Research ArticleStem Cell Biology

Autocrine Wnt regulates the survival and genomic stability of embryonic stem cells

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Science Signaling  10 Jan 2017:
Vol. 10, Issue 461, eaah6829
DOI: 10.1126/scisignal.aah6829

Genomic instability without Wnt

Unlike most cells in the body, embryonic stem cells renew themselves and can differentiate into almost any cell type. Although embryonic stem cells have been proposed to treat a myriad of human diseases, their use is fraught with the risk of the formation of noncancerous tumors called teratomas. The Wnt family of ligands promotes both the self-renewal and differentiation of embryonic stem cells. Augustin et al. either genetically ablated or overexpressed Evi, a protein that transports Wnts through the secretory pathway, in mouse embryonic stem cells, which would be expected to block or enhance the secretion of any of the Wnt family of ligands produced by these cells. Reducing Wnt secretion reduced the incidence of teratoma formation by Evi-deficient embryonic stem cells injected into mice. Furthermore, Wnt secretion ensured that proliferating embryonic stem cells segregated chromosomes properly and did not undergo apoptosis. Thus, enhancing Wnt signaling may prevent genomic instability in embryonic stem cells, which could help advance therapeutic application of stem cells.

Abstract

Wnt signaling plays an important role in the self-renewal and differentiation of stem cells. The secretion of Wnt ligands requires Evi (also known as Wls). Genetically ablating Evi provides an experimental approach to studying the consequence of depleting all redundant Wnt proteins, and overexpressing Evi enables a nonspecific means of increasing Wnt signaling. We generated Evi-deficient and Evi-overexpressing mouse embryonic stem cells (ESCs) to analyze the role of autocrine Wnt production in self-renewal and differentiation. Self-renewal was reduced in Evi-deficient ESCs and increased in Evi-overexpressing ESCs in the absence of leukemia inhibitory factor, which supports the self-renewal of ESCs. The differentiation of ESCs into cardiomyocytes was enhanced when Evi was overexpressed and teratoma formation and growth of Evi-deficient ESCs in vivo were impaired, indicating that autocrine Wnt ligands were necessary for ESC differentiation and survival. ESCs lacking autocrine Wnt signaling had mitotic defects and showed genomic instability. Together, our study demonstrates that autocrine Wnt secretion is important for the survival, chromosomal stability, differentiation, and tumorigenic potential of ESCs.

INTRODUCTION

Embryonic stem cells (ESCs) are a powerful model to study cell fate decisions because of their self-renewal and differentiation capacities. ESCs have the potential to differentiate into all cell types that form the three germ layers (1). Intrinsic and extrinsic signals are necessary to maintain ESCs in their pluripotent state (2), including leukemia inhibitory factor (LIF) [which signals through the transcription factor STAT3 (signal transducer and activator of transcription 3)] and bone morphogenetic protein (BMP) (which signals through the SMAD family of transcription factors) in mouse ESCs and transforming growth factor–β (TGF-β) (which signals through the Activin family of receptors) in human ESCs. To self-renew, ESCs must maintain their pluripotent state and undergo cell division. Rapid cell cycling of ESCs with a doubling time of 13 to 16 hours is characterized by a short G1 phase and a high proportion of cells in the S phase compared to somatic cells (3). Although proliferation of somatic cells is primarily controlled by G1-S checkpoint progression, ESCs fail to arrest in the G1 phase and do not show replicative senescence (1, 4, 5). Consequently, the initiation of chromosome replication can continue despite mitotic failures (6). Although maintenance of genome integrity is crucial for ESCs, little is known about signaling mechanisms that influence genomic stability in ESCs (7).

Wnt signaling is required for mouse as well as human ESC maintenance (8, 9). However, its role in ESC self-renewal versus differentiation has been debated (10). In mouse, the generation of germ line–competent ESCs from nonpermissive strains is facilitated by inhibition of glycogen synthase kinase 3 (GSK3) and an exogenous supply of Wnt3a (11, 12). Although a supply of exogenous Wnt3a supports ESC self-renewal, long-term treatment of mouse ESCs with Wnt3a results in differentiation toward the mesodermal lineage, supporting a prodifferentiation role of Wnt signaling (13). Blocking Wnt acylation by inhibition of Porcupine using the small-molecule inhibitor IWP2 (inhibitor of WNT production 2) enforces the development of epiblast-derived stem cells (EpiSCs) that are primed pluripotent stem cells, suggesting an essential role of Wnts in self-renewal (12). However, these results have not been supported by another study (14). Moreover, endogenous Wnt ligands can support loss of pluripotency in EpiSCs and favor spontaneous differentiation (15). Similarly, conversion of mouse EpiSCs to naïve-like pluripotent stem cells can be induced by inhibiting Porcupine with IWP2 (16). These partially conflicting reports indicate that Wnt signaling might perform complex stage-dependent roles in ESCs. Understanding the role of Wnt signaling in stem cell maintenance requires taking into account the variation in cell lines of different or same genomic backgrounds as well as heterogeneity within the culture dish and stem cell colony.

Here, we analyzed the role of autocrine Wnt proteins in mouse ESC maintenance and differentiation by genetically interfering with the secretion of Wnt ligands. An essential component of the Wnt secretion machinery is the conserved transmembrane protein Evi (also known as Wls and Gpr177) (17, 18). Genetic inactivation of Evi affects all Wnt signaling cascades, providing a means of modulating Wnt signaling through the regulation of Wnt ligand secretion. Evi knockout mice die during embryogenesis because of disruption of axial patterning, resulting in an absence of mesoderm and failure to form a primitive streak (19, 20). We showed in the present study that autocrine Wnt secretion did not affect the expression of the pluripotency genes Oct4, Sox2, and Nanog, but was required for ESC self-renewal in the absence of LIF. Basal Wnt signaling in ESCs prevented an increased rate of apoptosis, which was accompanied by chromosomal instability. In addition, we showed that Wnt secretion was essential for teratoma formation and lineage differentiation in vivo and in vitro. Evi overexpression in ESCs enforced cardiomyocyte differentiation, supporting a model in which canonical Wnt signaling drives mesoderm differentiation.

