Chemicals The small molecule compounds used were the follow‐ ing: LY 294002 (Wako Pure Chemical, Osaka, Japan), PS48 (Sigma‐Aldrich), PD0325901 (Stemgent), BI‐D1870 (Enzo, Farmingdale, NY, USA), SP600125 (Wako Pure Chemical), SB203580 (Wako Pure Chemical), SB431542 (Wako Pure Chemical), LDN193189 (Stemgenet), CHIR99021 (Stemgent), and Kempaullone (Wako Pure Chemical).
Induction of PGC‐like cells by ERK inhibition aformaldehyde (PFA) in phosphate‐buffered saline (PBS) for 10 min. The fixed cells were permeabilized with 0.1% Triton X‐100 in PBS for 5 min, and blocked with 10% normal goat serum (NGS) and 3% bovine serum albumin (BSA) in PBS for 1 h. To detect 5‐ methylcytosine (5meC), the cells were treated with 2 N HCl for 10 min and neutralized with 0.1 M Tris‐HCl, pH 8, for 10 min. The primary antibodies were diluted in 10% NGS and 3% BSA and incubated overnight at 4ºC. The primary antibodies used were the following: anti‐ 5meC (1:500 dilution, 162 33 D3; BD, Calbiochem, La Jolla, CA, USA), anti‐H3K9me2 (1:200, #07‐441; Up‐ state/Millipore, Billerica, MA, USA), anti‐H3K27me3 (1:200, #07‐449; Upstate/Millipore), anti‐Ddx4 (1:1,000; ab13840; Abcam, Cambridge, UK) and anti‐SSEA‐1 (1:50, TM13; Kyowa Medex, Tokyo, Japan). Appropriate sec‐ ondary antibodies were used to detect the primary an‐ tibody complexes (1:200; A11036, Invitrogen), and the specimens were incubated with 4 ,6‐diamidino‐2‐ phenylindole (DAPI) (1 μg/mL) for 1 h. Immunofluores‐ cence was observed using an LSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
Bisulfite Sequencing The genomic DNAs were bisulfite‐treated with the EpiTect Plus DNA Bisulfite Kit (Qiagen). Fully or semi‐ nested PCR was performed to amplify the promoter regions as described previously [28, 29]. Sequences of the PCR primers are listed in Supplementary Table S2. PCR amplification was carried out with Ex Taq (Takara Bio, Shiga, Japan) under the following conditions: 1 min at 94°C followed by 35 cycles of PCR consisting of 30 sec at 94°C, 30 sec at 60°C, and 30 sec at 68°C. The PCR products were purified using QIAEXII Gel Extraction Kit (Qiagen), cloned into the pGEM‐T Easy Vector (Promega, Madison, WI), and sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Fos‐ ter City, CA).
Transplantation Assay Quantitative Reverse‐Transcription Polyme‐ rase Chain Reaction (qRT‐PCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription (RT) was performed using the ThermoScript RT‐PCR System (Invitrogen, Carlsbad, CA, USA). Quantitative polymer‐ ase chain reaction (qPCR) was performed with ABI 7900HT‐3 (SDS 2.3) (Applied Biosystems, Foster City, CA, USA) and MX3000P (Aligent Technologies, Santa Clara, CA, USA). The expression levels of each gene were nor‐ malized by expression of the housekeeping gene Rplp0 (Arbp: ribosomal protein large P0). The primer sequenc‐ es are listed in Supplementary Table S1.
Immunohistochemistry The cells were cytospun at 800 rpm for 3 min onto 3‐ aminopropyltriethoxysilane‐coated slide glasses (Matsunami Glass, Osaka, Japan) and fixed with 4% par‐ www.StemCells.com
The BV+SC+ cells were induced upon OP9 feeder cells by treatment with PD0325901 and were collected on day 4 after differentiation induction (Exp. No.1 and No.2 in Table 1). The BV+ PGC‐like cells were also induced through epiblast‐like cells (EpiLCs) under chemically defined, serum‐free condition, as described previously [11] (Exp. No.2 in Table 1). The same BVSC ES cell line was used in both Exp. No.1 and No.2. The ESCs main‐ tained under serum‐containing medium or under se‐ rum‐free condition were used in Exp. No.1 or Exp. No.2, respectively. Ten thousand cells were injected into W/WV mutant mouse testes, as described previously [30]. Two months and 10 weeks after injection (Exp. No.1 and No.2, respectively), the recipients were killed and all seminiferous tubules of the testes were carefully examined under a dissecting microscope to examine whether spermatogenesis occurred.
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RESULTS In Vitro OP9 Mesodermal Differentiation In‐ duction System In this study, we used the BVSC ESC line, which carried two reporter genes, Blimp1 (Prdm1)‐promoter driven Venus and Stella (Dppa3)‐promoter driven CFP [11]. This ES cell line was established from the transgenic mice harboring the two reporters. Since Blimp1‐Venus (BV) and Stella‐CFP (SC) reporters were expressed in PGCs from E7.5 to E13.5 and from E8.5 to E15.5, respec‐ tively [31‐33], this ESC line allowed us to visualize PGC‐ like cells in culture. The BVSC ESCs were maintained in the medium supplemented with FCS, LIF, and 2i (Fig. 1A). The cells formed round and dome‐shaped colonies (Fig. 1B). The ESCs were negative for SC expression, but approximate‐ ly 80% of the cells expressed BV under this culture con‐ dition. Before differentiation induction, the ESCs were cultured with FCS and LIF, but without 2i, for 3 days. Morphology of the colonies changed to monolayered, epiblast‐like colonies (Fig. 1B). The expression of BV and SC was not altered by the removal of 2i. The cells were then seeded onto OP9 feeder layers without LIF and 2i, and induced for mesodermal differentiation (Fig. 1A). The BVSC ESCs formed the mesodermal colonies on day 3 after seeding and did not show upregulation of the SC reporter up to day 6 in the OP9 differentiation system (Fig. 1C, upper panel).
Induction of BV+SC+ Cells by the MEK Inhibi‐ tor Using this mesodermal differentiation system, we screened the small molecule compounds that inhibit mesodermal differentiation and induce PGC‐like cell fate in BVSC ESCs. The compounds included LY 294002 (PI3K inhibitor), PS48 (PDK1 activator), PD0325901 (MEK inhibitor), BI‐D1870 (S6K inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), SB431542 (TGF /Activin inhibitor, ALK4/5/7 inhibitor), LDN193189 (BMP inhibitor, ALK2/3 inhibitor), CHIR99021 (GSK3 inhibitor), and Kempaullone (inhibitor of various kinas‐ es). Among these molecules, the MEK inhibitor alone induced BV‐highly positive and SC‐positive (BV+SC+) cells (Fig. 1C, D). The BV+SC+ cells emerged on day 1 after induction and the intensity of SC progressively increased until day 3. The percentage of the BV+SC+ cells peaked on day 3, and the number of the BV+SC+ cells peaked on days 3–6. On the other hand, preculture of the ESCs without 2i before differentiation induction was essential for the induction of the BV+SC+ cells (Figs. 1A, 2A). Gene ex‐ pression analysis of the ESCs cultured with or without 2i showed that the markers of inner cell mass (ICM) cells decreased and those of epiblast increased after removal of 2i (Fig. 2B), suggesting that the transition from ground states to epiblast‐like states occurred in the absence of 2i. This result is in accordance with a recent www.StemCells.com
Induction of PGC‐like cells by ERK inhibition study showing that differentiation from ESCs to epiblast‐like cells (EpiLCs) is critical for induction of PGC‐ like cells in vitro [11]. In the above experiments, the BVSC ESCs were cul‐ tured in the media supplemented with FCS and LIF (Fig. 1, Condition 4 in Supplementary Fig.S1). To examine whether FCS has the priming effect on the induction of the BV+SC+ cells, the ESCs were cultured with the me‐ dia supplemented with LIF and 2i, but without FCS, be‐ fore the induction (Conditions 1‐3 in Supplementary Fig.S1). In this experiment again, when the ESCs were cultured with 2i (Condition 1; –FCS, +LIF, +2i), the BV+SC+ cells could not be induced upon OP9 cells by MEK inhibitor treatment. In contrast, the ESCs precultured without 2i for 3 days differentiated to the BV+SC+ cells (Condition 2; –FCS, +LIF, –2i). Next, to ex‐ amine the priming effect of LIF, EpiLCs were induced from the BVSC ESCs by treatment with Activin and bFGF in the absence of LIF and 2i (Condition 3; –FCS, –LIF, –2i, +Activin, +bFGF). The EpiLCs also generated the BV+SC+ cells upon OP9 cells in response to MEK inhibitor. Thus, priming with FCS and LIF is not essential for the MEK inhibitor‐induction of BV+SC+ cells.
