Differentiation 89 (2015) 42–50

Contents lists available at ScienceDirect

Differentiation journal homepage: www.elsevier.com/locate/diff

Critical role of Rgs19 in mouse embryonic stem cell proliferation and differentiation$ Young Rae Ji a,1, Hei Jung Kim a,1, Si Jun Park a,1, Ki Beom Bae a, Seo Jin Park a, Woo Young Jang a, Min-Cheol Kang a, Jain Jeong a, Yong Hun Sung a, Minjee Choi a, Wonyoung Lee a, Dong Gun Lee a, Sang-Joon Park b, Sanggyu Lee a, Myoung Ok Kim c, Zae Young Ryoo a,n,1 a School of Life Science, BK21 plus KNU Creative Bio Research Group, College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu, 702-701, Republic of Korea b Department of Histology, College of Veterinary Medicine, Kyungpook National University, Buk-ku, Daegu, Republic of Korea c Department of Animal Science, Kyungpook National University, Sangju, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 25 August 2014 Received in revised form 20 January 2015 Accepted 23 January 2015 Available online 9 March 2015

Mouse embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the ability to differentiate into the three germ layers required to form all embryonic tissues. These properties are maintained by both intrinsic and extrinsic factors. Many studies have contributed to the understanding of the molecular signal transduction required for pluripotency and controlled differentiation. Such an understanding is important in the potential application of stem cells to cell therapy for disease, and thus there is an interest in understanding the cell cycle regulation, pluripotency, and differentiation of ESCs. The regulator of G protein signaling (RGS) family consists of over 20 members. Rgs19, one such protein, specifically interacts with Gαi to enhance its GTPase activity. Growth factor receptors use Gi proteins for signal transduction, and Rgs19 may thus be involved in the regulation of cell proliferation. In a previous gain-of-function study, Rgs19 overexpression was found to enhance proliferation in various cell types. Our data demonstrate a role for Rgs19 in the regulation of ESC differentiation. Based on the presence of Rgs19 in ESCs, the morphological and molecular properties of wild-type and Rgs19 þ /  ESCs during LIF withdrawal, in vitro differentiation, and teratoma formation were compared. Our findings provide insight for the first time into the mechanisms involved in Rgs19 regulation of mouse ESC proliferation and differentiation. & 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Keywords: Rgs19 Embryonic stem cell Cell proliferation Cell cycle Cell differentiation Pluripotency

1. Introduction Mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts exhibit pluripotency and self-renewal properties. ESCs consequently have the ability to differentiate and form the tree germ layer tissues of the embryo (Evans and Kaufman, 1981; Martin, 1981). The pluripotent state and capacity for self-renewal of ESCs are maintained by extrinsic and intrinsic factors. The transcription factor Oct4 is a key regulator of the pluripotent state (Niwa et al., 2000). In cooperation with the transcription factor Sox2, Oct4 activates the transcription of self-renewal and pluripotency-related genes, while suppressing the expression of genes associated with differentiation (Masui et al., 2007; Niwa, 2007). Nanog is another

☆

Join the International Society for Differentiation (www.isdifferentiation.org). Corresponding author. Tel.: þ 82 53 950 7361; fax: þ82 53 943 6925. E-mail address: [email protected] (Z.Y. Ryoo). 1 These authors contributed equally to this work. n

important regulator which is specifically expressed in pluripotent cells including ESCs. The above-mentioned regulators were recently shown to reprogram differentiated somatic cells, together with other factors such as Klf4, c-Myc, and Lin28 (Kuroda et al., 2005; Yu et al., 2007). The elucidation of the molecular signal transduction mechanisms involved in the maintenance of pluripotency as well as the manipulated differentiation of ESCs for use in cell therapies for human disease is interesting and important research focuses. ESCs can also be used as a model of lineage determination in embryonic development, simplifying what is usually considered a complex and inaccessible process. Over the last few years, a number of extrinsic factors affecting ESC self-renewal have been identified. Further identification and characterization of the small molecules that allow for the precise regulation of ESC self-renewal would contribute to a greater understanding of the molecular mechanisms involved in maintaining ESC pluripotency. Although the critical requirements of a defined medium are understood, the full complements of ESC culture media

http://dx.doi.org/10.1016/j.diff.2015.01.002 Join the International Society for Differentiation (www.isdifferentiation.org) 0301-4681/& 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