RESULTS

Wnt secretion supports ESC self-renewal in the absence of LIF

ESCs express mRNAs encoding a broad repertoire of Wnt ligands, including Wnt2, Wnt3a, Wnt5a, Wnt6, Wnt7b, Wnt8a, Wnt9a, Wnt9b, Wnt10a, Wnt16, and those encoding potent enhancers (R-spondins) or inhibitors (Dickkopf-related proteins, DKKs), which influence Wnt signaling output in a dose-dependent manner (fig. S1A) (21, 22). To block autocrine signaling and to study the physiological consequences for stem cell maintenance and differentiation, we generated ESCs that lacked the pan-Wnt cargo receptor Evi (also known as Wls). We isolated 12 independent ESC lines from homozygous floxed Evi mouse blastocytes from a C57Black/6 background. ESCs were maintained on feeder cells in ESC medium containing serum and LIF. We then generated Evi-deficient ESC subclones (Evi-LOF) by in vitro Cre recombinase treatment. Several isogenic subclones (control and Evi-LOF lines) from the same parental clone were further analyzed in parallel to exclude clone-specific variation. Genomic polymerase chain reaction (PCR) of Evi-LOF verified efficient recombination, leading to the deletion of exon 3 (Fig. 1A and fig. S1B). Western blot analysis revealed a loss of Evi protein in Evi-LOF ESCs and a decrease in active β-catenin abundance; in addition, Wnt reporter activity was impaired in human embryonic kidney (HEK) 293T cells exposed to medium conditioned by these ESCs (Fig. 1B and fig. S1, C and D).

Fig. 1 Depletion of Evi in ESCs leads to reduced self-renewal in the absence of LIF.

(A) PCR genotyping of floxed Evi (control) and deleted Evi alleles (Evi-LOF) in the corresponding ESCs. (B) Western blot of control and Evi-LOF ESC lysates incubated with antibodies against Evi. β-Actin was used as a loading control. n = 2 independent experiments. (C) Bright-field image of control and Evi-LOF ESCs cultured without MEFs in ESC medium with LIF. Scale bar, 100 μm. n = 2 independent experiments. (D) RT-PCR analysis of control and Evi-LOF ESCs for the indicated genes. n = 3 biological replicates per group. (E) Analysis of AP-labeled ESC colonies in the presence and absence of LIF and/or Wnt3a 48 hours after seeding. n = 3 biological replicates per group. (F) Western blot of control and Evi-GOF ESC lysates incubated with antibodies against Evi. β-Actin was used as a loading control. The asterisk (*) denotes nonspecific band. n = 2 independent experiments. (G) RT-PCR analysis of control and Evi-GOF ESCs for the indicated genes. n = 3 biological replicates per group. (H) Analysis of AP-labeled ESC colonies in the absence of LIF 24, 48, and 72 hours after seeding. n = 3 biological replicates per group. *P < 0.05; P values were calculated by Student’s t tests. Error bars represent SD.

Morphologically, cultures of Evi-LOF ESCs resembled those of typical mouse ESCs (Fig. 1C). Gene expression analysis by reverse transcription (RT)–PCR revealed a significant reduction of the Wnt target Axin2, consistent with a down-regulation of canonical Wnt pathways. In contrast, expression of pluripotency genes including Oct4 (which encodes Octamer-binding transcription factor 4), Nanog, SSEA1 (stage-specific embryonic antigen-1), and Rex1 was unchanged (Fig. 1D). CD133 (which encodes prominin1) and the EpiSC marker Cldn6 (which encodes Claudin6) did not show significant differences in expression (12). In contrast, the expression of the primitive ectodermal marker Fgf5 (which encodes fibroblast growth factor 5) was increased in Evi-deficient ESCs, which is in line with the reduction in Fgf5 expression in adenomatous polyposis coli (APC)–deficient ESCs (23). Similarly, fluorescence-activated cell scanning (FACS)–based analysis of pluripotency-related proteins markers (Oct4, SSEA1, and Nanog) and of E-cadherin revealed no changes in abundance in Evi-LOF ESCs compared to controls (fig. S1E).

The undifferentiated state of ESCs was further confirmed by high alkaline phosphatase (AP) abundance, which labels cells with the potential to self-renew. When cultured with LIF, both control and Evi-LOF ESC cultures had similar numbers of AP-positive colonies (Fig. 1E and fig. S1F). Similarly, treatment with the Porcupine inhibitor LGK974 in the presence of LIF decreased the expression of the Wnt target gene Axin2 without altering the number of AP-positive colonies (fig. S1, G and H). However, culturing ESCs in the absence of LIF triggered a decrease in AP-positive colonies (Fig. 1E and fig. S1F). Evi-LOF ESCs showed a significant reduction in AP labeling compared to control cells, indicating that self-renewal of Evi-deficient ESCs in the absence of LIF was more strongly affected. The addition of recombinant Wnt3a significantly reversed the self-renewal phenotype (Fig. 1E).

To further investigate the role of Wnt secretion in ESC maintenance, we generated Evi gain-of-function (Evi-GOF) mouse ESCs through targeted integration of C-terminal tagged Evi–yellow fluorescent protein (YFP) into the ROSA26 locus. Cre-mediated deletion of the loxP sites was performed to induce constitutive ectopic Evi-YFP overexpression in ESCs as shown by Western blot (Fig. 1F). We have previously shown that Evi-YFP protein in these cells localizes to the secretory pathway and that Evi-YFP mRNA abundance is increased about sevenfold, resulting in increased Wnt reporter activity (24). The pluripotency genes Oct4, Nanog, and SSEA1 were expressed at similar amounts in control and Evi-GOF ESCs (Fig. 1G), but LIF withdrawal induced a much greater increase in AP staining of Evi-GOF ESCs than in control ESCs (Fig. 1H). Together, these data showed that Wnt signaling supported ESC self-renewal but was not essential for maintenance of the analyzed ESC line.

Evi-deficient ESCs have impaired in vitro differentiation

To further study the in vitro differentiation potential of Evi-LOF ESCs, we analyzed their ability to form embryoid bodies (EBs). Compared to control EBs, Evi-LOF EBs appeared smaller and more compact (Fig. 2A) and were reduced in volume (Fig. 2B). This reduction in size was rescued by adding recombinant Wnt3a (Fig. 2B). Although cell proliferation in Evi-LOF EBs was not altered, apoptosis was significantly increased, supporting a model whereby Wnt secretion promoted ESC survival (Fig. 2, C and D). In addition, recombinant Wnt3a significantly reduced the apoptosis rate of Evi-LOF EBs (Fig. 2D).

Fig. 2 Evi-LOF ESCs show impaired lineage differentiation in vitro.