Expression of Germ Cell and Mesodermal Marker Genes After the expression of Prdm1, Prdm14 and Tcfap2c commence, the downstream PGC‐specific genes such as Dppa3, Dnd1, and Nanos3 are upregulated in nascent and migrating PGCs [2‐6]. In contrast, mesodermal genes are upregulated transiently but downregulated rapidly by the actions of Prdm1, Prdm14, and Tcfap2c in nascent PGCs. Subsequently, gonadal germ cell marker genes including Ddx4 (Mvh), Dazl, and Piwil2 (Mili) are induced in germ cells after arriving at the gonads (Sup‐ plemental Fig. S2A). We collected the BV+SC+ cells and the BV–SC– cells from MEK inhibitor‐treated cultures on days 0, 4, and 6 and compared the expression of germ cell marker genes. As shown in Figure 3A, expression levels of all the PGC marker genes increased after differentiation induction in the BV+SC+ cells, whereas these genes ex‐ cept Nanos3 were downregulated in the BV–SC– cells. In addition, the expression of gonadal germ cell markers such as Ddx4 and Dazl, but not Mili, also increased in the BV+SC+ cells (Fig. 3B). About 14% of the BV+SC+ cells were positive for Ddx4 protein (Supplementary Fig.S2B), indicating that a fraction of the BV+SC+ cells acquired the characteristics of gonadal germ cells. Mes‐ odermal genes such as Hoxa1, Hoxb1, and Snail1 were efficiently upregulated when the BVSC ESCs were cul‐ tured without the MEK inhibitor (Fig. 3C). However, induction of these genes was suppressed in the BV+SC+ cells induced by the MEK inhibitor. These results showed that mesodermal differentiation was inhibited but germline differentiation was promoted in the MEK inhibitor‐induced BV+SC+ cells. We also examined expression of the marker genes for pluripotent (Oct4, Nanog), ectodermal (Ascl1, Sox1) ©AlphaMed Press 2014
Induction of PGC‐like cells by ERK inhibition
5 and endodermal cells (GATA4, GATA6) (Supplementary Fig.S3). The BV+SC+ cells expressed Oct4 and Nanog genes strongly but ectodermal and endodermal genes weakly, further showing the PGC‐like gene expression pattern. Meanwhile, expressions of Oct4 and Nanog in BV–SC–cells were comparable to those of ESCs. Alt‐ hough expressions of Ascl1, Sox1and GATA4 were at low levels, GATA6 we moderately induced in BV–SC– cells. These results suggest that BV–SC– cells may con‐ tain immature and endodermal cells.
Formation of Pluripotent Stem Cell Colonies from BV+SC+ Cells by RA Treatment To examine whether the BV+CV+ cells could form EGC‐ like colonies in response to RA, we cultured the BV+SC+ cells with the ESC culture medium containing LIF alone or with the EGC medium supplemented with RA, bFGF, and LIF. Reportedly, ESCs and PGCs show completely opposite responsiveness to RA [27]. Namely, RA treat‐ ment induces differentiation of ESCs but enhances de‐ differentiation of PGCs into pluripotent stem cells (EGCs) when cultured in the presence of bFGF, SCF, and LIF. As expected, the BVSC ESCs formed alkaline‐ phosphatase (ALP)‐positive, undifferentiated colonies in the ESC medium. However, the cells differentiated completely when seeded onto SCF‐expressing feeder cells and cultured with the EGC medium containing RA (Fig. 4A, B). In contrast, the BV+SC+ cells as well as the PGCs isolated from E11.5 embryos formed ALP‐positive EGC‐like colonies under the EGC culture condition (Fig. 4C, D). The efficiency of EGC‐like colony formation was approximately half that of the PGCs on E11.5 (Fig. 4E). Neither the BV+SC+ cells nor the E11.5 PGCs proliferat‐ ed to from the EGC colonies in the ESC medium. Thus, the BV+SC+ cells had PGC‐like responsiveness to RA.
Epigenetic Status and Ability to Contribute to Spermatogenesis PGCs undergo extensive epigenetic reprogramming, namely global DNA demethylation and acquisition of signature‐characteristic histone modifications [1]. Ex‐ pression of de novo DNA methyltransferases Dnmt3a/b is downregulated in PGCs [7]. In addition, expression of Uhrf1, which recruits Dnmt1 to hemi‐methylated DNA, also decreases in PGCs. Because Dnmt1 is required for the maintenance of methylation in newly synthesized DNA strand after replication, downregulation of Uhrf1 causes global DNA hypomethylation during PGC differ‐ entiation. Similar to PGCs in vivo, expression of Uhrf1 and Dnmt3b was downregulated but the expression of Dnmt1 was not altered in the BV+SC+ cells (Fig. 5A). Furthermore, global methylated cytosine levels were lower in the BV+SC+ cells than the ESCs (Fig. 5B), indi‐ cating that global DNA demethylation occurred in the BV+SC+ cells. In addition, consistent with gene expres‐ sion pattern, promoter methylation levels of germ line genes (Prdm14, Stella and Ddx4) were lower in in the www.StemCells.com
BV+SC+ cells than in the ESCs (Fig. 5C). Methylation lev‐ el of Nanog gene in the BV+SC+ cells was comparable to the ESCs. Levels of H3K27me3 increase and levels of H3K9me2 decrease during PGC differentiation [9]. However, significant increase of H3K27me3 and de‐ crease of H3K9m32 was not detected in the BV+SC+ cells (data not shown). The recent study has demonstrated that the PGC‐ like cells could be induced from BVSC ESCs through EpiLCs under chemically defined, serum‐free culture condition [11]. The PGC‐like cells induced under this culture condition successfully generated functional spermatozoa when transplanted into seminiferous tu‐ bules of newborn W/WV mutant mice. The develop‐ mental potential of the MEK‐inhibitor‐induced BV+SC+ cells was examined in transplantation assay (Fig. 6, Ta‐ ble 1). Although spermatogenesis colonies were ob‐ served in the testes grafted with the PGC‐like cells in‐ duced by the previously described method, the MEK‐ inhibitor‐induced BV+SC+ cells did not generate sper‐ matogenesis colonies. Instead, teratomas with large cysts formed in one of two transplantation experi‐ ments. Thus, although the BV+SC+ cells induced in this differentiation system acquired several characteristics of PGCs, specification to germ lineage appeared to be incomplete.
DISCUSSION In this study, we demonstrated that ERK signaling inhi‐ bition by a MEK inhibitor suppressed mesodermal dif‐ ferentiation but promoted germ cell differentiation in the OP9 mesodermal differentiation system. The BV+SC+ cells induced in this study exhibited PGC‐like gene expression, dedifferentiation to the EGC‐like cells in response to RA, and DNA hypomethylation. However, the cells could not acquire the signature‐characteristic histone modification of PGCs and germline differentia‐ tion potential in vivo. The OP9 feeder cells have been widely used to sup‐ port differentiation of mesodermal cell lineages such as various hematopoietic cells, endothelial cells, and cardiomyocytes [19‐24]. In a previous study, we showed that PGC‐like cells could be induced from ESCs upon Akt signaling activation in OP9 differentiation cultures [34]. However, similar to the BV+SC+ cells in this study, the germline commitment was incomplete in these cells. Thus, these results indicate that the factors for com‐ plete germline commitment are lacking in the OP9 dif‐ ferentiation system, although OP9 cells provide germline differentiation cues to some extent. Reportedly, the two signaling pathways, BMPs and Wnt3, play pivotal roles in PGC specification [10‐14]. However, these signals promote differentiation of both germline and mesodermal lineages [14‐17]. From this viewpoint, our finding highlights a unique role of ERK signaling: ERK signaling activation promotes mesoder‐ mal differentiation whereas inhibition promotes PGC differentiation. At present, it is difficult to exclude the ©AlphaMed Press 2014
Induction of PGC‐like cells by ERK inhibition
6 possibility that the effect of ERK signaling inhibition is indirectly mediated by non‐germ cells surrounding the differentiating germ cells. However, we favor the model that ERK signaling inhibition directly promotes germ cell differentiation as discussed below. Accumulating evidence shows that the FGF–ERK signaling axis promotes mesodermal differentiation. Studies in mice and Xenopus show that FGF signaling is required for mesoderm cell fate specification [35, 36]. In Xenopus mesoderm formation, SRF and Elk‐1, tran‐ scription factors activated by ERK downstream of FGF, are required for mesodermal gene induction [37]. SRF is also essential for mesoderm formation during mouse embryogenesis [38]. In addition to the evidence ob‐ tained from mice and Xenopus, the FGF–ERK signaling axis has been shown to promote mesodermal differen‐ tiation from mouse and human ESCs in vitro [39‐41]. Taken together with our findings, ERK signaling down‐ stream of FGF is critical for mesodermal specification in vivo and in vitro. In contrast, our results show that ERK signaling in‐ hibition not only antagonizes mesodermal differentia‐ tion but also promotes PGC cell fate under an in vitro mesodermal differentiation condition. Comprehensive gene expression analysis during PGC specification shows that the negative regulators of ERK signaling, such as Dusp6, Spry1, Spry2, and Spred1, are upregulated spe‐ cifically in PGC precursors and nascent PGCs [7]. In addi‐ tion, induction of these genes was severely impaired in the PGC‐like cells of Prdm1‐deficient embryos [2, 7], indicating that the genes were under Prdm1 control. Since mesodermal genes were not downregulated properly in the PGC‐like cells of Prdm1‐deficient embry‐ os, these negative regulators of ERK signaling may have a role in the suppression of mesodermal programming during PGC specification in vivo.
CONCLUSION Our results show that ERK signaling plays a critical role in suppressing mesoderm differentiation in PGC specifi‐ cation under the influence of mesodermal signals dur‐
REFERENCES 1 Saitou M, Kagiwada S, Kurimoto K. Epige‐ netic reprogramming in mouse pre‐ implantation development and primordial germ cells. Development 2012; 139:15‐31. 2 Magnusdottir E, Dietmann S, Murakami K et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat Cell Biol 2013; 15:905‐915. 3 Nakaki F, Hayashi K, Ohta H et al. Induc‐ tion of mouse germ‐cell fate by transcription factors in vitro. Nature 2013; 501:222‐226. 4 Ohinata Y, Payer B, O'Carroll D et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 2005; 436:207‐ 213.
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ing gastrulation. Roles of the FGF–ERK signaling axis in mouse PGC specification remain unknown. Further studies on whether inhibition of the FGF–ERK signaling axis and the negative regulators of EKR signaling are involved in suppressing the mesodermal program dur‐ ing PGC specification in vivo are needed.
ACKNOWLEDGMENT We thank Ms. N. Asada for technical assistance and Ms. M. Imaizumi for secretarial assistance.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.