remain to be uncovered, because of either their undefined composition or their suboptimal performance. In order to develop a defined culture medium that maintains ESC self-renewal, the signals and mechanisms controlling ESC fate must be understood. ESC pluripotency can be maintained in a feeder layer-free state by treatment with leukemia inhibitory factor (LIF) which signals via Stat-3 (Smith and Crompton, 1998; Williams et al., 1988). Many other signaling pathways (e.g., PI3 kinase/AKT, bone morphogenetic protein [BMP], MAPK-ERK kinases, and Wnt pathways) have also been suggested to be sufficient for the maintenance of ESC pluripotency; conversely, however, the regulation of these pathways has been reported to specify the differentiation of ESC into the three germ layers (Lee et al., 2009; Paling et al., 2004; Qi et al., 2004). To retain their identity and characteristics, ESCs must remain in a proliferative state. Progression through the mammalian cell cycle is driven by hetero-dimeric kinase complexes consisting of various subunits including cyclin and the catalytic subunit cyclin-dependent kinase (CDK). Various cyclin–CDK complexes differentially regulate cell cycle transitions. The complex of cyclin E and CDK2 or cyclin D and CDK4/6, for example, facilitates G1 progression, while the cyclinA– CDK2 complex facilitates S-phase progression, and cyclin A/B–Cdc2 facilitates M-phase entry. CDK2, in particular, plays a critical role in the cell cycle of ESCs (Neganova et al., 2009). ESCs exhibit unusual cell cycle features: the length of the S-phase is comparable to that of common cells, whereas, compared with common cells, ESCs have noticeably shorter G1 and G2 phases as well as a higher proliferation rate (Fujii-Yamamoto et al., 2005; Savatier et al., 2002; White et al., 2005). These properties may serve to suppress differentiation and maintain ESC pluripotency (Burdon et al., 2002; He et al., 2009; Lange and Calegari, 2010; Singh and Dalton, 2009). Unlike progenitors, ESCs do not exit the cell cycle during the early differentiation process. How cell cycle regulation is related to ESC differentiation is presently unclear; and plausible, critical evidence for this mechanism is limited and ambiguous. In a previous study on D3 mouse ESCs, treatment with CDK inhibitors was found to have no effect on the pluripotency marker genes in these cells (Stead et al., 2002). CDK2 is the main CDK regulating the G1/S state in ESCs, and in a study using CDK2-knockout mice, CDK2-knockout ESCs were found to be viable, indicating that these stem cells may not differentiate prematurely (Berthet et al., 2003; Ortega et al., 2003). On the other hand, a recent report demonstrated that a CDK inhibitor could induce transcript activation of marker genes for differentiation in mouse ESCs and that treatment with CDK2 siRNA could induce a morphology similar to that of differentiated cells (Koledova et al., 2010). In some reports human ESCs, CDK inhibitors have been found to result in decreased pluripotency marker expression; however, the results did not have consistency. These studies demonstrate that mouse ESCs can maintain their undifferentiated state despite a greatly prolonged G1 phase, and therefore that G1 elongation does not activate the progression of differentiation. A clear understanding of the relationship between cell cycle progression and differentiation is important for understanding the proper control of growth and differentiation of ESCs. The regulator of G protein signaling (RGS) family consists of over 20 members. RGS proteins are divided into subfamilies (RZ, R4, R7, R12, and RA) based on the sequence of the RGS domain (Ross and Wilkie, 2000). The A/RZ sub-family proteins, including Rgs19, have a cysteine string region in the N-terminus for binding to the plasma membrane and a PDZ binding domain in the C-terminus for protein interaction. Rgs19, a small protein of 217 amino acids, is highly expressed in the heart, lung, and liver, but not in the brain, skeletal muscle, or kidney (De Vries et al., 1995). RGS proteins have GTPase activity when they interact with GTP-Gα subunits, which facilitate regulation of their downstream signaling pathways and their lifespan (Hollinger and Hepler, 2002; Ross and Wilkie, 2000). Rgs19, which was one of the first RGS proteins to be discovered, selectively interacts with Gαi and Gαq to enhance its GTPase activity (De Vries et al., 1995).

43

Wnt signaling, one of the major signaling pathways in mouse ESCs, was shown to be inhibited by the interaction of Rgs19 with the Gαo subunit. Rgs19 was further found to modulate the phosphorylation of Dvl as well as the accumulation of β-catenin and Wnt-responsive gene transcription, and to inhibit the formation of primitive endoderm (Feigin and Malbon, 2007). Wnt/ β-catenin signaling has been related to the pluripotent state and differentiation of mouse ESCs (Kelly et al., 2011; Sato et al., 2004), and has been shown to enhance the induced pluripotency of somatic cells (Lluis et al., 2008; Marson et al., 2008). This effect of Rgs19 was not, however, observed in mouse ESCs. Moreover, Rgs19 overexpression has been shown to enhance the proliferation of various cell types and to deregulate cell cycle factors. These effects were not observed for other RGS proteins including Rgs4, Rgs10, and Rgs20 (Tso et al., 2010). In our previous study, Rgs19 was found to regulate palate development via cell proliferation and apoptosis (Sohn et al., 2012). Whether or not the cell cycle of mouse ESCs is regulated by Rgs19 remains to be determined, and at present, the role of Rgs19 in mouse ESCs is unknown. This study was therefore conducted to determine the precise role of Rgs19 in the processes of ESC selfrenewal, differentiation, and cell proliferation. Based on the availability of Rgs19 in ESCs, the role of Rgs19 in the maintenance of self-renewal was assessed. Furthermore, the morphological features and molecular events exhibited during LIF withdrawal, aggregation-induced differentiation, and teratoma formation were investigated in Rgs19 þ/  ESCs compared with wild-type (WT) ESCs. Our results demonstrate that Rgs19 is required for proliferation and differentiation of ESCs.

2. Materials and methods 2.1. Rgs19 þ /  ESCs For deletion of the Rgs19 gene, Rgs19-targeted mouse embryonic stem (mES) cells were purchased from the KOMP Repository, University of California Davis, USA. Briefly, the same flanking genomic sequences used in the Rgs19 targeting vector were used in the knockout vector, except that the sequence including exons 2–5 was deleted and replaced with a neo-LacZ cassette. The final construct contained 6.2 kb of 50 homology and 4 kb of 30 homology. For genotyping of the targeted gene, the LacZ gene was detected by polymerase chain reaction (PCR; denaturation for 5 min at 94 1C, followed by 30 cycles; denaturation for 30 s at 94 1C, annealing for 30 s at 62 1C and extension for 30 s at 72 1C) using the following primers to generate a 300 bp product: LacZ (forward) 50 -TACAACGTCGTGACTGGGAA-30 and LacZ (reverse) 50 ATGGATAGGTCACGTTGGT-30 . The 50 arm region of the Rgs19 allele was detected by PCR (annealing at 57 1C for 30 s) using the primer 50 arm (forward) 50 -AGGATTCTCCACATGGTGT-30 and En2 intron (reverse) 50 -TCCTCCTACATAGTTGGCAG-30 to generate a 300-bp product. The Rgs19 allele 30 arm region and the Neo gene were detected by PCR (annealing at 61 1C for 30 s) using the following primers to generate a 1 kb product: Neo (forward) 50 -AGGATCTC GTCGTGACCCAT-30 and 30 arm (reverse) 50 -TGAGTCTGACTCTGAGGTCC-30 . 2.2. Western blots ESC extracts (2  106 cells) were prepared using lysis buffer (iNtRON Biotechnology, Seoul, Korea). The protein concentration was determined by the Bradford protein assay, in which bovine serum albumin (BSA) was used as a standard. After blocking the membranes with 5% BSA or skim milk, the blots were incubated with primary antibody at 4 1C overnight, rinsed in TBS-Tween 20,

