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1Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky .... implantation stages as well as human placental development (Dvash.
Molecular Human Reproduction, Vol.21, No.3 pp. 271–280, 2015 Advanced Access publication on November 11, 2014 doi:10.1093/molehr/gau104

ORIGINAL RESEARCH

Human embryonic stem cells carrying an unbalanced translocation demonstrate impaired differentiation into trophoblasts: an in vitro model of human implantation failure A. Shpiz 1,2,†, Y. Kalma 1,†, T. Frumkin 1, M. Telias 1, A. Carmon 1, A. Amit 1, and D. Ben-Yosef 1,2,* 1 Wolfe PGD Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv, Israel 2Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel

*Correspondence address. Tel: +972-3-6925733; Fax: +972-3-6925687; E-mail: [email protected]

Submitted on June 12, 2014; resubmitted on October 7, 2014; accepted on October 23, 2014

abstract: Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT –PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring b-hCG secretion, by bhCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of b-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of b-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure. Key words: human embryonic stem cells / implantation failure / preimplantation genetic diagnosis / trophoblast differentiation / unbalanced translocation

Introduction Miscarriage occurs in 15–20% of all pregnancies, and 1–2% of fertile women experience recurring miscarriages (RMs) (Farquharson et al., 2005; van den Berg et al., 2012). The incidence of either of the parents being a carrier of a chromosomal abnormality, including translocation, has been reported to be between 2 and 8% in couples with RM (Elghezal et al., 2007; Dutta et al., 2011; Franssen et al., 2011), and 12.5% of such miscarriages were shown to be the result of embryos with unbalanced translocations (Kochhar and Ghosh 2012). Balanced chromosomal translocations †

are the result of the rearrangement of segments between non-homologous chromosomes (Evsikov et al., 2000; Vandeweyer and Kooy 2009). Carriers of balanced reciprocal translocations have an increased risk of producing repeated miscarriages and offspring with congenital malformations. The parental germinal cell carrying the balanced translocation can produce many chromosomal variants in gametes, but only two would result in a healthy child, and all of the others would give rise to unbalanced embryos. This can explain the high prevalence of RMs among translocation carriers. The study of human chromosomal translocations and their associated abnormal phenotypes is very challenging. Several research models have

These first authors contributed equally to this study.

& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

272 been suggested for studying the clinical implications of translocations. One model is based on tissues derived from aborted fetuses (Mademont-Soler et al., 2009; Begum et al., 2010). However, that model has several disadvantages, including the difficulty in retrieving pure biological material and the short lifespan of primary cell cultures extracted from aborted fetuses. Animal cells can be genetically manipulated to express specific chromosomal aberrations, and such an animal model may be applied for studying the formation of chromosomal breakpoints and rearrangements (Magnuson et al., 1985; Rabbitts 2001; Lobato et al., 2008; Weinstock et al., 2008). However, due to interspecies variations between mice and humans, it is less useful for investigating the clinical manifestations of a specific translocation. Another model that had been proposed for studying chromosomal translocations involves the establishment of human cell lines from tumor cells that carry translocations, but tumorigenic transformation is usually characterized by a myriad of non-specific chromosomal aberrations (Busch 1989). An alternative approach is to introduce monosomies/trisomies by perturbation of mitotic DNA topoisomerase II (Clarke et al., 1993, 1998), but it is ineffective for generating the partial monosomies/trisomies that characterize unbalanced translocations responsible for RMs. These suggested models carry translocations derived from abnormal mitotic divisions occurring in somatic cells unlike the abnormal meiotic division occurring only in gametogenesis. Human embryonic stem cells (hESCs) are an excellent in vitro tool for studying early stages of embryogenesis, including pre- and postimplantation stages as well as human placental development (Dvash et al., 2006; Vazin and Freed 2010). hESCs are derived from the inner cell mass (ICM) of blastocyst stage embryos, and can be cultured in vitro indefinitely without losing their pluripotency (Thomson et al., 1998; Reubinoff et al., 2000). On the other hand they can be differentiated into virtually any cell type in the body, including extra-embryonic lineages (cyto- and syncytiotrophoblasts) (Drukker et al., 2012; Sudheer et al., 2012; Giakoumopoulos and Golos, 2013), and can therefore serve as a highly effective tool for studying early stages of implantation. hESCs that carry chromosomal aberrations can be derived directly from affected embryos following preimplantation genetic diagnosis (PGD) (Eiges et al., 2007; Ben-Yosef et al., 2008; Deleu et al., 2009; Frumkin et al., 2010; Seriola et al., 2011) or from karyotypically normal hESCs by inducing DNA double-strand breaks at specified loci using zinc finger nucleases (Brunet et al., 2009). These translocated hESCs can then serve as a human in vitro model for studying genetic disorders. Numerous mutated hESCs have been derived from affected embryos following PGD for monogenic disorders (Strelchenko et al., 2004; Frumkin et al., 2010; Mateizel et al., 2010; Ben-Yosef et al., 2011). In contrast, only a few studies have thus far reported the derivation of a hESC line carrying unbalanced translocations (Sun et al., 2008; Frydman et al., 2009; Narwani et al., 2010; Stephenson and Braude 2010). Apart from the derivation and characterization of these pluripotent hESCs, none of the currently translocated hESC lines have been investigated for determining the cause of implantation failure that characterizes almost all translocated embryos. Translocation t(11;22) is the most common recurrent nonRobertsonian constitutional reciprocal translocation in humans, and it has been reported in .160 unrelated families who have experienced repeated implantation failure (Fraccaro et al., 1980; Zackai and Emanuel 1980; Iselius et al., 1983; Armstrong et al., 2000; Hill et al., 2000).