AUTHOR CONTRIBUTIONS T.K.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writ‐ ing, Final approval of manuscript.; Y.K.: Collection and assembly of data, Data analysis and interpretation.; H.O.: Performed transplantation experiments, Data analysis and interpretation.; M.O.: Collection and as‐ sembly of data, Data analysis and interpretation.; Y.S.: Data analysis and interpretation.; K.L.: Collection and assembly of data, Data analysis and interpretation.; N.Y.: Collection and assembly of data, Data analysis and interpretation.; K.F.: Collection and assembly of data, Data analysis and interpretation.; A.I.: Performed trans‐ plantation experiments, Data analysis and interpreta‐ tion.; N.S.: Collection and assembly of data, Data analy‐ sis.; M.T.: Collection and assembly of data, Data analy‐ sis.; K.H.: Provision of study materials, Data analysis and interpretation.; M.O.: Performed transplantation exper‐ iments, Data analysis and interpretation.; T.S.: Per‐ formed transplantation experiments, Data analysis and interpretation.; M.S.: Provision of study materials, Data analysis and interpretation.; T.N.: Conception and de‐ sign, Data analysis and interpretation, Financial support, Manuscript writing, Final approval of manuscript.
5 Vincent SD, Dunn NR, Sciammas R et al. The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Develop‐ ment 2005; 132:1315‐1325. 6 Yamaji M, Seki Y, Kurimoto K et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet 2008; 40:1016‐1022. 7 Kurimoto K, Yabuta Y, Ohinata Y et al. Complex genome‐wide transcription dynam‐ ics orchestrated by Blimp1 for the specifica‐ tion of the germ cell lineage in mice. Genes Dev 2008; 22:1617‐1635. 8 Yabuta Y, Kurimoto K, Ohinata Y et al. Gene expression dynamics during germline specification in mice identified by quantita‐
tive single‐cell gene expression profiling. Biol Reprod 2006; 75:705‐716. 9 Seki Y, Yamaji M, Yabuta Y et al. Cellular dynamics associated with the genome‐wide epigenetic reprogramming in migrating pri‐ mordial germ cells in mice. Development 2007; 134:2627‐2638. 10 Lawson KA, Dunn NR, Roelen BA et al. Bmp4 is required for the generation of pri‐ mordial germ cells in the mouse embryo. Genes Dev 1999; 13:424‐436. 11 Hayashi K, Ohta H, Kurimoto K et al. Re‐ constitution of the mouse germ cell specifica‐ tion pathway in culture by pluripotent stem cells. Cell 2011; 146:519‐532. 12 Ohinata Y, Ohta H, Shigeta M et al. A signaling principle for the specification of the germ cell lineage in mice. Cell 2009; 137:571‐ 584.
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7 13 Ying Y, Qi X, Zhao GQ. Induction of pri‐ mordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signal‐ ing pathways. Proc Natl Acad Sci U S A 2001; 98:7858‐7862. 14 Ying Y, Zhao GQ. Cooperation of endo‐ derm‐derived BMP2 and extraembryonic ectoderm‐derived BMP4 in primordial germ cell generation in the mouse. Dev Biol 2001; 232:484‐492. 15 Liu P, Wakamiya M, Shea MJ et al. Re‐ quirement for Wnt3 in vertebrate axis for‐ mation. Nat Genet 1999; 22:361‐365. 16 Winnier G, Blessing M, Labosky PA et al. Bone morphogenetic protein‐4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995; 9:2105‐2116. 17 Ying Y, Liu XM, Marble A et al. Require‐ ment of Bmp8b for the generation of primor‐ dial germ cells in the mouse. Mol Endocrinol 2000; 14:1053‐1063. 18 Plotnikov A, Zehorai E, Procaccia S et al. The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta 2011; 1813:1619‐1633. 19 Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid‐myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002; 109:29‐37. 20 Nakano T, Kodama H, Honjo T. Genera‐ tion of lymphohematopoietic cells from em‐ bryonic stem cells in culture. Science 1994; 265:1098‐1101. 21 Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Sci‐ ence 1996; 272:722‐724. 22 Oike Y, Takakura N, Hata A et al. Mice homozygous for a truncated form of CREB‐ binding protein exhibit defects in hematopoi‐ esis and vasculo‐angiogenesis. Blood 1999; 93:2771‐2779. 23 Schroeder T, Fraser ST, Ogawa M et al. Recombination signal sequence‐binding pro‐
tein Jkappa alters mesodermal cell fate deci‐ sions by suppressing cardiomyogenesis. Proc Natl Acad Sci U S A 2003; 100:4018‐4023. 24 Yanagi K, Takano M, Narazaki G et al. Hyperpolarization‐activated cyclic nucleotide‐ gated channels and T‐type calcium channels confer automaticity of embryonic stem cell‐ derived cardiomyocytes. Stem Cells 2007; 25:2712‐2719. 25 Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70:841‐847. 26 Kimura T, Tomooka M, Yamano N et al. AKT signaling promotes derivation of embry‐ onic germ cells from primordial germ cells. Development 2008; 135:869‐879. 27 Koshimizu U, Taga T, Watanabe M et al. Functional requirement of gp130‐mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells. Development 1996; 122:1235‐1242. 28 Imamura M, Miura K, Iwabuchi K et al. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells. BMC Dev Biol 2006; 6:34. 29 Maatouk DM, Kellam LD, Mann MR et al. DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell line‐ ages. Development 2006; 133:3411‐3418. 30 Chuma S, Kanatsu‐Shinohara M, Inoue K et al. Spermatogenesis from epiblast and primordial germ cells following transplanta‐ tion into postnatal mouse testis. Develop‐ ment 2005; 132:117‐122. 31 Ohinata Y, Sano M, Shigeta M et al. A comprehensive, non‐invasive visualization of primordial germ cell development in mice by the Prdm1‐mVenus and Dppa3‐ECFP double transgenic reporter. Reproduction 2008; 136:503‐514. 32 Saitou M, Barton SC, Surani MA. A mo‐ lecular programme for the specification of
germ cell fate in mice. Nature 2002; 418:293‐ 300. 33 Sato M, Kimura T, Kurokawa K et al. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech Dev 2002; 113:91‐94. 34 Yamano N, Kimura T, Watanabe‐ Kushima S et al. Metastable primordial germ cell‐like state induced from mouse embryonic stem cells by Akt activation. Biochem Biophys Res Commun 2010; 392:311‐316. 35 Ciruna B, Rossant J. FGF signaling regu‐ lates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell 2001; 1:37‐49. 36 Kimelman D, Kirschner M. Synergistic induction of mesoderm by FGF and TGF‐beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 1987; 51:869‐877. 37 Nentwich O, Dingwell KS, Nordheim A et al. Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk‐1 and Egr‐1. Dev Biol 2009; 336:313‐326. 38 Arsenian S, Weinhold B, Oelgeschlager M et al. Serum response factor is essential for mesoderm formation during mouse embryo‐ genesis. EMBO J 1998; 17:6289‐6299. 39 Bernardo AS, Faial T, Gardner L et al. BRACHYURY and CDX2 mediate BMP‐induced differentiation of human and mouse pluripo‐ tent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 2011; 9:144‐155. 40 Kraushaar DC, Rai S, Condac E et al. Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells. J Biol Chem 2012; 287:22691‐22700. 41 Yu P, Pan G, Yu J et al. FGF2 sustains NANOG and switches the outcome of BMP4‐ induced human embryonic stem cell differen‐ tiation. Cell Stem Cell 2011; 8:326‐334.
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Figure 1. Induction of the BV+SC+ cells from ESCs by the MEK inhibitor. (A) Scheme of differentiation induction. The BVSC ESCs were maintained in the medium containing FCS, LIF, and 2i. Before induction, the cells were precultured without 2i for 3 days. The cells were then seeded onto OP9 feeder cells and induced for differentiation with or without the MEK inhibitor in the absence of LIF. (B) The BVSC ESCs cultured with or without 2i formed dome‐shaped or monolayered colonies, respectively. The ESCs cultured under both conditions expressed BV weakly but not SC. (C) Flow cytometric analysis of the BV+SC+ cells. Expression levels of BV and SC reporters were examined from day 0 to day 6 after differentiation induction. The BV+SC+ cells were detected in the culture with MEKi, but not in the cul‐ ture without the MEK inhibitor. (D) Representative pictures of the BV+SC+ cells induced by the MEK inhibitor. Scale bar, 100 μm. (E) Percentages of the BV+SC+ cells after differentiation induction (n = 4, mean ± SD).
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Figure 2. Effects of preculture without 2i. (A) Induction of the BV+SC+ cells. The BVSC ESCs cultured with or without 2i before induction for 3 days were seeded onto OP9 cells. The expression of BV and SC reporters was examined on day 0 and day 3 after differentiation induc‐ tion. (B) Expression of inner cell mass (ICM) marker genes (Klf4, Prdm14, Tbx3, and Tcl1) and epiblast marker genes (Dnmt3b, Fgf5 and Otx2) in the BVSC ESCs cultured with or without 2i. The gene mRNA levels were quantified by qRT‐ PCR analysis. Representative results of two independent experiments are shown (mean ± SD).
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Figure 3. Expression of germ cell and mesoderm marker genes in the BV+SC+ cells. The mRNA levels of the PGC marker genes (A; Prdm1, Prdm14, Dppa3, Dnd1, Nanos3, and Tcfap2c), gonocyte marker genes (B; Ddx4, Dazl, and Piwil2) and mesodermal marker genes (C; Hoxa1, Hoxb1, and Snail1) were quantified by qRT‐PCR analysis. The BV+SC+ cells and the BV–SC– cells were collected on day 4 and day 6 after differentiation in‐ duction. The ESCs cultured without 2i for 3 days were used as the control on day 0. The cells induced without MEKi were also used as a positive control for mesodermal differentiation in (C). The average of two independent experi‐ ments are shown with SDs.