44

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

and exposed to horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The blots were probed for β-actin as a loading control. Immunoreactivity was determined using an enhanced chemiluminescence detection system (ECL; GE Healthcare, Waukesha, WI, USA). Anti-Rgs19 antibody was purchased from Santa Cruz and antibodies against phospho-STAT3, STAT3, OCT4, Sox2, Cdk2, Cdk4, Cdk6, CyclinD1, CyclinE1, and Cdc25a were obtained from Cell Signaling Technology (Beverly, MA, U.S.A). Nanog and p53 antibodies were purchased from Abcam (Cambridge, UK), and CyclinA and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

diaminobenzidine tetrahydrochloride (DAB) reagent kit (Zymed, San Francisco, CA, USA). 2.7. Propidium iodide (PI) staining After 1 day in culture, JM8A3.N1 ESCs were harvested, washed twice in PBS, and then fixed in 70% ethanol at  20 1C for 20 min. After two additional washes in PBS, the fixed cells were suspended in PBS containing 100 μg/mL RNase A, and then incubated for 1 h at 37 1C. The cells were subsequently stained with propidium iodide (PI; 1000 μg/mL) for 30 min and analyzed by flow cytometry (FACSCalibur; BD Biosciences).

2.3. Cell culture and cell proliferation assay 2.8. Teratoma assay WT ESCs (JM8A3.N1 cell line) were used for Rgs19 gene deletion. Mouse ESCs were cultured on primary mouse embryonic fibroblasts (MEFs), treated with Mitomycin C, in KO Dulbecco's Modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with ESC grade 15% Knockout SR (Invitrogen/Gibco-BRL), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 1000 units/mL ESGRO LIF (Millipore, Watford, UK). ESCs were grown in the presence of LIF to maintain their undifferentiated state for 48 h. For alkaline phosphatase (AP) staining, cells were stained using an Alkaline Phosphatase Detection kit (Millipore). For cell proliferation assays, 2  104 cells/well were seeded into six-well plates in ES cell culture medium. Triplicate samples were prepared for each cell type. Media was changed every 2 days and cells were counted at 2, 4 and 6 days. To examine the pluripotency of mouse ESCs, the cells were cultivated in conditioned media with 5% serum and in the absence of LIF for 48 and 72 h. 2.4. Quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA was then revers-transcribed into cDNA using an RT-PCR kit (Takara, Tokyo, Japan) and subjected to qPCR with SYBR Premix Ex Taq (Takara). Real-time PCR was performed using a Step One Plus PCR system (Applied Biosystems, Foster City, CA, USA). The relative abundance of target cDNA was quantified and normalized by dividing the relative target gene expression by the relative β-actin cDNA expression in each sample. The sequences of the primers used are shown in Supplemental Table 1. 2.5. Embryoid body (EB) formation WT and Rgs19 þ/  ESCs were trypsinized into small clumps of three to five cells. The cell suspensions were then applied to gelatin-coated dishes and incubated at 37 1C for 45 min to allow feeder cells to attach to the dishes. EBs formation was induced by suspension of ESCs in ESC culture medium lacking LIF, after which EBs were harvested at the indicated time points. 2.6. Histology and immunohistochemistry Teratomas were fixed in 4% paraformaldehyde (PFA) at 4 1C overnight, and then embedded in paraffin. Specimens were sectioned to a thickness of 7 mm. Teratoma sections were stained with hematoxylin and eosin (H&E), after which they were assessed and photographed using light microscopy. Primary antibodies against Ki67 (Thermo scientific, Fremon, CA, USA), Cdc25a (Santa Cruz), NeuN (Millipore), and CD31 (BD Bioscience, San Jose, CA, USA) were used for immunohistochemistry. Biotinylated goat anti-rabbit and anti-mouse IgG were used as secondary antibodies, which were visualized using a

All animal experiments were carried out in accordance with the Kyungpook National University guidelines for animal experimentation and with permission from the Kyungpook National University. BALB/C nude mice were anesthetized and 200 mL cell suspension (1  107 cells) in DMEM (Invitrogen) was injected subcutaneously. At four weeks after injection, the resulting teratomas were enucleated and subjected to histological analysis. 2.9. Statistical analysis A sample size of n Z3 was used for all experiments, and results are expressed as mean 7standard deviation (SD). Statistical analyses consisted of an analysis of variance (ANOVA) assessment followed by Student's t-test. Differences with po0.05 considered statistically significant.

3. Results 3.1. Rgs19 expression levels profiles in mES cells and in EB development or monolayer culture Rgs19 has previously been shown to regulate proliferation and tumorigenesis (Tso et al., 2010, 2011), and thus the role of Rgs19 in ECSs was examined in this study. To quantify Rgs19 mRNA in ESCs during development, mES cells and EBs were harvested and the Rgs19 transcript levels were determined by real-time PCR. It was shown that, during EB formation and development, Rgs19 expression is initially (after 3 days) upregulated by about two fold, is further increased by 6 days, and is decreased again by 9 days (Fig. 1A, p o0.05). Using another method of ESC differentiation, namely monolayer differentiation, changes in Rgs19 expression levels similar to those induced during EB formation were observed (Fig. 1B, p o0.05). These findings suggest that Rgs19 may be required for EB development and monolayer differentiation. 3.2. Confirmation of Rgs19 þ/  mouse ES cells For deletion of the Rgs19 allele, a targeting vector containing a neo-LacZ cassette inserted into the Rgs19 exon 2–5 site (Fig. 2A) was constructed. The neo gene within the neo cassette was used for positive selection of transfected ESCs exhibiting disrupted expression of the target gene. Constructs were designed based on the Rgs19 genomic sequence and gene targeting was confirmed by genomic PCR. Three primer pairs were confirmed by PCR, and all products were detected by genomic PCR in Rgs19 þ/  ESCs (Fig. 2B). Western blotting revealed that the Rgs19 protein expression in Rgs19 þ/  ESCs was lower than that in WT ESCs (Fig. 2C). Similarly, real-time PCR indicated that the Rgs19 transcript level was reduced in Rgs19 þ /  ESCs compared with WT ESCs (Fig. 2D, po 0.05). Karyotype analysis was used to confirm that the Rgs19