(A) Images of Evi-LOF and control EBs at day 4 after EB initiation. Scale bar, 200 μm. n = 3 independent experiments. (B) Analysis of relative EB volume in the presence or absence of Wnt3a at day 4 after EB initiation. n = 3 biological replicates per group. (C) EdU-based proliferation analysis of control and Evi-LOF EBs at day 4 after EB initiation. n = 3 biological replicates per group. (D) Analysis of annexin V–positive, propidium iodide (PI)–negative cells in the presence or absence of Wnt3a at day 4 after EB initiation. n = 3 biological replicates per group. (E) RT-PCR analysis of markers of lineage differentiation of control and Evi-LOF EBs 4 days after EB start. n = 3 biological replicates per group. (F) RT-PCR analysis of markers of lineage differentiation of Evi-LOF EBs compared to Evi-LOF EBs cultured in the presence of recombinant Wnt3a 4 days after EB initiation. n = 3 biological replicates per group. (G) Images of Evi-GOF and control EBs at days 3 and 6 after EB initiation. Scale bars, 100 μm (top) and 300 μm (bottom). n = 3 independent experiments. (H) Analysis of relative EB size at days 4 and 6 after EB initiation. n = 3 biological replicates per group. (I) Relative quantification of contracting EBs of control and Evi-GOF clones at indicated time points. n = 3 biological replicates per group. (J) RT-PCR analysis of markers of lineage differentiation of control and Evi-GOF EBs 12 days after EB start. n = 3 biological replicates per group. (K) RT-PCR analysis of Evi expression of EBs 12 days after EB initiation. n = 3 biological replicates per group. *P < 0.05; ***P < 0.001; P values were calculated by Student’s t test. Error bars represent SD.

Evi-LOF embryos die around embryonic day 8.5 because of gastrulation defects, mimicking the phenotype of Wnt3-deficient mouse embryos (19). To address whether all three germ layers were represented in Evi-LOF EBs, we analyzed selected markers in EBs generated from day 13 ESCs by quantitative reverse transcription PCR. Transcript abundance of the ectodermal marker Sox1 (which encodes Sex-determining region Y-box 1) was slightly increased and Gbx2 (which encodes gastrulation brain homeobox 2) was not changed in Evi-LOF EBs relative to controls (Fig. 2E). Among the tested early mesodermal markers, we observed lower expression of Pdgfra (which encodes platelet-derived growth factor receptor α) and the Wnt target gene Brachyury (T) in Evi-LOF EBs, suggesting that Wnt signaling drove mesoderm formation (Fig. 2E) (25). Moreover, the expression of endodermal markers Foxa2 (which encodes Forkhead box A2) and Cer1 (which encodes Cerberus 1) was strongly reduced, indicating impaired endodermal differentiation (Fig. 2E). Our results were consistent with in vitro differentiation experiments of Porcupine-deficient ESCs showing a comparable inhibitory effect of Porcupine and Evi on the response of ESCs to Wnt (26). Culturing Evi-LOF EBs with recombinant Wnt3a rescued the expression of select mesodermal and endodermal marker genes (T, Foxa2, and Cer1), whereas it had only a small effect on the expression of Sox1, Gbx2, and Pdgfra (Fig. 2F). These data suggest that autocrine Wnt secretion was essential for the in vitro differentiation of mesodermal and endodermal lineages but not for that of ectodermal lineage.

Next, the effect of Evi overexpression on mesodermal differentiation, particularly cardiogenic specification, was analyzed by counting the frequency of beating EBs and characterizing cardiac gene expression. EBs of 3 and 6 days of age were similar in size, suggesting similar growth rates of both ESC lines (Fig. 2, G and H). There were more contracting EBs after 10 days of EB initiation in Evi-GOF EB cultures (Fig. 2I). Almost all Evi-GOF EBs were beating after 12 days of culture, whereas only 50% of control EBs showed contracting foci, indicating amplified cardiomyocyte differentiation in Evi-GOF cultures. Similarly, enhanced cardiomyocyte differentiation correlated with increased gene expression of Mlc2v (which encodes myosin light chain 2v) and Nkx2.5 (which encodes NK2 homeobox 5) (Fig. 2J). Because mesodermal differentiation can be directed toward hematopoietic, endothelial, or cardiomyocyte lineages, we analyzed the expression of additional mesodermal lineage markers related to hemangioblast differentiation (CD31, CD34, and Tie2) (Fig. 2J). However, only cardiomyocyte markers showed increased expression, whereas hemangioblast lineage markers remained unchanged, consistent with previous findings that β-catenin–dependent Wnt signaling antagonizes cardiomyogenesis compared to hematopoiesis depending on the time point during EB formation (27). Furthermore, expression of endodermal and ectodermal differentiation-related genes (Fgf5, CD133, Foxa2, and Gata6) was not significantly altered, suggesting that ectopic Evi-mediated Wnt secretion promoted differentiation toward the cardiomyocyte lineage in vitro. Evi expression was increased 100-fold during EB differentiation (Fig. 2K), supporting previous results that show that Wnt signaling represents an important driving force during early ESC differentiation (27). Together, constitutive overexpression of Evi in ESCs favored cardiomyocyte differentiation during EB formation.

Evi-deficient ESCs have aberrant in vivo differentiation

Pluripotent ESCs form benign teratomas composed of derivatives from endodermal, mesodermal, and ectodermal origin (2). We tested the ability of Evi-LOF ESCs to form all three germ layers by analyzing teratomas formed from ESCs subcutaneously injected into mice. Tumor initiation of Evi-LOF ESCs was reduced with only 20% of Evi-LOF ESC–injected mice developing tumors as compared to 90% of control ESC–injected mice (Fig. 3A). Evi-LOF ESC–derived tumors showed an impaired growth rate over a period of 27 days, which led to reduced tumor weight (Fig. 3, B and C). Proliferation was increased twofold in Evi-LOF teratomas, indicating that these tumors contained an increased proportion of proliferating stem and progenitor cells compared to lower cycling differentiated cells (Fig. 3D). Evi-LOF teratomas also showed a 10-fold increase in apoptotic cells (Fig. 3E). Whereas histological analysis of teratoma sections of control ESCs revealed the expected morphological structures of ectodermal lineages such as neural rosettes, mesodermal-derived lineages such as bone and cartilage, and endodermal lineages such as gut epithelial cells, these structures were poorly developed or absent in Evi-LOF ESCs (Fig. 3F). Instead, we observed a high degree of infiltrating erythrocytes, consistent with a hemorrhagic phenotype (Fig. 3F).

Fig. 3 Teratoma growth and lineage differentiation depend on Evi expression.

(A) Tumor initiation after transplantation of control and Evi-LOF ESCs in C57Black/6 mice (control, n = 20; Evi-LOF, n = 28). (B) Measurement of tumor growth over time (control, n = 14; Evi-LOF, n = 10). (C) Tumor weight after 27 days of transplantation (control, n = 14; Evi-LOF, n = 10; top). Images of control and Evi-LOF teratomas after 27 days of initiation (bottom; scale bar, 1 cm). (D) EdU-based proliferation analysis of single-cell tumor suspensions (control, n = 14; Evi-LOF, n = 10). (E) Measurement of apoptosis by staining of single-cell tumor suspensions for annexin V (control, n = 14; Evi-LOF, n = 10). (F) Hematoxylin and eosin staining of control and Evi-LOF teratomas. Evi-LOF teratomas did not reveal matched lineage differentiation but rather show amorphic pattern as shown by the three Evi-LOF teratoma pictures (right) (scale bar, 100 μm). n = 4 mice per group. (G) Fold change expression data of candidate genes (with P < 0.01) related to Wnt and Hh signaling, ESC and EpiSC maintenance, and mesodermal-lineage differentiation. n = 4 biological replicates per genotype, *P < 0.05; P values were calculated by Student’s t test. Error bars represent SD.