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Figure 4. Responsiveness of the BV+SC+ cells to RA. (A, B) The ESCs cultured in the ESC or the EGC medium. The BVSC ESCs cultured in the ESC medium containing LIF alone formed ALP‐positive, undifferentiated colonies (A). However, when seeded onto SCF‐expressing feeder cells and cultured with the EGC medium containing RA, bFGF, and LIF, the ESCs differentiated and formed ALP‐negative colonies (B). Scale bar, 100 μm. (C) ALP‐positive, EGC colonies generated from the PGCs on E11.5. When cultured in the EGC medium containing RA, the PGCs gave rise to ALP‐positive EGC colonies. (D) ALP‐positive EGC‐like cell colonies generated from the BV+SC+ cells. Similar to the PGCs on E11.5, ALP‐positive colonies were formed from the BV+SC+ cells when cultured with the EGC medium containing RA. Neither the PGCs on E11.5 nor the BV+SC+ cells proliferated in the ESC media. (E) The efficiency of forming the EGC‐like cell colonies from the BV+SC+ cells. The PGCs on E11.5 and the BV+SC+ cells were cultured under an EGC culture condition for 5 days. The EGC or EGC‐like cell colonies visualized by ALP staining were counted. The percentage of the number of EGC or EGC‐like cell colonies per number of seeded cells is shown (n = 3, mean ± SD).
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Figure 5. DNA methylation status of the BV+SC+ cells. (A) Expression of Uhrf1, Dnmt3b, and Dnmt1 genes in the BVSC ESCs, the BV+SC+ cells, and the BV–SC– cells. The gene mRNA levels were quantified by qRT‐PCR analysis. Representative results of two independent experiments are shown (mean ± SD). (B) Immunostaining with anti‐5mC antibody. The gonad cells on E13.5, the BVSC ESCs, and the BV+SC+ cells were cytospun and stained with the anti‐5meC antibody. The gonocytes were outlined in the gonad sample. The nuclei were counterstained with DAPI. Scale bar, 10 μm. (C) Promoter methylation of germ line genes (Prdm14, Stella and Ddx4) and pluripotency gene (Nanog), as deter‐ mined by bisulfite sequencing analysis.
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Figure 6. Transplantation assay. (A) Testes grafted with the PGC‐like cells. Left and middle pictures show the testes grafted with the BV+ PGC‐like cells induced under chemically defined, serum‐free culture condition (Hayashi’s method). The testes contained spermato‐ genesis colonies as shown in (B). The testis grafted with the MEK‐inhibitor induced BV+SC+ cells (right picture) showed teratomas with large cyst (white arrowhead). Bar; 2 mm. (B) Spermatogenesis colonies. Black arrows show spermatogenesis colonies generated from the PGC‐like cells in‐ duced under chemically defined, serum‐free culture condition (Hayashi’s method). Bars; 0.5 mm. (C) Testicular sperm (arrows) and round spermatids (arrow heads) isolated from the tubules containing spermatogen‐ esis colonies. Bars; 50 μm. (D) Tissues of the three germ layers generated in teratomas. Keratinized epithelium (arrow, ectoderm), muscle (mes‐ oderm), and mucosal glands (arrow, endoderm) are shown at the left, middle, and right panels, respectively. Bars; 50 μm.
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Table 1 Differentiation ability of the PGC‐like cells in transplantation assay
6
No. of tes‐ tes with teratomas 0
No. of testes with spermato‐ genesis 0
No. of spermato‐ genesis colonies in testis 0
4
0
3
1, 2, 3
3
6
0
3
1, 1, 2
5
10
4
0
0
Exp. No.
Donor cells
No. of re‐ cipients
No. of testes transplanted
1
OP9‐MEKi method, day4
3
Hayashi’s method, day4
2
Hayashi’s method, day6 OP9‐MEKi method, day4
2
The BV+SC+ PGC‐like cells were induced upon OP9 feeder cells in FCS‐containing medium with MEKi as described in the text (OP9‐MEKi method in Exp. No.1 and No.2). The BV+ PGC‐like cells were also induced through epiblast‐like cells under chemically defined, serum‐free condition, as described previously (Hayashi’s method in Exp. No.2) [11]. Ten thousand PGC‐like cells were transplanted into a testis of newborn W/Wv mice. Two months (Exp. No.1) or 10 weeks (Exp. No.2) later, the testes were isolated (see Fig. 6A). The number of spermatogenesis colonies was counted under a dissecting microscope (see Fig. 6B). The same BVSC ES cell line was used in Exp. No.1 and No.2. The ESCs maintained under serum‐containing media or under serum‐free condition were used in the Exp. No.1 or the Exp. No.2, respectively.
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Graduate School of Frontier Biosciences, 2 and Department of Pathology, Medical 6 School, Research Institute for Microbial Diseases, Osaka University, 2‐2 Yamada‐oka, 3 Suita, Osaka 565‐0871, Japan; Laboratory 4 of Molecular Embryology, Laboratory of Stem Cell Biology, Department of Bioscienc‐ es, Kitasato University School of Science, 1‐ 15‐1, Kitasato, Minami‐ku, Sagamihara, 5 Kanagawa 252‐0373, Japan; Department of 9 Anatomy and Cell Biology, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida‐kanoe‐ cho, Sakyo‐ku, Kyoto 606‐8501, Japan; 7 Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Sakaecho 35‐2, Itabashi‐ku, Tokyo 173‐0015, Japan.; 8 Precursory Research for Embryonic Science 10 and Technology (PRESTO), Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), Yoshida‐kanoe‐cho, Sakyo‐ku, 11 Kyoto 606‐8501, Japan; Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara‐cho, Shogoin Yoshida, Sakyo‐ 12 ku, Kyoto 606‐8507, Japan.; Institute for Integrated Cell‐Material Sciences, Kyoto University, Yoshida‐Ushinomiya‐cho, Sakyo‐ 13 ku, Kyoto 606‐8501, Japan.; Core Research for Evolutional Science and Technology (CREST), JST, 7 Gobancho, Chiyoda‐ku, To‐ kyo 102‐0075, Japan. Correspondence: Tohru KIMURA, PhD, Laboratory of Molecular Embryology, La‐ boratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, 1‐15‐1, Kitasato, Minami‐ku, Sagamihara, Kanagawa 252‐0373, Japan, Tel: +81‐042‐778‐8864; Fax: +81‐042‐778‐ 8864, E‐mail: tkimura@ kitasato‐u.ac.jp; Grant support: Supported in part by the Ministry of Education, Science, Sports and Culture, and by PRESTO, ERATO, CREST of JST.; The English in this document has been checked by at least two professional edi‐ tors, both native speakers of English.
Induction of Primordial Germ Cell‐Like Cells From Mouse Embryonic Stem Cells by ERK Sig‐ nal Inhibition TOHRU KIMURA1,2,3,4, YOSHIAKI KAGA1, HIROSHI OHTA5, MIKA ODAMOTO3,4, YOICHI SEKITA2, KUNPENG LI1, NORIKO YAMANO1, KEITA FUJIKAWA3,4, AYAKO ISOTANI6, NORIHIKO SASAKI7, MASASHI TOYODA7, KATSUHIKO HAYASHI5,8, MASARU OKABE6, TAKASHI SHINOHARA9, MITINORI SAITOU5,10,11,12, AND TORU NAKANO1,2,13 Key Words. Primordial Germ Cells • In Vitro Differentiation • Embryonic Stem cells • Mesoderm • ERK Signal
ABSTRACT Primordial germ cells (PGCs) are embryonic germ cell precursors. Specifica‐ tion of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces meso‐ dermal differentiation in vitro. Using this mesodermal differentiation sys‐ tem, we demonstrated that PGC‐like cells were efficiently induced from mouse ESCs by ERK signaling inhibition. Inhibition of ERK signaling by a MEK inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC‐like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK sig‐ naling suppresses mesodermal differentiation but activates germline dif‐ ferentiation program in this mesodermal differentiation system. Our find‐ ings provide a new insight into the signaling networks regulating PGC spec‐ ification. STEM CELLS 2014; 00:000–000
Received August 31, 2013; accepted for publication June 06, 2014 ©AlphaMed Press 1066‐5099/2014/$30.00/0 This article has been accepted for publica‐ tion and undergone full peer review but has not been through the copyediting, typeset‐ ting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/stem.1781
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INTRODUCTION Gametes are derived from a founder population of pri‐ mordial germ cells (PGCs) [1], and the specification of PGCs occurs under the influence of mesoderm‐inducing signals. On embryonic day (E) 7.0 in mice, PGCs emerge from a subset of epiblast cells that migrate to the extraembryonic region together with future extraembryonic mesoderm cells during gastrulation. PGCs return to the embryonic region on E7.75 and col‐ onize the genital ridges by E11.5. After settlement into the genital ridges, germ cells undergo spermatogenesis and oogenesis to produce sperm and oocytes, respec‐ tively. During specification to germ lineage cells, the gene expression profiles and epigenetic status of the cells are drastically altered by Prdm1 (Blimp1), Prdm14, and Tcfap2c (AP2 ), transcription factors essential for PGC specification [2‐6]. Germ cell‐specific genes, such as Dppa3 and Dnd1, are induced in the nascent PGCs, i.e., the PGCs that are just emerging in the extraembryonic region on E7.0 [7]. In contrast, mesoderm‐related genes are repressed in the nascent PGCs, although they are transiently induced by the mesoderm‐inducing signals [7, 8]. PGCs undergo epigenetic reprogramming, which involves genome‐wide alterations of repressive histone modification and DNA methylation patterns [9]. All the‐ se changes are presumed crucial for the determination of cell fate toward PGCs. Two signaling pathways, bone morphogenetic pro‐ tein (BMP) and Wnt play important roles in PGC specifi‐ cation. BMP4 secreted from extraembryonic ectoderm is critical for PGC formation in vivo [10]. In addition, BMP4 is sufficient to induce Prdm1 and Prdm14 in the epiblast and to promote the formation of PGC‐like cells from the epiblast [11, 12]. While BMP8b emitted from extraembryonic ectoderm is also important for PGC formation [13], its function is to restrict the inhibitory signals against BMP4 outside posterior and proximal epiblast cells [12]. In addition, BMP2 from visceral en‐ doderm appears to augment the role of BMP4 to ensure the generation of a sufficient number of PGCs [14]. Fi‐ nally, Wnt3 enables epiblast cells to respond to BMP4 to form PGCs [12]. In addition to the roles in PGC specification, these signals are involved in mesodermal differentiation. For example, Wnt3 is required for formation of the primi‐ tive streak, mesoderm, and node [15]. Mice deficient for BMP4 have little or no mesodermal tissues, truncat‐ ed or disorganized posterior structures, and a reduction in extraembryonic mesoderm [16]. Similar abnormali‐ ties in embryonic and extraembryonic mesoderm are also observed in mice lacking BMP2 or BMP8b [14, 17]. Thus, BMPs and Wnt3 regulate differentiation of both germline and mesodermal lineages in embryos from the egg cylinder to gastrulation stages. ERK signaling is a canonical mitogen‐activated pro‐ tein kinase (MAPK) signal [18]. This signaling pathway www.StemCells.com
initiates with activation of Ras GTPase by growth fac‐ tors. Ras then activates the kinase cascade, which se‐ quentially activates MAPKKK (Raf), MAPKK (MEK), and MAPK (ERK). Finally, ERK phosphorylated by MEK trans‐ locates into the nucleus and activates several transcrip‐ tion factors such as SRF and Elk‐1. The ERK signal axis is involved in various biological processes including prolif‐ eration, survival, and differentiation. In this study, we demonstrated that PGC‐like cells were induced from mouse embryonic stem cells (ESCs) in an in vitro mesodermal differentiation system using OP9 feeder cells. When seeded onto OP9 feeder cells without leukemia inhibitor factor (LIF), ESCs differenti‐ ate into a variety of mesodermal lineage cells [19‐24]. Using this mesodermal culture system, we demonstrat‐ ed that ERK signaling inhibition suppressed mesoderm differentiation but promoted induction of PGC‐like cells. A potential role of ERK signaling is discussed in the con‐ text of suppressing mesodermal programming during PGC specification.