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

45

Fig. 1. Expression profiles of Rgs19 during ESC differentiation. (A) Rgs19 mRNA expression during EB formation as determined by qRT-PCR. EBs derived from wild-type ESCs were harvested on days 0, 3, 6, and 9. (B) Rgs19 mRNA expression was assayed during monolayer differentiation. Monolayer-differentiated cells derived from wild-type ES cells were harvested on days 0, 3, 6, and 9. Quantitative data and representative results of three independent experiments are shown. *p o 0.05 compared with day 0.

Fig. 2. Construction and confirmation of Rgs19 þ/  mouse ESCs. (A) Configuration of the wild-type, targeted vector, and Rgs19 mutant alleles in the Rgs19 gene. The Rgs19 gene contains six exons with start and stop codons located on exons 2 and 6, respectively. (B) Genomic PCR analysis of various ESC genotypes was conducted using three sets of primers. (C) Western blotting of purified ESC colonies of various genotypes. β-actin was used as a loading control. Data were obtained from three independent experiments. (D) qRT-PCR analysis of purified ESC colonies of various genotypes. β-actin was used as a normalization control. Rgs19 mRNA expression was assayed, and quantitative data and representative results of three independent experiments are shown. *p o0.05 compared with WT expression levels.

þ/  ESC line was normal (data not shown). The Rgs19 þ/  ESC line lacking the Rgs19 gene was successfully established. 3.3. Rgs19 does not play a role in maintaining pluripotency in mouse ESCs ESCs have remarkable self-renewal ability and can indefinitely produce pluripotent daughter cells. To determine the effect of Rgs19 gene knockout on the self-renewal ability of ESCs, ESCs were stained with AP. AP staining in WT and Rgs19 þ/  ESCs did not differ; however, the colonies formed by Rgs19 þ/  ESCs were smaller than those formed by WT ESCs (Fig. 3A and B). qRT-PCR was used to detect the mRNA expression levels of the pluripotency markers Oct4, Nanog, and Sox2; and it was found that the expression levels of Oct4, Nanog, and Sox2 did not differ between WT and Rgs19 þ/  ESCs (Fig. 3C). Moreover, the protein levels of Oct4, Sox2, and Nanog were similar in WT and Rgs19 þ/  ESCs (Fig. 3C). p-STAT3, a known LIF signal transcription factor, was also

detected at similar levels in WT and Rgs19 þ/  ES cells by western blotting (Fig. 3D). Short-term differentiation experiments were conducted to identify potential early loss of pluripotency, and it was found that, after 48 h and 72 h in differentiation medium, the mRNA levels of Oct4, Nanog, and Sox2 in Rgs19 þ/  ESCs were similar to those in WT ESCs (data not shown). These results demonstrate that, in LIF culture with MEF feeder cells, Rgs19 does not play a role in ESC pluripotency. 3.4. Rgs19 affects EB formation and the expression of differentiation markers of the three lineages The Rgs19 mRNA expression profile in Fig. 1 is suggestive of a critical role for Rgs19 in EB formation, and thus the role of Rgs19 during EB formation was further examined. After 6–9 days, the EBs formed by Rgs19 þ/ ESCs was found to be significantly smaller in diameter than those formed by WT ESCs (Fig. 4A, po0.05). To assess the differentiation of Rgs19 þ/ ESC-derived EBs, the mRNA

46

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

Fig. 3. Rgs19 is not required for self-renewal in mouse embryonic stem cells. (A) AP staining was performed to detect self-renewal in mouse embryonic stem cells. Scale bars: 100 mm. (B) The mRNA expression levels of the pluripotency markers Oct4, Sox2, and Nanog were assayed by qRT-PCR. β-actin was used as a normalization control. (C) Cell lysates from ESCs cultured in media supplemented with LIF were subjected to western blotting with antibodies against Oct4, Sox2, Nanog, and p-Stat3 (Thr705, activated form). The results of three independent experiments are shown. β-actin was used as a loading control.

levels of three markers of each of the three embryonic germ layers were measured at day 9 of EB formation: βIII-tubulin, MAP2, and Pax6 (ectoderm); Gata6, Hand1, and Msx1 (mesoderm); and α-fetoprotein, and Sox17 (endoderm) were assayed by qRT-PCR. As shown in Fig. 4B, the expression levels of seven of the eight marker genes were significantly lower in EBs of Rgs19 þ/ ESCs than in EBs of WT ESCs. Only the expression levels of Msx1 did not differ between the Rgs19 þ/ and WT EBs (Fig. 4B, po0.05). These results indicate that ablation of Rgs19 leads to abnormal expression of germ layer markers, suggesting that Rgs19 may be required for in vitro differentiation of ESCs. 3.5. Rgs19 regulates cell proliferation and the cell cycle To determine the role of Rgs19 in ESC proliferation, cells were counted during in vitro culture. Briefly, cells were seeded at 2  103 cells/well in a six-well plate at day 0 and counted at days 2, 4, and 6. The cell density of Rgs19 þ/  ESCs at days 4 and 6 was decreased compared with WT ES cells (Fig. 5A, p o0.05), indicative of reduced proliferation of Rgs19 þ/  ESCs. These findings indicate that Rgs19 regulates the proliferation of ES cells. Cell proliferation may be important for the maintenance of self-renewal and pluripotency in ESCs; however, little is known about the regulation of cell cycle fate in ESCs. Reduced proliferation is typically associated with cell cycle arrest. In this study, therefore, ESCs were starved and then cultured for 24 h, after which cell cycle analyses were conducted using PI staining and FACS analysis. Compared with WT ESCs, a substantial accumulation of Rgs19 þ/  ESCs in the sub G1 and G1 phases of the cell cycle was observed. It was furthermore found that the G2/M phase population was significantly reduced in Rgs19 þ/  ESCs compared with WT ESCs (Fig. 5B, p o0.05). These findings indicate that loss of