To dissect the changes in gene expression underlying the observed morphological differences in Evi-LOF teratomas, we performed transcriptome analysis of Evi-LOF teratomas and corresponding control tumors. We found altered expression of 488 genes (log2 fold changes ≥1 and adjusted P values <0.05) with 186 genes showing increased expression and 302 showing decreased expression in Evi-LOF teratomas as compared to controls (fig. S2A). Pathway analysis of gene expression in Evi-LOF teratomas revealed that genes with decreased expression were involved in muscle contraction and development (fig. S2B). Accordingly, immunohistological stainings with antibodies against Acta2 and Desmin revealed reduced labeling in Evi-LOF teratoma sections (fig. S2C). The gene expression and immunohistochemistry data supported a model whereby differentiation of ESCs into the mesodermal lineage was disturbed when autocrine Wnt secretion was impaired. We also found that Evi and Axin2 transcript abundance was decreased in Evi-LOF teratomas, suggesting that stromal cells did not compensate for the genetic ablation of Evi (Fig. 3G). Moreover, TCF3 expression was increased, consistent with a previous report demonstrating that TCF3 expression is inhibited by Wnt signaling (28).

Transcripts showing increased abundance in Evi-LOF teratomas belonged to the gene ontology classes of cell cycle, DNA/nucleotide metabolism and replication, cell proliferation and differentiation, and developmental processes (fig. S2D), indicating that Evi-LOF teratomas were proliferative but immature tumors (29, 30). Nanog, Oct4, Sall4, and the EpiSC factors Cldn6 and Fgf5 showed significantly increased expression, underscoring the embryonic phenotype of the Evi-LOF teratomas (Fig. 3G). Immunohistochemical staining of Nanog revealed a uniform distribution of Nanog-positive cells in the Evi-LOF teratomas as compared to control teratomas, in which Nanog staining was restricted to a few clusters of cells (fig. S2E). Evi-LOF teratoma profiling revealed increased Shh (Sonic hedgehog) expression (Fig. 3G). Shh and Wnt signaling antagonize each other during the generation of dopaminergic neurons by mouse ESCs, which is promoted by β-catenin–dependent Wnt signaling and inhibited by high Shh (31). In line with these findings, the expression of genes related to neuronal differentiation and maturation was significantly reduced in Evi-LOF teratoma. The expression of candidate genes such as Syp (which encodes Synaptobrevin), Syt4 (which encodes Synaptotagmin), Nsg2 (which encodes Neuron specific gene family member 2), and Des (which encodes Desmin) was decreased in Evi-LOF teratomas (Fig. 3G). Together, Wnt secretion supported ESC survival in vivo, which was necessary for teratoma formation, and the ablation of Wnt secretion impaired cardiomyocyte and neuronal differentiation and instead contributed to a proliferating population of undifferentiated cells.

Evi supports ESC survival

Next, we investigated the survival phenotype of ESCs upon loss of basal Wnt signaling. Annexin V labeling of ESCs cultured in the presence or absence of LIF revealed that loss of Evi increased apoptosis, a phenotype that was rescued by the GSK3 inhibitor SB216763 (Fig. 4A). Cell death was also reduced after the addition of recombinant Wnt3a independently of the presence of LIF, suggesting that canonical Wnt signaling supported ESC survival (Fig. 4A and fig. S1, I and J). Accordingly, the addition of the Porcupine inhibitor LGK974 increased apoptosis, which was attenuated by the addition of recombinant Wnt3a (fig. S1K). Culturing ESCs in the presence of medium conditioned by mouse L cells either expressing Wnt3a or Wnt5a reduced cell death by at least 50% independently of the presence of LIF (Fig. 4B), indicating that both canonical and noncanonical Wnt ligands support ESC survival.

Fig. 4 Wnt secretion supports ESC survival.

(A) Analysis of annexin V labeling in ESCs cultured in the presence or absence of LIF, SB216763 (SB), or Wnt3a for 72 hours. n = 4 biological replicates per group. (B) Analysis of annexin V labeling in ESCs cultured in medium conditioned by parental, Wnt3-overexpressing (Wnt3a L cell medium), or Wnt5a-overexpressing (Wnt5a L cell medium) mouse L cells for 72 hours. n = 3 biological replicates per group. (C) RT-PCR analysis of control and Evi-LOF ESCs for the indicated genes. n = 3 biological replicates per group. (D) Western blot of control and Evi-LOF ESC lysates incubated with antibodies against the indicated proteins. β-Actin was used as a loading control. n = 2 independent experiments. (E) Immunofluorescence images for Mdc1 of Evi-LOF and control ESCs. (F) Quantification of Mdc1 foci per nucleus. n = 3 sets of cells imaged in three independent experiments. (G) RT-PCR analysis of control and Evi-LOF ESCs against Mdc1 after 2 days of LGK947 treatment. n = 3 biological replicates per group. (H) Plot of DigiWest analysis (left). Western blot intensities of the indicated proteins on Evi-LOF and control ESC lysates based on DigiWest analysis (right). n = 1 independent experiment with four biological replicates per genotype. (I) Western blot of control and Evi-LOF ESC lysates incubated with the indicated antibodies. β-Actin was used as a loading control. n = 2 independent experiments. *P < 0.05; **P < 0.01; P values were calculated by Student’s t test. Error bars represent SD.

To dissect the apoptotic phenotype, we first analyzed the expression of trp53, which encodes the apoptosis-promoting protein p53. We found that the expression of both Trp53 and the p53 target gene and apoptotic activator Bax (32) was comparable in Evi-LOF ESCs and control cells (Fig. 4C). In contrast, the antiapoptotic protein Birc5 (also known as Survivin), which is transcriptionally regulated by p53 and Rb signaling (33), was significantly reduced at the protein level in Evi-LOF ESCs (Fig. 4D). Birc5 interacts with Mdc1 (also known as Nfbd1) and other DNA damage response proteins such as γ-H2AX (34). Mdc1 is a protein with antiapoptotic properties, is concentrated in DNA damage foci, and directly inhibits p53 by blocking the transactivator domain of p53 or reducing the phosphorylation of Ser15 in p53 (35). In contrast to γ-H2AX, Mdc1 abundance was increased in Evi-LOF ESCs and might be involved in the survival fate of ESCs (Fig. 4D) (36). Immunostaining for Mdc1 revealed more foci per nucleus in Evi-LOF ESCs compared to control cells (Fig. 4, E and F). Mdc1 transcript abundance was slightly lower in Evi-LOF ESCs, and short-term treatment of ESCs with LGK974 decreased Mdc1 expression (Fig. 4G), suggesting that the increase in protein abundance was not due to transcriptional up-regulation but protein stabilization.