MATERIALS AND METHODS Animals ICR mice were purchased from Nihon SLC (Shizuoka, Japan) and used for preparation of the PGCs. WBB6F1‐ W/Wv mice (Nihon SLC) were used for transplantation assay. Animal care was in accordance with the guide‐ lines of Osaka University and Kyoto University for ani‐ mal welfare.
Cell Lines The male Blimp1‐mVenus and stella‐ECFP (BVSC) ESCs were established from the embryo carrying Blimp1 (Prdm1)‐promoter driven Venus and Stella (Dppa3)‐ promoter driven CFP, which was backcrossed to C57/BL6 mice more than six times [11]. The OP9 feeder cells were used for mesodermal differentiation of the ESCs [20, 21]. The Sl/Sl‐m220 feeder cells, which ex‐ press a membrane bound form of stem cell factor (SCF), were used for derivation of embryonic germ cells (EGCs) and EGC‐like cells [25].
Differentiation Induction The BVSC ESCs were maintained with Glasgow Mini‐ mum Essential Medium (Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (JRH, Lenexa, KS, USA), 1% nonessential amino acids solution (NEAA) (Gibco, Gaithersburg, MD, USA), 1 mM sodium pyruvate (Gibco), 2.75 g/L sodium bicarbonate, 75 mg/L penicillin G, 50 mg/L streptomycin, 1000 U/mL LIF, 1 μM 2‐mercaptoethanol, and 2 inhibitors (2i; 1 μM of the MEK inhibitor PD0325901 and 3 μM of the GSK3 inhibitor CHIR99021) (Stemgent, Cambridge, MA, USA). The ESCs were cultured without 2i for 3 days before differentiation induction. The ESCs were cultured on gelatin‐coated dishes. OP9 stromal cells were cultured ©AlphaMed Press 2014
3 in Minimum Essential Medium‐alpha (Gibco) supple‐ mented with 20% FCS (JRH), 1% NEAA (Gibco), 2.2 g/L sodium bicarbonate, 2 mM L‐glutamine (Gibco), 75 mg/L penicillin G, and 50 mg/L streptomycin. The mes‐ odermal differentiation induction of ESCs on OP9 stro‐ mal cells was described previously [20, 21]. The cells were analyzed using a BD FACSAria system (BD Biosci‐ ences, Franklin Lakes, NJ, USA) and photographed under an Olympus IX70 and IX71 inverted microscope (Olym‐ pus, Tokyo, Japan).
Derivation of EGCs Derivation of EGCs was performed as described previ‐ ously [25, 26]. Briefly, gonadal cell suspension was pre‐ pared from E11.5 embryos. The cells induced from the BVSC ESCs were sorted using a BD FACSAria system. The cells were seeded onto mitomycin C‐treated Sl/Sl‐m220 feeder cells and cultured with Dulbecco's modified Ea‐ gle’s medium (Gibco) supplemented with 15% knockout serum replacement (Gibco), 1% NEAA, 1 mM sodium pyruvate, 1000 U/mL LIF, 20 ng/mL bFGF (R&D Systems, Minneapolis, MN, USA) and 2 μM retinoic acid (RA; Sigma‐Aldrich) for 5 days. The EGC colonies were visual‐ ized by staining with an Alkaline Phosphatase Staining Kit (Sigma‐Aldrich). The number of adherent PGCs at 8 h post‐seeding was defined as the number of seeded PGCs. Multilayered colonies with more than 20 cells were considered to be EGC colonies, as described pre‐ viously [26, 27].
Chemicals The small molecule compounds used were the follow‐ ing: LY 294002 (Wako Pure Chemical, Osaka, Japan), PS48 (Sigma‐Aldrich), PD0325901 (Stemgent), BI‐D1870 (Enzo, Farmingdale, NY, USA), SP600125 (Wako Pure Chemical), SB203580 (Wako Pure Chemical), SB431542 (Wako Pure Chemical), LDN193189 (Stemgenet), CHIR99021 (Stemgent), and Kempaullone (Wako Pure Chemical).
Induction of PGC‐like cells by ERK inhibition aformaldehyde (PFA) in phosphate‐buffered saline (PBS) for 10 min. The fixed cells were permeabilized with 0.1% Triton X‐100 in PBS for 5 min, and blocked with 10% normal goat serum (NGS) and 3% bovine serum albumin (BSA) in PBS for 1 h. To detect 5‐ methylcytosine (5meC), the cells were treated with 2 N HCl for 10 min and neutralized with 0.1 M Tris‐HCl, pH 8, for 10 min. The primary antibodies were diluted in 10% NGS and 3% BSA and incubated overnight at 4ºC. The primary antibodies used were the following: anti‐ 5meC (1:500 dilution, 162 33 D3; BD, Calbiochem, La Jolla, CA, USA), anti‐H3K9me2 (1:200, #07‐441; Up‐ state/Millipore, Billerica, MA, USA), anti‐H3K27me3 (1:200, #07‐449; Upstate/Millipore), anti‐Ddx4 (1:1,000; ab13840; Abcam, Cambridge, UK) and anti‐SSEA‐1 (1:50, TM13; Kyowa Medex, Tokyo, Japan). Appropriate sec‐ ondary antibodies were used to detect the primary an‐ tibody complexes (1:200; A11036, Invitrogen), and the specimens were incubated with 4 ,6‐diamidino‐2‐ phenylindole (DAPI) (1 μg/mL) for 1 h. Immunofluores‐ cence was observed using an LSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
Bisulfite Sequencing The genomic DNAs were bisulfite‐treated with the EpiTect Plus DNA Bisulfite Kit (Qiagen). Fully or semi‐ nested PCR was performed to amplify the promoter regions as described previously [28, 29]. Sequences of the PCR primers are listed in Supplementary Table S2. PCR amplification was carried out with Ex Taq (Takara Bio, Shiga, Japan) under the following conditions: 1 min at 94°C followed by 35 cycles of PCR consisting of 30 sec at 94°C, 30 sec at 60°C, and 30 sec at 68°C. The PCR products were purified using QIAEXII Gel Extraction Kit (Qiagen), cloned into the pGEM‐T Easy Vector (Promega, Madison, WI), and sequenced using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Fos‐ ter City, CA).
Transplantation Assay Quantitative Reverse‐Transcription Polyme‐ rase Chain Reaction (qRT‐PCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription (RT) was performed using the ThermoScript RT‐PCR System (Invitrogen, Carlsbad, CA, USA). Quantitative polymer‐ ase chain reaction (qPCR) was performed with ABI 7900HT‐3 (SDS 2.3) (Applied Biosystems, Foster City, CA, USA) and MX3000P (Aligent Technologies, Santa Clara, CA, USA). The expression levels of each gene were nor‐ malized by expression of the housekeeping gene Rplp0 (Arbp: ribosomal protein large P0). The primer sequenc‐ es are listed in Supplementary Table S1.