Rgs19 function in ESCs results in decreased cellular proliferation via cell cycle arrest. 3.6. Rgs19 deregulates cell cycle gene expression and signaling To investigate the effect of Rgs19 on the expression of cell cycle genes, the mRNA levels of Cdks, Cyclins, and p53 were assayed by qRTPCR. Compared with expression levels in WT ESCs, the expression of Cdk2 and Cdk4 was found to be decreased in Rgs19 þ / ESCs, while Cdk6 expression was increased. Moreover, Cdc25a, CyclinD1, and CyclinE1 were downregulated in Rgs19 þ / ESCs, while p53 expression was elevated (Fig. 5C, po0.05). The expression of cell cycle genes was further assessed on a protein level using western blotting. In Rgs19 þ/ ESCs compared with WT ESCs, Cdk2 and Cdk4 protein levels were reduced while Cdk6 expression was increased. CyclinD1, CyclinE1, and Cdc25a protein levels were also reduced in Rgs19 þ / ESCs, and the protein expression of p53 was increased in Rgs19 þ / ES cells (Fig. 5D). Rgs19 downregulation in ESCs was thus shown to result in dysregulation of cell cycle gene expression and cellular signaling. 3.7. Rgs19 decreases ESC tumorigenicity and differentiation in teratoma formation To investigate ESC tumorigenicity and differentiation during teratoma formation in vivo, ESCs were subcutaneously injected into BALB/C nude mice. Initially, 2  106 ESCs were injected; however, teratoma formation by Rgs19 þ/ ESCs was not detected after 9 weeks. A higher cell number (5  106 ESCs) was then injected; however, teratoma formation was still not detected in mice injected with Rgs19 þ/ ESCs (data not shown). Finally, 1  107 ESCs were injected, which resulted in teratoma formation after 4 weeks. Rgs19

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

47

Fig. 4. Rgs19 is required for differentiation and EB formation of mouse ESCs. (A) Morphology of EBs derived from WT and Rgs19 þ /  ESCs. EBs were photographed on days 3, 6 and 9 (40  magnification). The EB diameter was measured. (B) The mRNA expression of βIII-tubulin, MAP2, and Pax6 (ectoderm markers); Gata6, Hand1, and Msx1 (mesoderm markers); and α-fetoprotein, and Sox17 (endoderm markers) was measured after 9 days of EB differentiation of WT and Rgs19 þ /  ESCs. β-actin was used as a normalization control. The results of three independent experiments are shown. *po 0.05 compared with WT expression levels.

þ/ ESC-derived teratomas were found to be smaller than WT ESCderived teratomas: teratoma weight and volume were significantly lower in Rgs19 þ/ ESC-injected mice than in WT ESC-injected mice (Fig. 6A, po0.05). Immunohistochemistry using NeuN as a neuronal marker was thus conducted to identify neural differentiation. Fewer NeuN-positive cells were detected in the Rgs19 þ/ teratoma than in the WT teratoma. To detect cell proliferation, teratoma specimens were stained for Ki67, an M phase maker, as well as Cdc25a. Ki67 and Cdc25a staining intensities were reduced in the Rgs19 þ / teratoma compared with the WT teratoma. Vessels were not visible in the Rgs19 þ/ teratoma. CD31 was used as an angiogenesis marker, and no CD31-positive cells were detected in the Rgs19 þ/ teratoma (Fig. 6B). These results demonstrate that Rgs19 regulates teratoma formation by decreasing cell proliferation, differentiation, and angiogenesis in nude mice.

4. Discussion The role of Rgs19 underlying molecular mechanisms involved in cell cycle regulation remains unknown. In our assessment of the function of Rgs19 in ESC cell cycle regulation, we examined whether Rgs19 promotes cell growth mediated by cell cycle regulation. Our findings demonstrate that Rgs19 þ/ dramatically arrests the cell cycle in ESCs and regulates the expression of CDKs, cyclins, and cellular signaling. The role of Rgs19 in ESC proliferation and differentiation was also investigated in this study, and it was found that the deletion of Rgs19 in ESCs markedly disturbs normal differentiation processes,