Next, we performed a multiplex Western blot analysis (DigiWest) with antibodies against 167 proteins involved in signaling (such as the Wnt, TGF-β, and mitogen-activated protein kinase pathways), cell cycle control, apoptosis, and cytoskeletal and chromatin regulation. Log2 fold changes were plotted against P values to visualize the abundance of the candidate proteins (Fig. 4H). Gadd45α (which encodes Growth arrest and DNA damage–inducible protein α) and Dapk3 (which encodes Death-associated protein kinase 3) had the greatest log2 fold changes that were highly significant (Fig. 4H). Western blot analysis confirmed the increase in Dapk3 and Gadd45α protein abundance in Evi-LOF ESCs (Fig. 4I). In contrast, neither gene was transcriptionally regulated in Evi-LOF and control ESCs (fig. S3A). Dapk3 has several functions: It is proapoptotic in various cell types but it is also involved in autophagy, necrosis, and non–death-related signaling events. Dapk3 can trigger the intrinsic apoptosis pathway independently of p53 signaling (37). Similarly to Dapk3, Gadd45α regulates many cellular functions including DNA repair, cell cycle control, cell death, and genomic stability (38). The gene encoding Gadd45α is transcriptionally regulated by p53 and other factors, such as Atf4 or Foxo3, after DNA stress induction (38). We found that the transcription of Gadd45α in Evi-LOF ESCs was not altered, arguing for posttranscriptional stabilization of Gadd45α (fig. S3A). These results indicated that cell death in Evi-LOF ESCs relied on a complex network of multifunctional protein components implicated in apoptosis and genomic stability.

Evi deficiency in ESCs leads to impaired mitotic progression and chromosomal instability

We sought to dissect the survival defect of Evi-LOF ESCs. Analysis of 5-ethynyl-2′-deoxyuridine (EdU) incorporation in the presence or absence of LIF and Wnt3a revealed comparable DNA replication rates of Evi-LOF ESCs and control cells, indicating that lack of Wnt secretion has no effect on S-phase progression (fig. S3B). Similarly, Evi-GOF ESCs incorporated EdU in the presence and absence of LIF as well as under high-serum (10%) and low-serum (5%) conditions at rates indistinguishable from control cells (fig. S3C). Checkpoint controls during the cell cycle of eukaryotic cells are necessary to coordinate and prevent the accumulation of DNA damage, and cells undergo apoptosis when severe stress occurs during the cell cycle. Next, we investigated the cell cycle distribution of Evi-LOF and control ESC lines. We observed ~40% of all cells in the S phase for both Evi-LOF and control lines, indicative of a high number of continuously cycling cells (Fig. 5, A and B). Evi-LOF cells and the corresponding control lines did not show significant differences in the proportion of cells in different stages of the cell cycle, indicating that cell cycle progression in mouse ESCs was not dependent on basal Wnt activity.

Fig. 5 Wnt secretion supports anaphase progression and genomic stability.

(A) FACS-based analysis of the cell cycle distribution of Evi-LOF and control ESCs. Cells were gated for cycling cells. n = 4 biological replicates per group. (B) Representative FACS profiles of control and Evi-LOF ESCs. Cycling populations are highlighted in gray. (C) Representative picture of normal anaphase and anaphase with lagging chromosome. Scale bar, 5 μm. (D) Quantification of aberrant chromosome segregation during anaphase in Evi-LOF and control ESCs [control, n = 386 cells; Evi-LOF, n = 259 cells; for LGK974 (LGK)–treated cultures: control, n = 141 cells; Evi-LOF, n = 109 cells; Wnt control, n = 120 cells; Evi-LOF, n = 152 cells; for SB-treated cultures: control, n = 129 cells; Evi-LOF, n = 101 cells]. n = 3 independent experiments. (E) Quantification of aberrant chromosome segregation of Evi-LOF ESCs in the presence of mouse L cell conditioned medium (control, n = 297 cells; Evi-LOF, n = 203 cells). n = 3 independent experiments. (F) Images showing metaphase spread preparations of control and Evi-LOF cells. n = 3 independent experiments. Scale bar, 4 μm. (G) Percentage of euploid Evi-LOF and control cells in metaphase (control, n = 163 cells; Evi-LOF, n = 90 cells). n = 3 independent experiments. (H) Chromosomes from metaphase spread preparations were counted and cells were grouped according to the diagram (control, n = 307 cells; Evi-LOF, n = 409 cells). n = 3 independent experiments. Data represent means ± SD of four independent experiments and four independent clones per genotype, *P < 0.05, **P < 0.001. Cells were analyzed at passages 5, 10, and 15. (I) Schematic illustration on the requirement of autocrine Wnt ligands by ESCs. Basal Wnt activity is necessary to support ESC survival and genomic stability and for lineage differentiation but not for self-renewal. *P < 0.05; ***P < 0.001; P values were calculated by Student’s t test. Error bars represent SD.

Next, we analyzed the mitotic progression of Evi-LOF ESCs, because several factors with differential abundance, including Birc5, Mdc1, and Gadd45α, have been implicated in the regulation of genomic stability (3941). Segregation errors, such as lagging chromosomes or anaphase bridges, are typical disruptions taking place during anaphases, which are signs of chromosomal loss associated with aneuploidy (Fig. 5C) (42). We analyzed mitotic defects in four independent control and four independent Evi-LOF lines, which were subcloned in parallel after treatment with Cre recombinase (table S1). The percentage of Evi-LOF ESCs in anaphase with aberrant chromosome segregation was significantly increased (Fig. 5, C and D). We also observed an increase in mis-segregation in the presence of LGK974 (Fig. 5D). Mis-segregation was attenuated after the addition of recombinant Wnt3a or SB216763 to the medium, supporting the concept that basal Wnt activity was necessary to maintain proper mitotic progression (Fig. 5D). Similar to the effects of adding recombinant Wnt3a, culturing Evi-LOF ESCs with media conditioned by Wnt3a- or Wnt5a-expressing mouse L cells reduced anaphase aberrations and supported anaphase progression (Fig. 5E). Wnt5a-conditioned medium had no effect on axin2 expression in ESCs, indicating that Wnt5a signaled through a noncanonical signaling route (fig. S3D). Accordingly, both canonical and noncanonical Wnt signaling promote survival and mitotic transition in ESCs.