Immunohistochemistry The cells were cytospun at 800 rpm for 3 min onto 3‐ aminopropyltriethoxysilane‐coated slide glasses (Matsunami Glass, Osaka, Japan) and fixed with 4% par‐ www.StemCells.com
The BV+SC+ cells were induced upon OP9 feeder cells by treatment with PD0325901 and were collected on day 4 after differentiation induction (Exp. No.1 and No.2 in Table 1). The BV+ PGC‐like cells were also induced through epiblast‐like cells (EpiLCs) under chemically defined, serum‐free condition, as described previously [11] (Exp. No.2 in Table 1). The same BVSC ES cell line was used in both Exp. No.1 and No.2. The ESCs main‐ tained under serum‐containing medium or under se‐ rum‐free condition were used in Exp. No.1 or Exp. No.2, respectively. Ten thousand cells were injected into W/WV mutant mouse testes, as described previously [30]. Two months and 10 weeks after injection (Exp. No.1 and No.2, respectively), the recipients were killed and all seminiferous tubules of the testes were carefully examined under a dissecting microscope to examine whether spermatogenesis occurred.
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RESULTS In Vitro OP9 Mesodermal Differentiation In‐ duction System In this study, we used the BVSC ESC line, which carried two reporter genes, Blimp1 (Prdm1)‐promoter driven Venus and Stella (Dppa3)‐promoter driven CFP [11]. This ES cell line was established from the transgenic mice harboring the two reporters. Since Blimp1‐Venus (BV) and Stella‐CFP (SC) reporters were expressed in PGCs from E7.5 to E13.5 and from E8.5 to E15.5, respec‐ tively [31‐33], this ESC line allowed us to visualize PGC‐ like cells in culture. The BVSC ESCs were maintained in the medium supplemented with FCS, LIF, and 2i (Fig. 1A). The cells formed round and dome‐shaped colonies (Fig. 1B). The ESCs were negative for SC expression, but approximate‐ ly 80% of the cells expressed BV under this culture con‐ dition. Before differentiation induction, the ESCs were cultured with FCS and LIF, but without 2i, for 3 days. Morphology of the colonies changed to monolayered, epiblast‐like colonies (Fig. 1B). The expression of BV and SC was not altered by the removal of 2i. The cells were then seeded onto OP9 feeder layers without LIF and 2i, and induced for mesodermal differentiation (Fig. 1A). The BVSC ESCs formed the mesodermal colonies on day 3 after seeding and did not show upregulation of the SC reporter up to day 6 in the OP9 differentiation system (Fig. 1C, upper panel).
Induction of BV+SC+ Cells by the MEK Inhibi‐ tor Using this mesodermal differentiation system, we screened the small molecule compounds that inhibit mesodermal differentiation and induce PGC‐like cell fate in BVSC ESCs. The compounds included LY 294002 (PI3K inhibitor), PS48 (PDK1 activator), PD0325901 (MEK inhibitor), BI‐D1870 (S6K inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), SB431542 (TGF /Activin inhibitor, ALK4/5/7 inhibitor), LDN193189 (BMP inhibitor, ALK2/3 inhibitor), CHIR99021 (GSK3 inhibitor), and Kempaullone (inhibitor of various kinas‐ es). Among these molecules, the MEK inhibitor alone induced BV‐highly positive and SC‐positive (BV+SC+) cells (Fig. 1C, D). The BV+SC+ cells emerged on day 1 after induction and the intensity of SC progressively increased until day 3. The percentage of the BV+SC+ cells peaked on day 3, and the number of the BV+SC+ cells peaked on days 3–6. On the other hand, preculture of the ESCs without 2i before differentiation induction was essential for the induction of the BV+SC+ cells (Figs. 1A, 2A). Gene ex‐ pression analysis of the ESCs cultured with or without 2i showed that the markers of inner cell mass (ICM) cells decreased and those of epiblast increased after removal of 2i (Fig. 2B), suggesting that the transition from ground states to epiblast‐like states occurred in the absence of 2i. This result is in accordance with a recent www.StemCells.com
Induction of PGC‐like cells by ERK inhibition study showing that differentiation from ESCs to epiblast‐like cells (EpiLCs) is critical for induction of PGC‐ like cells in vitro [11]. In the above experiments, the BVSC ESCs were cul‐ tured in the media supplemented with FCS and LIF (Fig. 1, Condition 4 in Supplementary Fig.S1). To examine whether FCS has the priming effect on the induction of the BV+SC+ cells, the ESCs were cultured with the me‐ dia supplemented with LIF and 2i, but without FCS, be‐ fore the induction (Conditions 1‐3 in Supplementary Fig.S1). In this experiment again, when the ESCs were cultured with 2i (Condition 1; –FCS, +LIF, +2i), the BV+SC+ cells could not be induced upon OP9 cells by MEK inhibitor treatment. In contrast, the ESCs precultured without 2i for 3 days differentiated to the BV+SC+ cells (Condition 2; –FCS, +LIF, –2i). Next, to ex‐ amine the priming effect of LIF, EpiLCs were induced from the BVSC ESCs by treatment with Activin and bFGF in the absence of LIF and 2i (Condition 3; –FCS, –LIF, –2i, +Activin, +bFGF). The EpiLCs also generated the BV+SC+ cells upon OP9 cells in response to MEK inhibitor. Thus, priming with FCS and LIF is not essential for the MEK inhibitor‐induction of BV+SC+ cells.
Expression of Germ Cell and Mesodermal Marker Genes After the expression of Prdm1, Prdm14 and Tcfap2c commence, the downstream PGC‐specific genes such as Dppa3, Dnd1, and Nanos3 are upregulated in nascent and migrating PGCs [2‐6]. In contrast, mesodermal genes are upregulated transiently but downregulated rapidly by the actions of Prdm1, Prdm14, and Tcfap2c in nascent PGCs. Subsequently, gonadal germ cell marker genes including Ddx4 (Mvh), Dazl, and Piwil2 (Mili) are induced in germ cells after arriving at the gonads (Sup‐ plemental Fig. S2A). We collected the BV+SC+ cells and the BV–SC– cells from MEK inhibitor‐treated cultures on days 0, 4, and 6 and compared the expression of germ cell marker genes. As shown in Figure 3A, expression levels of all the PGC marker genes increased after differentiation induction in the BV+SC+ cells, whereas these genes ex‐ cept Nanos3 were downregulated in the BV–SC– cells. In addition, the expression of gonadal germ cell markers such as Ddx4 and Dazl, but not Mili, also increased in the BV+SC+ cells (Fig. 3B). About 14% of the BV+SC+ cells were positive for Ddx4 protein (Supplementary Fig.S2B), indicating that a fraction of the BV+SC+ cells acquired the characteristics of gonadal germ cells. Mes‐ odermal genes such as Hoxa1, Hoxb1, and Snail1 were efficiently upregulated when the BVSC ESCs were cul‐ tured without the MEK inhibitor (Fig. 3C). However, induction of these genes was suppressed in the BV+SC+ cells induced by the MEK inhibitor. These results showed that mesodermal differentiation was inhibited but germline differentiation was promoted in the MEK inhibitor‐induced BV+SC+ cells. We also examined expression of the marker genes for pluripotent (Oct4, Nanog), ectodermal (Ascl1, Sox1) ©AlphaMed Press 2014
Induction of PGC‐like cells by ERK inhibition
5 and endodermal cells (GATA4, GATA6) (Supplementary Fig.S3). The BV+SC+ cells expressed Oct4 and Nanog genes strongly but ectodermal and endodermal genes weakly, further showing the PGC‐like gene expression pattern. Meanwhile, expressions of Oct4 and Nanog in BV–SC–cells were comparable to those of ESCs. Alt‐ hough expressions of Ascl1, Sox1and GATA4 were at low levels, GATA6 we moderately induced in BV–SC– cells. These results suggest that BV–SC– cells may con‐ tain immature and endodermal cells.
Formation of Pluripotent Stem Cell Colonies from BV+SC+ Cells by RA Treatment To examine whether the BV+CV+ cells could form EGC‐ like colonies in response to RA, we cultured the BV+SC+ cells with the ESC culture medium containing LIF alone or with the EGC medium supplemented with RA, bFGF, and LIF. Reportedly, ESCs and PGCs show completely opposite responsiveness to RA [27]. Namely, RA treat‐ ment induces differentiation of ESCs but enhances de‐ differentiation of PGCs into pluripotent stem cells (EGCs) when cultured in the presence of bFGF, SCF, and LIF. As expected, the BVSC ESCs formed alkaline‐ phosphatase (ALP)‐positive, undifferentiated colonies in the ESC medium. However, the cells differentiated completely when seeded onto SCF‐expressing feeder cells and cultured with the EGC medium containing RA (Fig. 4A, B). In contrast, the BV+SC+ cells as well as the PGCs isolated from E11.5 embryos formed ALP‐positive EGC‐like colonies under the EGC culture condition (Fig. 4C, D). The efficiency of EGC‐like colony formation was approximately half that of the PGCs on E11.5 (Fig. 4E). Neither the BV+SC+ cells nor the E11.5 PGCs proliferat‐ ed to from the EGC colonies in the ESC medium. Thus, the BV+SC+ cells had PGC‐like responsiveness to RA.