although the self-renewal capacity of the cells is not overtly affected. Compared with WT ESCs, Rgs19 þ/ ESCs reduce teratoma formation and exhibit delayed differentiation. Overall, this study provides the first evidence for the role and potential mechanism of Rgs19 in the control of ESCs proliferation and differentiation. Numerous Rgs proteins have been shown to be involved in the regulation of cell growth and survival (Sethakorn et al., 2010), as well as in the promotion or inhibition of tumorigenesis. Our findings of inhibited proliferation in Rgs19 þ/ ESCs in the undifferentiated state are thus not surprising. In a previous study, Rgs19 overexpression was shown to enhance cell proliferation via interaction of its C-terminal PDZ motif (Tso et al., 2010), to increase cell proliferation by deregulation of cell cycle-related genes such as Cdk6 and CyclinD1 (Tso et al., 2011). In our study, the expression of Cdks and cyclins was lower in Rgs19 þ/ ESCs compared with WT cells. In our previous study, Rgs19 was found to regulate palate development via cell proliferation and apoptosis (Sohn et al., 2012). Together, these findings indicate that Rgs19 may play an important role in cell cycle regulation. Moreover, at least 23 RGS proteins have been shown to be differentially expressed in various cancers (Hurst and Hooks, 2009), and Rgs19 in particular is upregulated in ovarian cancer (Hurst et al., 2009). Previous study indicated that, serum induced MAPK pathways, which are JNK and p38, is suppressed by Rgs19 (Ip et al., 2012), these pathways are known to regulate negatively cell proliferation (Perdiguero et al., 2007; Wada et al., 2008). So suppression of MAPK by Rgs19 could increase cell proliferation. Notch signal is also regulated by Rgs19 via Akt signaling (Sangphech et al., 2014), this mechanism regulates the cell cycle and apoptosis. These

48

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

Fig. 5. Rgs19 þ /  deregulates cell proliferation and cell cycle gene expression in mouse ESCs. (A) Cells were counted after 0, 2, 4, and 6 days in culture to assess proliferation of mouse ESCs. (B) Cell cycle analysis was carried out by PI staining of WT and Rgs19 þ /  ESCs. (C) mRNA levels of the cell cycle-related Cdks (Cdk1, Cdk2, Cdk4, Cdk6), cyclins (CyclinA1, cdc25a, CyclinD1, CyclinE1), and p53 were assayed by qRT-PCR. β-actin was used as a normalization control. (D) Cellular signaling was detected by western blotting of cell cycle-related genes. β-actin was used as a loading control. The results of three independent experiments are shown. *p o 0.05 compared with WT expression levels.

regulations of Rgs19 might be involved in proliferation of ES cells. Further studies on Rgs19 will reveal the role of Rgs19 in cell proliferation and apoptosis in various cancers. In this study, increased Cdk6 expression in Rgs19 þ/ ESCs was detected by qRT-PCR and western blotting. The cell cycle of ESCs is especially short due to the absence of the G1/S checkpoint (Becker et al., 2006; Savatier et al., 1996). Moreover, there is little kinase activity of the associated Cdk4 and Cdk6 molecules in the cell cycle of mouse ESCs, and high activity of Cdk2 and the cyclinA/E complex (Neganova et al., 2009). Our findings in this study may thus represent a compensatory effect that occurs via Cdk2 and Cdk4. It has been shown that Cdk2 and Cdk4 KO neural stem cells have increased Cdk6 expression, while MEFs do not (Lim and Kaldis, 2012). Upregulation of Cdk6 may therefore be occurring as a compensatory mechanism in ESCs and in neural stem cells possessing stemness. Our results show that Rgs19 regulates the cell cycle but not the pluripotency of mouse ESCs. Rgs19, however, regulated differentiation during EB and teratoma formation of ESCs. It is of great interest to clarify the link between cell cycle control and differentiation of ESCs. Recently, pluripotency genes were found to be regulated during cell cycle control by an interaction complex of CDK6 and CDC25A (Zhang et al., 2009). Downregulation of CDK2 may also affect differentiation in mouse ESCs (Koledova et al., 2010) and, importantly, the kinase activity of CDK2 is significantly decreased upon differentiation (Faast et al., 2004). Our results indicate that Rgs19 þ/  decreases differentiation via cell cycle deregulation. Another possible explanation for the reduced differentiation observed involves GIPC, which is a binding partner of Rgs19. GIPC plays a role in TGF signaling for epithelial

mesenchymal transition (EMT) in cancer (Blobe et al., 2001; Lee et al., 2010). EMT is important for ESC differentiation and Rgs19 has previously been shown to regulate the expression of TGF ligand for EMT in palate development (Sohn et al., 2012). Impaired differentiation in Rgs19 þ/  ESCs may therefore involve TGF signaling. A third possible explanation for the differentiation suppression observed in Rgs19 þ /  ESCs involves Wnt and NGF signaling, which play a critical role in ESC differentiation. Rgs19 has previously been shown to regulate Wnt and NGF signaling (Feigin and Malbon, 2007; Ji et al., 2010). Suppression of ESC differentiation in Rgs19 þ/ cells may thus be regulated by signaling of ligands. In further study, we will examine the role of Res19 in ESC differentiation by regulation of EMT events or signaling of ligands such as Wnt and NGF. Angiogenesis was not observed in Rgs19 þ/  teratomas, as CD31, an angiogenesis marker, was not detected by immunohistochemistry in Rgs19 þ/  teratomas. Angiogenesis occurs during hyper-proliferation of cells, such as in teratomas and cancer, and we propose two possible reasons for the observed lack of angiogenesis here. Firstly, cell proliferation was decreased due to cell cycle deregulation; and secondly, Rgs19 regulates angiogenesis molecules such as vascular endothelial growth factor (VEGF). In a previous study, Rgs19 was shown to be recruited by VEGF receptor (VEGFR) and thus Rgs19 may play a role in the VEGF pathway (Lahteenvuo et al., 2009). Future studies will therefore include investigations into whether the role of Rgs19 is affected by cell proliferation or VEGF signaling in angiogenesis. Synectin (also known as GIPC) is a binding partner of Rgs19. Synectin regulates not only VEGF signaling, but also angiogenesis and artery

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

49

Fig. 6. Rgs19 regulates teratoma formation via cell proliferation, differentiation, and angiogenesis. (A) Teratomas were isolated 4 weeks after subcutaneous injection of WT and Rgs19 þ /  ESCs into nude mice. Teratoma weight and volume were determined. (B) Histological analyses were performed by H&E staining and immunohistochemistry of WT and Rgs19 þ /  teratomas. Quantification of cell number was graphed. Scale bars: Ki67, Cdc25a: 50 mm; H&E, NeuN, and CD31: 100 mm.

branching (Chittenden et al., 2006; Horowitz and Seerapu, 2012; Paye et al., 2009). The lack of angiogenesis in Rgs19 þ/  ESCderived teratomas may therefore be an effect of VEGF signaling via binding of Synectin and Rgs19. This potential mechanism will also be included in future studies on Rgs19. Rgs19 plays a number of important roles in ESCs, affecting processes ranging from cell cycle regulation to differentiation. Because the mechanism underlying the effects of Rgs19 on these processes in ESCs remains poorly understood, additional studies in this area are being conducted. We believe that a better understanding of these cellular events will facilitate a clearer understanding of the mechanisms by which ESCs coordinate differentiation from pluripotent cells to the three germ layer cells, as well as cell cycle regulation in in vitro ESC culture.