Metaphase chromosome counting revealed shifts in chromosome number distributions (Fig. 5, F to H). We observed a 50% reduction of euploid metaphases in multiple independent subclones of Evi-LOF ESCs (Fig. 5G). Specifically, the proportion of Evi-LOF ESCs in metaphase with chromosome numbers ranging between 36 and 44 was significantly decreased. At the same time, the number of metaphases with more than 44 chromosomes doubled, indicating that loss of autocrine Wnt signaling led to a disposition to aneuploidy (Fig. 5H). We propose that Wnt signaling in ESCs (i) was essential for lineage differentiation; (ii) supported ESC self-renewal, although this effect was only apparent in LIF-depleted conditions; and (iii) was necessary for mitotic stability and survival of ESCs (Fig. 5I).

DISCUSSION

The precise role of Wnt signaling in self-renewal and differentiation of ESCs has remained a matter of debate. Here, we demonstrated by using loss-of-function or gain-of-function approaches that autocrine Wnt signaling was required for ESC maintenance and for preventing mitotic defects and chromosomal instability. ESCs that lacked the Wnt cargo receptor Evi (also known as Wls) had an increased rate of apoptosis accompanied by chromosomal stability defects. Moreover, ESC-derived Wnt ligands supported ESC self-renewal in a LIF-dependent manner. However, they affected the expression of Wnt target genes without affecting that of key pluripotency genes in the presence of LIF. Mesodermal and endodermal differentiation of ESCs depended on autocrine Wnt ligands during EB differentiation, and Wnt ligands were essential for teratoma formation and differentiation. In addition, we showed that Evi overexpression in ESCs enhanced cardiomyocyte differentiation, supporting the model that Wnt signaling drives mesoderm differentiation (Fig. 5I).

Autocrine Wnt secretion in mitosis and survival

Several components of the Wnt signaling pathway, including Dvl, Axin2, β-catenin, and APC have been implicated in proper centrosome function and regulating spindle microtubules during mitosis (27, 4345). Excessive Wnt signaling also correlates with genomic instability and tumorigenesis of colorectal cancers (46). Blocking Wnt signaling in colonic tumor cells triggers increased microtubule assembly, which is followed by impaired chromosomal segregation and aneuploidy (47). Wnt/STOP signaling, which induces Wnt-dependent protein stabilization independent of β-catenin signaling, has been suggested as a pathway that regulates microtubule dynamics and chromosomal separation (47, 48). In line with these reports, Evi-depleted ESCs showed genomic instability, indicating that tight control of basal Wnt activity is necessary to maintain chromosomal stability in ESCs. In addition, our results suggested that noncanonical Wnt signaling also contributed to ESC survival and proper mitotic progression. Thus, Wnt activity that is extremely high or extremely low may lead to genomic instability, which impairs survival or supports tumor development. Whether both conditions affect overlapping mitotic signaling routes remains to be shown.

Evi-LOF ESCs showed no changes in p53 expression, consistent with analysis of β-catenin–deficient ESCs (49). Instead, Mdc1 abundance was increased in Evi-LOF ESCs, which might help to balance p53 activation (35). Mdc1 is also involved in the regulation of genomic stability (41) and interacts with Birc5 and other DNA damage response proteins (34). Birc5 abundance peaks in a cell cycle–dependent manner during the G2-M phase, during which it specifically localizes to the mitotic spindle (39). In addition, Birc5 is a target gene of β-catenin–dependent Wnt signaling (50). Likewise, Gadd45α mutant mice show impaired genomic stability (40). In summary, we observed aberrant expression of genes in Evi-LOF ESCs encoding factors that are involved in survival and mitosis. Overall, our results support the concept that signaling pathways responsible for stem and tumor cell growth also act as regulators of chromosomal instability (51).

Wnt ligands and their role in maintaining pluripotency

Wnt signaling plays a pivotal role in ESC maintenance and lineage differentiation (52). Delineating the role of Wnt signaling in these opposing processes has been challenging because many components of the Wnt signaling pathway exist as multiple isoforms or have additional functions in other signaling pathways and cellular processes, such as in the case of β-catenin and GSK3 (23, 29, 30, 5356). In many cases, only limited insights have been obtained to distinguish between Wnt-dependent and Wnt-independent functions (44, 45, 5759). The analysis is further complicated by the repertoire of different Wnt ligands released by ESCs, meaning that autocrine Wnt responses in ESCs may reflect the outcome of all Wnt proteins present. By manipulating the pan-Wnt secretion factor Evi, all Wnt signaling branches can be affected in the same cell at the same time. Our Evi loss-of-function and gain-of function ESC approaches provide research avenues to investigate the role of Wnt ligands in stem cell self-renewal and differentiation. We showed in the present study that basal Wnt activity did not appear to be required for the maintenance of pluripotent genes, including Oct4, Nanog, and Sox2, which agrees with analysis of β-catenin–deficient ESCs (28, 56, 58). Analysis of ESCs cultured in the presence of the Porcupine inhibitor IWP2, which globally interferes with Wnt secretion, has suggested that Wnt signaling is essential for the maintenance of ESC pluripotency by preventing their transition to EpiSCs (12). However, our expression profiling of Evi-LOF ESCs is not fully in line with this conclusion. Our results instead argue for a minor role of Wnt ligands in ESC self-renewal, because only in the absence of LIF do Wnt ligands support ESC self-renewal and maintenance. The data might also indicate that Wnt dependency may vary between different genetic backgrounds and experimental setups or that Porcupine might be involved in the acylation of proteins different from Wnt ligands. Moreover, IWP2 treatment or silencing of components that mediate β-catenin–dependent transcriptional activity has little effect on the activity of a Nanog reporter under conditions that promote self-renewal (60). Similar results have been shown with human ESCs (61). These results suggest that active β-catenin is retained at the plasma membrane and unable to mediate β-catenin–dependent transcription under pluripotent conditions. Upon exit from pluripotency, the amount of active β-catenin in the cytosol is increased, which triggers the induction of Wnt target genes. Our model is also consistent with other studies showing that transcriptionally competent β-catenin is not required for self-renewal under pluripotent culture conditions (namely, when LIF is present) (28, 55, 58).

The role of Wnt in ESC differentiation and teratoma formation

Autocrine Wnt secretion by ESCs is required for multilineage differentiation because ablation of Wnt secretion maintained teratoma cells in their undifferentiated and progenitor-like state. At the same time, markers for mesodermal and neuronal differentiation were reduced. Evi overexpression in ESCs facilitated cardiomyocyte differentiation, supporting a model that constitutively enforced Wnt secretion in ESCs drives the induction of cardiac progenitors. β-Catenin–dependent Wnt signaling has been proposed to have a dual role during cardiogenesis, either promoting or inhibiting cardiomyocyte differentiation depending on timing (27, 62, 63). Early treatment of EBs with Wnt3a stimulates mesoderm induction, leading to the enhanced induction of cardiac progenitors. Activation of Wnt signaling at later time points reduces the induction of cardiac progenitors, indicating that persistent β-catenin–dependent Wnt signaling attenuates cardiomyocyte maturation. In this context, Wnt11 is necessary to restrain Wnt/β-catenin signaling and support cardiac differentiation at later time points. Constitutive overexpression of Wnt1 increases the induction of cardiac progenitors during EB differentiation (25), which agrees with our finding that persistent canonical Wnt signaling through Evi overexpression enforced the early cardiogenic differentiation route.