Epigenetic Status and Ability to Contribute to Spermatogenesis PGCs undergo extensive epigenetic reprogramming, namely global DNA demethylation and acquisition of signature‐characteristic histone modifications [1]. Ex‐ pression of de novo DNA methyltransferases Dnmt3a/b is downregulated in PGCs [7]. In addition, expression of Uhrf1, which recruits Dnmt1 to hemi‐methylated DNA, also decreases in PGCs. Because Dnmt1 is required for the maintenance of methylation in newly synthesized DNA strand after replication, downregulation of Uhrf1 causes global DNA hypomethylation during PGC differ‐ entiation. Similar to PGCs in vivo, expression of Uhrf1 and Dnmt3b was downregulated but the expression of Dnmt1 was not altered in the BV+SC+ cells (Fig. 5A). Furthermore, global methylated cytosine levels were lower in the BV+SC+ cells than the ESCs (Fig. 5B), indi‐ cating that global DNA demethylation occurred in the BV+SC+ cells. In addition, consistent with gene expres‐ sion pattern, promoter methylation levels of germ line genes (Prdm14, Stella and Ddx4) were lower in in the www.StemCells.com
BV+SC+ cells than in the ESCs (Fig. 5C). Methylation lev‐ el of Nanog gene in the BV+SC+ cells was comparable to the ESCs. Levels of H3K27me3 increase and levels of H3K9me2 decrease during PGC differentiation [9]. However, significant increase of H3K27me3 and de‐ crease of H3K9m32 was not detected in the BV+SC+ cells (data not shown). The recent study has demonstrated that the PGC‐ like cells could be induced from BVSC ESCs through EpiLCs under chemically defined, serum‐free culture condition [11]. The PGC‐like cells induced under this culture condition successfully generated functional spermatozoa when transplanted into seminiferous tu‐ bules of newborn W/WV mutant mice. The develop‐ mental potential of the MEK‐inhibitor‐induced BV+SC+ cells was examined in transplantation assay (Fig. 6, Ta‐ ble 1). Although spermatogenesis colonies were ob‐ served in the testes grafted with the PGC‐like cells in‐ duced by the previously described method, the MEK‐ inhibitor‐induced BV+SC+ cells did not generate sper‐ matogenesis colonies. Instead, teratomas with large cysts formed in one of two transplantation experi‐ ments. Thus, although the BV+SC+ cells induced in this differentiation system acquired several characteristics of PGCs, specification to germ lineage appeared to be incomplete.
DISCUSSION In this study, we demonstrated that ERK signaling inhi‐ bition by a MEK inhibitor suppressed mesodermal dif‐ ferentiation but promoted germ cell differentiation in the OP9 mesodermal differentiation system. The BV+SC+ cells induced in this study exhibited PGC‐like gene expression, dedifferentiation to the EGC‐like cells in response to RA, and DNA hypomethylation. However, the cells could not acquire the signature‐characteristic histone modification of PGCs and germline differentia‐ tion potential in vivo. The OP9 feeder cells have been widely used to sup‐ port differentiation of mesodermal cell lineages such as various hematopoietic cells, endothelial cells, and cardiomyocytes [19‐24]. In a previous study, we showed that PGC‐like cells could be induced from ESCs upon Akt signaling activation in OP9 differentiation cultures [34]. However, similar to the BV+SC+ cells in this study, the germline commitment was incomplete in these cells. Thus, these results indicate that the factors for com‐ plete germline commitment are lacking in the OP9 dif‐ ferentiation system, although OP9 cells provide germline differentiation cues to some extent. Reportedly, the two signaling pathways, BMPs and Wnt3, play pivotal roles in PGC specification [10‐14]. However, these signals promote differentiation of both germline and mesodermal lineages [14‐17]. From this viewpoint, our finding highlights a unique role of ERK signaling: ERK signaling activation promotes mesoder‐ mal differentiation whereas inhibition promotes PGC differentiation. At present, it is difficult to exclude the ©AlphaMed Press 2014
Induction of PGC‐like cells by ERK inhibition
6 possibility that the effect of ERK signaling inhibition is indirectly mediated by non‐germ cells surrounding the differentiating germ cells. However, we favor the model that ERK signaling inhibition directly promotes germ cell differentiation as discussed below. Accumulating evidence shows that the FGF–ERK signaling axis promotes mesodermal differentiation. Studies in mice and Xenopus show that FGF signaling is required for mesoderm cell fate specification [35, 36]. In Xenopus mesoderm formation, SRF and Elk‐1, tran‐ scription factors activated by ERK downstream of FGF, are required for mesodermal gene induction [37]. SRF is also essential for mesoderm formation during mouse embryogenesis [38]. In addition to the evidence ob‐ tained from mice and Xenopus, the FGF–ERK signaling axis has been shown to promote mesodermal differen‐ tiation from mouse and human ESCs in vitro [39‐41]. Taken together with our findings, ERK signaling down‐ stream of FGF is critical for mesodermal specification in vivo and in vitro. In contrast, our results show that ERK signaling in‐ hibition not only antagonizes mesodermal differentia‐ tion but also promotes PGC cell fate under an in vitro mesodermal differentiation condition. Comprehensive gene expression analysis during PGC specification shows that the negative regulators of ERK signaling, such as Dusp6, Spry1, Spry2, and Spred1, are upregulated spe‐ cifically in PGC precursors and nascent PGCs [7]. In addi‐ tion, induction of these genes was severely impaired in the PGC‐like cells of Prdm1‐deficient embryos [2, 7], indicating that the genes were under Prdm1 control. Since mesodermal genes were not downregulated properly in the PGC‐like cells of Prdm1‐deficient embry‐ os, these negative regulators of ERK signaling may have a role in the suppression of mesodermal programming during PGC specification in vivo.
CONCLUSION Our results show that ERK signaling plays a critical role in suppressing mesoderm differentiation in PGC specifi‐ cation under the influence of mesodermal signals dur‐
REFERENCES 1 Saitou M, Kagiwada S, Kurimoto K. Epige‐ netic reprogramming in mouse pre‐ implantation development and primordial germ cells. Development 2012; 139:15‐31. 2 Magnusdottir E, Dietmann S, Murakami K et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat Cell Biol 2013; 15:905‐915. 3 Nakaki F, Hayashi K, Ohta H et al. Induc‐ tion of mouse germ‐cell fate by transcription factors in vitro. Nature 2013; 501:222‐226. 4 Ohinata Y, Payer B, O'Carroll D et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 2005; 436:207‐ 213.
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ing gastrulation. Roles of the FGF–ERK signaling axis in mouse PGC specification remain unknown. Further studies on whether inhibition of the FGF–ERK signaling axis and the negative regulators of EKR signaling are involved in suppressing the mesodermal program dur‐ ing PGC specification in vivo are needed.
ACKNOWLEDGMENT We thank Ms. N. Asada for technical assistance and Ms. M. Imaizumi for secretarial assistance.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.
AUTHOR CONTRIBUTIONS T.K.: Conception and design, Collection and assembly of data, Data analysis and interpretation, Manuscript writ‐ ing, Final approval of manuscript.; Y.K.: Collection and assembly of data, Data analysis and interpretation.; H.O.: Performed transplantation experiments, Data analysis and interpretation.; M.O.: Collection and as‐ sembly of data, Data analysis and interpretation.; Y.S.: Data analysis and interpretation.; K.L.: Collection and assembly of data, Data analysis and interpretation.; N.Y.: Collection and assembly of data, Data analysis and interpretation.; K.F.: Collection and assembly of data, Data analysis and interpretation.; A.I.: Performed trans‐ plantation experiments, Data analysis and interpreta‐ tion.; N.S.: Collection and assembly of data, Data analy‐ sis.; M.T.: Collection and assembly of data, Data analy‐ sis.; K.H.: Provision of study materials, Data analysis and interpretation.; M.O.: Performed transplantation exper‐ iments, Data analysis and interpretation.; T.S.: Per‐ formed transplantation experiments, Data analysis and interpretation.; M.S.: Provision of study materials, Data analysis and interpretation.; T.N.: Conception and de‐ sign, Data analysis and interpretation, Financial support, Manuscript writing, Final approval of manuscript.