Acknowledgments This work was supported by a grant from the Next-Generation BioGreen21 Program (No. PJ 009573), a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008-0062618), and an NRF grant funded by the Ministry of Education, Science and Technology (No. 2013R1A1A2060793). Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.diff.2015.01.002. References

Disclosure statement The authors have nothing to disclose. Conflict of interest The authors declare no potential conflicts of interest.

Becker, K.A., Ghule, P.N., Therrien, J.A., Lian, J.B., Stein, J.L., van Wijnen, A.J., Stein, G.S., 2006. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J. Cell. Physiol. 209, 883–893. Berthet, C., Aleem, E., Coppola, V., Tessarollo, L., Kaldis, P., 2003. Cdk2 knockout mice are viable. Curr. Biol. 13, 1775–1785. Blobe, G.C., Liu, X., Fang, S.J., How, T., Lodish, H.F, 2001. A novel mechanism for regulating transforming growth factor beta (TGF-beta) signaling. Functional modulation of type III TGF-beta receptor expression through interaction with the PDZ domain protein, GIPC. J. Biol. Chem. 276, 39608–39617.

50

Y.R. Ji et al. / Differentiation 89 (2015) 42–50

Burdon, T., Smith, A., Savatier, P., 2002. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432–438. Chittenden, T.W., Claes, F., Lanahan, A.A., Autiero, M., Palac, R.T., Tkachenko, E.V., Elfenbein, A., Ruiz de Almodovar, C., Dedkov, E., Tomanek, R., Li, W., Westmore, M., Singh, J.P., Horowitz, A., Mulligan-Kehoe, M.J., Moodie, K.L., Zhuang, Z.W., Carmeliet, P., Simons, M., 2006. Selective regulation of arterial branching morphogenesis by synectin. Dev. Cell 10, 783–795. De Vries, L., Mousli, M., Wurmser, A., Farquhar, M.G., 1995. GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. Proc. Natl. Acad. Sci. USA 92, 11916–11920. Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156. Faast, R., White, J., Cartwright, P., Crocker, L., Sarcevic, B., Dalton, S., 2004. Cdk6–cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a). Oncogene 23, 491–502. Feigin, M.E., Malbon, C.C., 2007. RGS19 regulates Wnt-beta-catenin signaling through inactivation of Galpha(o). J. Cell Sci. 120, 3404–3414. Fujii-Yamamoto, H., Kim, J.M., Arai, K., Masai, H., 2005. Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells. J. Biol. Chem. 280, 12976–12987. He, S., Nakada, D., Morrison, S.J., 2009. Mechanisms of stem cell self-renewal. Annu. Rev. Cell Dev. Biol. 25, 377–406. Hollinger, S., Hepler, J.R., 2002. Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol. Rev. 54, 527–559. Horowitz, A., Seerapu, H.R., 2012. Regulation of VEGF signaling by membrane traffic. Cell. Signal. 24, 1810–1820. Hurst, J.H., Hooks, S.B., 2009. Regulator of G-protein signaling (RGS) proteins in cancer biology. Biochem. Pharmacol. 78, 1289–1297. Hurst, J.H., Mendpara, N., Hooks, S.B., 2009. Regulator of G-protein signalling expression and function in ovarian cancer cell lines. Cell. Mol. Biol. Lett. 14, 153–174. Ip, A.K., Tso, P.H., Lee, M.M., Wong, Y.H., 2012. Elevated expression of RGS19 impairs the responsiveness of stress-activated protein kinases to serum. Mol. Cell. Biochem. 362, 159–168. Ji, Y.R., Kim, M.O., Kim, S.H., Yu, D.H., Shin, M.J., Kim, H.J., Yuh, H.S., Bae, K.B., Kim, J.Y., Park, H.D., Lee, S.G., Hyun, B.H., Ryoo, Z.Y., 2010. Effects of regulator of G protein signaling 19 (RGS19) on heart development and function. J. Biol. Chem. 285, 28627–28634. Kelly, K.F., Ng, D.Y., Jayakumaran, G., Wood, G.A., Koide, H., Doble, B.W., 2011. betacatenin enhances Oct-4 activity and reinforces pluripotency through a TCFindependent mechanism. Cell Stem Cell 8, 214–227. Koledova, Z., Kafkova, L.R., Calabkova, L., Krystof, V., Dolezel, P., Divoky, V., 2010. Cdk2 inhibition prolongs G1 phase progression in mouse embryonic stem cells. Stem Cells Dev. 19, 181–194. Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H., Nakatsuji, N., Tada, T., 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25, 2475–2485. Lahteenvuo, J.E., Lahteenvuo, M.T., Kivela, A., Rosenlew, C., Falkevall, A., Klar, J., Heikura, T., Rissanen, T.T., Vahakangas, E., Korpisalo, P., Enholm, B., Carmeliet, P., Alitalo, K., Eriksson, U., Yla-Herttuala, S., 2009. Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor1-dependent mechanisms. Circulation 119, 845–856. Lange, C., Calegari, F., 2010. Cdks and cyclins link G1 length and differentiation of embryonic, neural and hematopoietic stem cells. Cell Cycle 9, 1893–1900. Lee, J.D., Hempel, N., Lee, N.Y., Blobe, G.C., 2010. The type III TGF-beta receptor suppresses breast cancer progression through GIPC-mediated inhibition of TGF-beta signaling. Carcinogenesis 31, 175–183. Lee, M.Y., Lim, H.W., Lee, S.H., Han, H.J., 2009. Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal. Stem Cells 27, 1858–1868. Lim, S., Kaldis, P., 2012. Loss of Cdk2 and Cdk4 induces a switch from proliferation to differentiation in neural stem cells. Stem Cells 30, 1509–1520. Lluis, F., Pedone, E., Pepe, S., Cosma, M.P., 2008. Periodic activation of Wnt/betacatenin signaling enhances somatic cell reprogramming mediated by cell fusion. Cell Stem Cell 3, 493–507. Marson, A., Foreman, R., Chevalier, B., Bilodeau, S., Kahn, M., Young, R.A., Jaenisch, R., 2008. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3, 132–135. Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638. Masui, S., Nakatake, Y., Toyooka, Y., Shimosato, D., Yagi, R., Takahashi, K., Okochi, H., Okuda, A., Matoba, R., Sharov, A.A., Ko, M.S., Niwa, H., 2007. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat. Cell Biol. 9, 625–635. Neganova, I., Zhang, X., Atkinson, S., Lako, M., 2009. Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells. Oncogene 28, 20–30.