Historically, the regulatory network of pluripotent stem cells has been analyzed in mouse ESCs. Although key pluripotency circuits appear to be conserved, several differences regarding molecular signatures exist, which makes it necessary to consider species-specific, developmental stage–specific, or epigenetic variations (64). EpiSCs, which are postimplantation-derived stem cells, are the murine counterpart of human ESCs (65). Blocking Wnt signaling improves the pluripotency of human ESCs and EpiSCs and prevents their spontaneous differentiation (15). In addition, the conversion of mouse EpiSCs to ESC-like cells is improved upon repression of the Wnt signaling pathway by IWP2 (16). However, it should be taken into account that stem cell survival and genomic integrity can be affected by blocking Wnt signaling. The identification of ESC-derived Wnt ligands as regulators for genomic stability and survival as well as lineage differentiation is important for understanding the molecular basis of the processes regulating stem cell maintenance and may also predict how dysregulated Wnt signaling affects genomic stability in tumor cells.

MATERIALS AND METHODS

Cell culture, cell lines, constructs, and generation of ESC lines

Floxed Evi (Evi-LOF) mouse ESCs were isolated from the inner cell mass of preimplantation embryos and clonally expanded (genetic background: C57BL/6). Floxed Evi-GOF mouse lines have been previously described (genetic background: 129Ola) (20). ESCs were cultured on mitomycin C–inactivated feeder cells [mouse embryonic fibroblasts (MEFs)] in LIF-supplemented ESC medium [Dulbecco’s minimum essential medium (DMEM) high glucose (Gibco) supplemented with 15% fetal calf serum (FCS) (PAN-Biotech), 1% penicillin-streptomycin (Invitrogen), 2 mM glutamine (Invitrogen), 100 μM β-mercaptoethanol (Sigma), 1 mM MEM–sodium pyruvate (Invitrogen), 1% nonessential amino acids (Invitrogen), and 1000 U/ml LIF (Millipore)] (66). Conditioned medium was generated by culturing confluent mouse L cells (parental, Wnt3a-overexpressing, or Wnt5a-overexpressing) or ESCs for 3 days in ESC medium. Conditioned medium was then diluted 1:2 with fresh ESC medium and used in assays. For Cre recombinase treatment, floxed ESC lines were seeded at a density of 5 × 105 per 6-well. Tat Cre recombinase (Excellgen) was applied at a concentration of 100 μg/ml in DMEM containing 1% bovine serum albumin (BSA) for 90 min. Subsequently, cells were washed to remove unbound protein, trypsinized, and replated at clonal density on MEFs in ESC medium. Individual colonies were picked and expanded on MEFs. Picked clones were genetically confirmed for deletion of Evi exon 3 by PCR genotyping (23 of 72 clones were correctly recombined). Recombinant mouse Wnt3a was obtained from PeproTech and used at a concentration of 100 ng/ml. SB216763 was purchased from Biomol, dissolved in dimethyl sulfoxide (DMSO), and used at 10 μM. LGK974 (Wuxi) was dissolved in DMSO and used at the indicated concentrations. Drug-treated cells were compared to DMSO-treated reference cells. To deplete MEFs, ESCs were passaged on gelatin-coated plates and used for experiments after three to five passages on gelatin-coated plates. ESCs were cultured at 37°C at 5% CO2 and passaged 1:3 every 2 to 3 days.

Animal experiments

For teratoma formation, 3 × 106 viable ESCs in 100 μl of phosphate-buffered saline (PBS) were injected into the dorsal flank of C57Black/6 or 129/Ola mice. All mice were at the age of 6 to 10 weeks when transplanted. Tumor growth was monitored every 2 or 3 days, and tumor volume was calculated using the following formula: volume = 0.5 × length × width2. Animals were sacrificed on day 27 after transplantation. For in vivo analysis, mice were injected intraperitoneally with EdU (20 μg/g mouse) 2 hours before sacrifice. Animal welfare and experimental procedures were performed in accordance to German animal protection law and were approved by the Regierungspräsidium Karlsruhe, Germany (AZ 35-9185.81/230/11).

Genotyping, RNA and RT-PCR, and plasmid transfection

DNA was isolated from cell pellets and prepared according to the manufacturer’s protocol (Qiagen). PCR reactions were performed with Taq DNA Polymerase (Qiagen). Total RNA was extracted from ESCs and teratomas using the RNeasy extraction kit (Qiagen) according to the manufacturer’s instructions. RT-PCR and quantitative PCR were performed with 25 ng of cDNA and LightCycler 480 Probes Master as described (Roche). Relative mRNA expression was calculated as a fold change compared to control. SDHA (which encodes succinate dehydrogenase complex subunit A) was used as a housekeeping gene. Primer sequences are listed in tables S3 and S4.

Immunohistochemistry

After deparaffination, antigen retrieval was performed by heating sections at 98°C for 10 min in 10 mM citrate buffer (pH 6.0). To block endogenous peroxidase, sections were treated with 1% H2O2 for 30 min. After blocking with avidin/biotin blocking solution at room temperature for 1 hour, sections were incubated with indicated primary antibodies in blocking solution overnight at 4°C. After washing with PBS, sections were incubated with labeled secondary antibodies for 3 hours at room temperature. Incubation with the avidin/biotin complex was carried out for 30 min at room temperature, followed by incubation with diaminobenzidine substrate for horseradish peroxidase (HRP)–labeled antibodies. Counterstaining was performed with hematoxylin. Sections were mounted in Faramount (Dako) and photographed using a Zeiss Axioskop 40 Microscope.

Imaging

For immunofluorescence microscopy, cells were plated on 10-mm coverslips and harvested 2 days after seeding. Cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.25% Triton X-100. Coverslips were incubated with primary antibodies overnight at 4°C in PBS containing 5% BSA and 3% goat serum. After washing, secondary antibodies were applied for 1 hour at room temperature. Slides were mounted with ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole (Molecular Probes). Antibodies and antibody concentrations are listed in table S2.

Chromosome analysis

ESCs at passages 5, 10, and 15 were analyzed for mitotic defect 2 days after seeding. Cells were harvested after incubation in colcemid (20 μl/ml, Biochrom) containing medium for 4 hours. Cells were then treated with 75 mM KCl for 10 min, centrifuged, and slowly fixed in methanol/acetic acid (3:1). The fixation procedure was repeated two times. Chromosome spreads were incubated at 50°C for 1 hour and stained with Wright and Giemsa (Sigma) according to the manufacturer’s protocol.