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7 13 Ying Y, Qi X, Zhao GQ. Induction of pri‐ mordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signal‐ ing pathways. Proc Natl Acad Sci U S A 2001; 98:7858‐7862. 14 Ying Y, Zhao GQ. Cooperation of endo‐ derm‐derived BMP2 and extraembryonic ectoderm‐derived BMP4 in primordial germ cell generation in the mouse. Dev Biol 2001; 232:484‐492. 15 Liu P, Wakamiya M, Shea MJ et al. Re‐ quirement for Wnt3 in vertebrate axis for‐ mation. Nat Genet 1999; 22:361‐365. 16 Winnier G, Blessing M, Labosky PA et al. Bone morphogenetic protein‐4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995; 9:2105‐2116. 17 Ying Y, Liu XM, Marble A et al. Require‐ ment of Bmp8b for the generation of primor‐ dial germ cells in the mouse. Mol Endocrinol 2000; 14:1053‐1063. 18 Plotnikov A, Zehorai E, Procaccia S et al. The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta 2011; 1813:1619‐1633. 19 Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid‐myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002; 109:29‐37. 20 Nakano T, Kodama H, Honjo T. Genera‐ tion of lymphohematopoietic cells from em‐ bryonic stem cells in culture. Science 1994; 265:1098‐1101. 21 Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Sci‐ ence 1996; 272:722‐724. 22 Oike Y, Takakura N, Hata A et al. Mice homozygous for a truncated form of CREB‐ binding protein exhibit defects in hematopoi‐ esis and vasculo‐angiogenesis. Blood 1999; 93:2771‐2779. 23 Schroeder T, Fraser ST, Ogawa M et al. Recombination signal sequence‐binding pro‐
tein Jkappa alters mesodermal cell fate deci‐ sions by suppressing cardiomyogenesis. Proc Natl Acad Sci U S A 2003; 100:4018‐4023. 24 Yanagi K, Takano M, Narazaki G et al. Hyperpolarization‐activated cyclic nucleotide‐ gated channels and T‐type calcium channels confer automaticity of embryonic stem cell‐ derived cardiomyocytes. Stem Cells 2007; 25:2712‐2719. 25 Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70:841‐847. 26 Kimura T, Tomooka M, Yamano N et al. AKT signaling promotes derivation of embry‐ onic germ cells from primordial germ cells. Development 2008; 135:869‐879. 27 Koshimizu U, Taga T, Watanabe M et al. Functional requirement of gp130‐mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells. Development 1996; 122:1235‐1242. 28 Imamura M, Miura K, Iwabuchi K et al. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells. BMC Dev Biol 2006; 6:34. 29 Maatouk DM, Kellam LD, Mann MR et al. DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell line‐ ages. Development 2006; 133:3411‐3418. 30 Chuma S, Kanatsu‐Shinohara M, Inoue K et al. Spermatogenesis from epiblast and primordial germ cells following transplanta‐ tion into postnatal mouse testis. Develop‐ ment 2005; 132:117‐122. 31 Ohinata Y, Sano M, Shigeta M et al. A comprehensive, non‐invasive visualization of primordial germ cell development in mice by the Prdm1‐mVenus and Dppa3‐ECFP double transgenic reporter. Reproduction 2008; 136:503‐514. 32 Saitou M, Barton SC, Surani MA. A mo‐ lecular programme for the specification of
germ cell fate in mice. Nature 2002; 418:293‐ 300. 33 Sato M, Kimura T, Kurokawa K et al. Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells. Mech Dev 2002; 113:91‐94. 34 Yamano N, Kimura T, Watanabe‐ Kushima S et al. Metastable primordial germ cell‐like state induced from mouse embryonic stem cells by Akt activation. Biochem Biophys Res Commun 2010; 392:311‐316. 35 Ciruna B, Rossant J. FGF signaling regu‐ lates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell 2001; 1:37‐49. 36 Kimelman D, Kirschner M. Synergistic induction of mesoderm by FGF and TGF‐beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 1987; 51:869‐877. 37 Nentwich O, Dingwell KS, Nordheim A et al. Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk‐1 and Egr‐1. Dev Biol 2009; 336:313‐326. 38 Arsenian S, Weinhold B, Oelgeschlager M et al. Serum response factor is essential for mesoderm formation during mouse embryo‐ genesis. EMBO J 1998; 17:6289‐6299. 39 Bernardo AS, Faial T, Gardner L et al. BRACHYURY and CDX2 mediate BMP‐induced differentiation of human and mouse pluripo‐ tent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 2011; 9:144‐155. 40 Kraushaar DC, Rai S, Condac E et al. Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells. J Biol Chem 2012; 287:22691‐22700. 41 Yu P, Pan G, Yu J et al. FGF2 sustains NANOG and switches the outcome of BMP4‐ induced human embryonic stem cell differen‐ tiation. Cell Stem Cell 2011; 8:326‐334.
See www.StemCells.com for supporting information available online. STEM CELLS ; 00:000–000
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Figure 1. Induction of the BV+SC+ cells from ESCs by the MEK inhibitor. (A) Scheme of differentiation induction. The BVSC ESCs were maintained in the medium containing FCS, LIF, and 2i. Before induction, the cells were precultured without 2i for 3 days. The cells were then seeded onto OP9 feeder cells and induced for differentiation with or without the MEK inhibitor in the absence of LIF. (B) The BVSC ESCs cultured with or without 2i formed dome‐shaped or monolayered colonies, respectively. The ESCs cultured under both conditions expressed BV weakly but not SC. (C) Flow cytometric analysis of the BV+SC+ cells. Expression levels of BV and SC reporters were examined from day 0 to day 6 after differentiation induction. The BV+SC+ cells were detected in the culture with MEKi, but not in the cul‐ ture without the MEK inhibitor. (D) Representative pictures of the BV+SC+ cells induced by the MEK inhibitor. Scale bar, 100 μm. (E) Percentages of the BV+SC+ cells after differentiation induction (n = 4, mean ± SD).
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Figure 2. Effects of preculture without 2i. (A) Induction of the BV+SC+ cells. The BVSC ESCs cultured with or without 2i before induction for 3 days were seeded onto OP9 cells. The expression of BV and SC reporters was examined on day 0 and day 3 after differentiation induc‐ tion. (B) Expression of inner cell mass (ICM) marker genes (Klf4, Prdm14, Tbx3, and Tcl1) and epiblast marker genes (Dnmt3b, Fgf5 and Otx2) in the BVSC ESCs cultured with or without 2i. The gene mRNA levels were quantified by qRT‐ PCR analysis. Representative results of two independent experiments are shown (mean ± SD).
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Induction of PGC‐like cells by ERK inhibition
Figure 3. Expression of germ cell and mesoderm marker genes in the BV+SC+ cells. The mRNA levels of the PGC marker genes (A; Prdm1, Prdm14, Dppa3, Dnd1, Nanos3, and Tcfap2c), gonocyte marker genes (B; Ddx4, Dazl, and Piwil2) and mesodermal marker genes (C; Hoxa1, Hoxb1, and Snail1) were quantified by qRT‐PCR analysis. The BV+SC+ cells and the BV–SC– cells were collected on day 4 and day 6 after differentiation in‐ duction. The ESCs cultured without 2i for 3 days were used as the control on day 0. The cells induced without MEKi were also used as a positive control for mesodermal differentiation in (C). The average of two independent experi‐ ments are shown with SDs.
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Figure 4. Responsiveness of the BV+SC+ cells to RA. (A, B) The ESCs cultured in the ESC or the EGC medium. The BVSC ESCs cultured in the ESC medium containing LIF alone formed ALP‐positive, undifferentiated colonies (A). However, when seeded onto SCF‐expressing feeder cells and cultured with the EGC medium containing RA, bFGF, and LIF, the ESCs differentiated and formed ALP‐negative colonies (B). Scale bar, 100 μm. (C) ALP‐positive, EGC colonies generated from the PGCs on E11.5. When cultured in the EGC medium containing RA, the PGCs gave rise to ALP‐positive EGC colonies. (D) ALP‐positive EGC‐like cell colonies generated from the BV+SC+ cells. Similar to the PGCs on E11.5, ALP‐positive colonies were formed from the BV+SC+ cells when cultured with the EGC medium containing RA. Neither the PGCs on E11.5 nor the BV+SC+ cells proliferated in the ESC media. (E) The efficiency of forming the EGC‐like cell colonies from the BV+SC+ cells. The PGCs on E11.5 and the BV+SC+ cells were cultured under an EGC culture condition for 5 days. The EGC or EGC‐like cell colonies visualized by ALP staining were counted. The percentage of the number of EGC or EGC‐like cell colonies per number of seeded cells is shown (n = 3, mean ± SD).
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Figure 5. DNA methylation status of the BV+SC+ cells. (A) Expression of Uhrf1, Dnmt3b, and Dnmt1 genes in the BVSC ESCs, the BV+SC+ cells, and the BV–SC– cells. The gene mRNA levels were quantified by qRT‐PCR analysis. Representative results of two independent experiments are shown (mean ± SD). (B) Immunostaining with anti‐5mC antibody. The gonad cells on E13.5, the BVSC ESCs, and the BV+SC+ cells were cytospun and stained with the anti‐5meC antibody. The gonocytes were outlined in the gonad sample. The nuclei were counterstained with DAPI. Scale bar, 10 μm. (C) Promoter methylation of germ line genes (Prdm14, Stella and Ddx4) and pluripotency gene (Nanog), as deter‐ mined by bisulfite sequencing analysis.
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Induction of PGC‐like cells by ERK inhibition
Figure 6. Transplantation assay. (A) Testes grafted with the PGC‐like cells. Left and middle pictures show the testes grafted with the BV+ PGC‐like cells induced under chemically defined, serum‐free culture condition (Hayashi’s method). The testes contained spermato‐ genesis colonies as shown in (B). The testis grafted with the MEK‐inhibitor induced BV+SC+ cells (right picture) showed teratomas with large cyst (white arrowhead). Bar; 2 mm. (B) Spermatogenesis colonies. Black arrows show spermatogenesis colonies generated from the PGC‐like cells in‐ duced under chemically defined, serum‐free culture condition (Hayashi’s method). Bars; 0.5 mm. (C) Testicular sperm (arrows) and round spermatids (arrow heads) isolated from the tubules containing spermatogen‐ esis colonies. Bars; 50 μm. (D) Tissues of the three germ layers generated in teratomas. Keratinized epithelium (arrow, ectoderm), muscle (mes‐ oderm), and mucosal glands (arrow, endoderm) are shown at the left, middle, and right panels, respectively. Bars; 50 μm.
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Table 1 Differentiation ability of the PGC‐like cells in transplantation assay
6
No. of tes‐ tes with teratomas 0
No. of testes with spermato‐ genesis 0
No. of spermato‐ genesis colonies in testis 0
4
0
3
1, 2, 3
3
6
0
3
1, 1, 2
5
10
4
0
0
Exp. No.
Donor cells
No. of re‐ cipients
No. of testes transplanted
1
OP9‐MEKi method, day4
3
Hayashi’s method, day4
2
Hayashi’s method, day6 OP9‐MEKi method, day4
2
The BV+SC+ PGC‐like cells were induced upon OP9 feeder cells in FCS‐containing medium with MEKi as described in the text (OP9‐MEKi method in Exp. No.1 and No.2). The BV+ PGC‐like cells were also induced through epiblast‐like cells under chemically defined, serum‐free condition, as described previously (Hayashi’s method in Exp. No.2) [11]. Ten thousand PGC‐like cells were transplanted into a testis of newborn W/Wv mice. Two months (Exp. No.1) or 10 weeks (Exp. No.2) later, the testes were isolated (see Fig. 6A). The number of spermatogenesis colonies was counted under a dissecting microscope (see Fig. 6B). The same BVSC ES cell line was used in Exp. No.1 and No.2. The ESCs maintained under serum‐containing media or under serum‐free condition were used in the Exp. No.1 or the Exp. No.2, respectively.
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