Niwa, H., 2007. How is pluripotency determined and maintained? Development 134, 635–646. Niwa, H., Miyazaki, J., Smith, A.G., 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376. Ortega, S., Prieto, I., Odajima, J., Martin, A., Dubus, P., Sotillo, R., Barbero, J.L., Malumbres, M., Barbacid, M., 2003. Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat. Genet. 35, 25–31. Paling, N.R., Wheadon, H., Bone, H.K., Welham, M.J., 2004. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J. Biol. Chem. 279, 48063–48070. Paye, J.M., Phng, L.K., Lanahan, A.A., Gerhard, H., Simons, M., 2009. Synectindependent regulation of arterial maturation. Dev. Dyn. 238, 604–610. Perdiguero, E., Ruiz-Bonilla, V., Gresh, L., Hui, L., Ballestar, E., Sousa-Victor, P., BaezaRaja, B., Jardi, M., Bosch-Comas, A., Esteller, M., Caelles, C., Serrano, A.L., Wagner, E.F., Munoz-Canoves, P., 2007. Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation. EMBO J. 26, 1245–1256. Qi, X., Li, T.G., Hao, J., Hu, J., Wang, J., Simmons, H., Miura, S., Mishina, Y., Zhao, G.Q., 2004. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc. Natl. Acad. Sci. USA 101, 6027–6032. Ross, E.M., Wilkie, T.M., 2000. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827. Sangphech, N., Osborne, B.A., Palaga, T., 2014. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19). Immunobiology 219, 653–660. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., Brivanlou, A.H., 2004. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63. Savatier, P., Lapillonne, H., Jirmanova, L., Vitelli, L., Samarut, J., 2002. Analysis of the cell cycle in mouse embryonic stem cells. Methods Mol. Biol. 185, 27–33. Savatier, P., Lapillonne, H., van Grunsven, L.A., Rudkin, B.B., Samarut, J., 1996. Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor upregulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells. Oncogene 12, 309–322. Sethakorn, N., Yau, D.M., Dulin, N.O., 2010. Non-canonical functions of RGS proteins. Cell. Signal. 22, 1274–1281. Singh, A.M., Dalton, S., 2009. The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming. Cell Stem Cell 5, 141–149. Smith, P.D., Crompton, M.R., 1998. Expression of v-src in mammary epithelial cells induces transcription via STAT3. Biochem. J. 331 (Pt 2), 381–385. Sohn, W.J., Ji, Y.R., Kim, H.S., Gwon, G.J., Chae, Y.M., An, C.H., Park, H.D., Jung, H.S., Ryoo, Z.Y., Lee, S., Kim, J.Y., 2012. Rgs19 regulates mouse palatal fusion by modulating cell proliferation and apoptosis in the MEE. Mech. Dev. 129, 244–254. Stead, E., White, J., Faast, R., Conn, S., Goldstone, S., Rathjen, J., Dhingra, U., Rathjen, P., Walker, D., Dalton, S., 2002. Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities. Oncogene 21, 8320–8333. Tso, P.H., Wang, Y., Wong, S.Y., Poon, L.S., Chan, A.S., Wong, Y.H., 2010. RGS19 enhances cell proliferation through its C-terminal PDZ motif. Cell. Signal. 22, 1700–1707. Tso, P.H., Yung, L.Y., Wang, Y., Wong, Y.H., 2011. RGS19 stimulates cell proliferation by deregulating cell cycle control and enhancing Akt signaling. Cancer Lett. 309, 199–208. Wada, T., Stepniak, E., Hui, L., Leibbrandt, A., Katada, T., Nishina, H., Wagner, E.F., Penninger, J.M., 2008. Antagonistic control of cell fates by JNK and p38-MAPK signaling. Cell Death Differ. 15, 89–93. White, J., Stead, E., Faast, R., Conn, S., Cartwright, P., Dalton, S., 2005. Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation. Mol. Biol. Cell 16, 2018–2027. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., Gough, N.M., 1988. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., Thomson, J.A., 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920. Zhang, X., Neganova, I., Przyborski, S., Yang, C., Cooke, M., Atkinson, S.P., Anyfantis, G., Fenyk, S., Keith, W.N., Hoare, S.F., Hughes, O., Strachan, T., Stojkovic, M., Hinds, P.W., Armstrong, L., Lako, M., 2009. A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A. J. Cell Biol. 184, 67–82.