FACS analysis

Teratomas were mechanically minced into small pieces using scalpels, followed by a digestion step for 2 hours at 37°C in collagenase D at a concentration of 2 mg/ml in RPMI medium supplemented by 10% FCS. Gentle pipetting was performed during digestion. Cell suspension was filtered through a 70-μm nylon filter mesh (BD), washed in PBS once, counted, and processed for antibody staining and FACS analysis. Single-cell suspensions were stained with antibodies diluted in PBS containing 2% FCS for 45 min at 4°C. Secondary antibody staining was performed for nonfluorescent conjugated primary antibodies. After staining, cells were fixed with 4% PFA for 5 min, washed, and subsequently processed for FACS analysis. For intracellular labeling, cells were fixed and permeabilized with 0.2% Triton X-100/PBS for 5 min before incubation with conjugated primary antibodies. Proliferation assays were performed 2 days after seeding using EdU Click iT reagent (Invitrogen), according to the manufacturer’s instruction. Briefly, EdU at a final concentration of 20 μM was added to the cell culture and incubated at 37°C for 2 hours. Cells were fixed for 15 min with 4% PFA and permeabilized for 15 min at room temperature. Fixed cells were stained with the Click iT Alexa Fluor 647 azide for 30 min at room temperature. For annexin V and PI stainings, cultures were incubated in medium as indicated. Supernatant and attached cells were harvested 3 days (annexin V) after seeding and stained according to the manufacturer’s protocol (Roche). Fluorescence data were obtained with a FACSCanto II (BD) with FACSDIVA software. Data were subsequently analyzed using FlowJo 8.8.7 software.

In vitro differentiation

Feeder-free ESCs were harvested and resuspended in ESC medium without LIF at a concentration of 50,000 cells/ml. Hanging drop culture method was performed to generate EBs. A suspension (20 μl) containing 500 cells was pipetted on the lid of a 10-cm culture dish. After 2 days, EBs were transferred to an ultralow-attachment plate and cultured for 2 days. On day 4 of differentiation, EBs were transferred to 48-well plates for subsequent differentiation. On day 13 of differentiation, cells were harvested and pooled for analyses. For AP staining, 200,000 ESCs were seeded on coverslips in 24-well plates in the indicated medium and stained according to the manufacturer’s protocol (Millipore) after 2 days. At least eight random sites per slide were imaged using a Zeiss Axioskop 40 and analyzed by the image processing and analysis software package Fiji.

Western blot analysis and DigiWest

Cell pellets were lysed in urea buffer containing 8 M urea in PBS. Lysates were incubated on ice for 15 min and centrifuged at 13,000 rpm for 10 min at 4°C. Supernatants were collected and protein concentrations were determined by the bicinchoninic acid (BCA) kit (Perbio, Thermo Scientific) according to the manufacturer’s instruction. Proteins (15 to 40 μg) were supplemented with 5× Laemmli buffer and heated for 10 min at 80°C. Cell lysates were separated on 4 to 12% NuPAGE SDS–polyacrylamide gel electrophoresis bis/tris gradient gels (Life Technologies) and transferred to polyvinylidene difluoride membranes (Millipore, Merck Biosciences). Membranes were blocked in 5% nonfat dry milk/PBS incubated with primary antibodies overnight at 4°C. Blots were then incubated with corresponding HRP-conjugated secondary antibodies (Sigma-Aldrich) as listed in table S2. DigiWest analysis was performed as previously described (67).

Expression profiling

Teratomas were harvested 27 days after ESC injection and cut into small pieces. The poly(A) fraction was isolated from each of the eight teratoma samples using the RNeasy kit (Qiagen) and labeled complementary RNA (cRNA) were prepared according to the Illumina TotalPrep RNA amplification protocol (P/N IL1791M). cRNA (1.5 μg) were used to probe an Illumina Mouse Sentrix-8v2 beadchip according to the standard Illumina hybridization protocol (#11322355). These arrays have, on average, 15 beads per probe and cover more than 47,000 transcripts and known splice variants. Gene expression microarrays were scanned using the Illumina iScan Scanner according to the standard Illumina scanning protocol. The complete data set contained eight samples. Using BeadStudio software (v3.2), the limma package (v3.2.1), part of the Bioconductor/R package, was used to test for differential expression (68). This test assumes a linear model for gene expression. The differential expression test between Evi-LOF and control teratoma samples is based on the null hypothesis that the expression values of a gene in the samples come from the same distribution and results in P values for each gene and sample pair. A simple design matrix was created to fit a linear model to each gene expression value. An empirical Bayes method was used to moderate standard errors and estimate log fold change from the data, and a moderated t statistic was used to assess differential expression. Genes that had an adjusted (Benjamini-Hochberg) P value < 0.01 with respect to the third contrast listed above were regarded as differentially expressed and used for further PANTHER (69) (http://www.pantherdb.org/) analysis.

Statistical analysis

Unless otherwise indicated, data are expressed as means ± SD. Statistical significance was calculated by a two-tailed Student’s t test with unequal variance. A P value of <0.05 was considered statistically significant and marked in comparisons by asterisks. Two asterisks represent P values of <0.01. Three asterisks mark P values of <0.001.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/10/461/eaah6829/DC1

Fig. S1. Characterization of Evi-LOF ESCs.

Fig. S2. Expression analysis of teratomas formed from Evi-LOF ESCs.

Fig. S3. Proliferation and expression analysis of Evi-LOF ESC and controls.

Table S1. Lagging chromosome and karyotype analysis of Evi-LOF ESCs.

Table S2. Antibodies.

Table S3. Primers for qPCR.

Table S4. Primers for genotyping.

REFERENCES AND NOTES

Acknowledgments: We thank S. Leible and T. Miersch for help with the RNA sequencing experiments and G. Vollert and N. Tuysuz for support in ESC culture. We acknowledge the support by the DKFZ FACS core facility, the DKFZ Genomics and Proteomics core Facility for the array-based expression analysis, and the DKFZ animal department and transgenic unit for animal support and support to generate ESCs. We thank D. Kranz, G. Pereira, B. Kyewski, H. R. Rodewald, H. Augustin, and members of the Boutros lab for fruitful discussions. We also thank M. Templin for support of the DigiWest analysis, as well as C. Niehrs and S. Acebron for exchanging ideas on the ESC survival phenotype. We acknowledge T. Holland-Letz for statistical advice. Funding: This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB873 “Stem Cells in Development and Disease”). Author contributions: I.A. and M.B. designed the study. I.A., D.L.D., and J.H. performed research and analyzed and interpreted the data. G.E. performed DigiWest experiments. G.K. analyzed the expression-profiling data. I.A. and M.B. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Expression profiling data have been deposited in ArrayExpress (accession number E-MTAB-2927).
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