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The aim of the present study was to report the derivation of a novel hESC line that carries the unbalanced reciprocal translocation t(11;22) and harbors a partial monosomy der(11)t(11;22)(q23;q12). We show that our Lis05_t(11,22) hESC line is pluripotent and that it can differentiate into derivatives of the three germ layers both in vitro and in vivo. Interestingly, these unbalanced translocated hESCs showed a delayed and reduced ability to develop into trophoblast cells, as demonstrated by their decreased and delayed secretion of b-hCG and their impaired expression of trophoblastic genes, compared with control hESCs. We suggest that the unbalanced chromosomal constitution in translocation t(11;22) cells leads to an impaired trophoblastic differentiation that leads to implantation failure of unbalanced translocation t(11;22) embryos.

Methods PGD for translocation t(11;22) Peripheral lymphocytes obtained from the parental carrier were used to calibrate the fluorescent in situ hybridization (FISH) analysis for translocation t(11;22). Extracted lymphocytes were stimulated to proliferate using phytohemagglutinin-M (PHA-M, Biological Industries, Beit Haemek, Israel, 0.2 ml/5 ml peripheral blood karyotyping medium) for 72 h, and metaphase spreads were prepared using 0.2 ml of colcemide solution (10 mg/ml, Biological Industries, Beit Haemek, Israel) following standard cytogenetic techniques (Rooney and Czepulkowski 1992). FISH analysis was performed using probes for chromosomes 11 and 22: CEP 11—Aqua Spectrum for the chromosome 11 a-satellite region and LSI DiGeorge/VCFS—a twocolor probe mixture containing TUPLE 1 Orange Spectrum probe for 22q11.2; ARSA Green Spectrum for 22q13.3 region (Vysis, Inc., Downer’s Grove, IL, USA). Ovulation induction for the PGD procedure as well as fertilization by intracytoplasmic sperm injection (ICSI) and embryo culture were performed as described previously (Malcov et al., 2004). Embryo biopsy was performed on 6 – 8 cell embryos. Individual blastomeres were spread onto a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany) using 0.01 M HCl/0.1% Tween 20 solution as previously described (Barbash-Hazan et al., 2009). Embryos diagnosed with a balanced translocation were transferred back to the uterus for implantation. Embryos diagnosed as having unbalanced translocation were donated by the parents for derivation of hESC lines after they signed an informed consent form. The procedure abided by the approved protocol by the Israeli National Ethics Committee (7/04-043).

Human embryonic stem cells hESCs were derived as previously described (Frumkin et al., 2010) and grown on mitomycin-treated mouse embryonic fibroblasts (MEFs) in standard hESC medium composed of KO-DMEM (Gibco, Surrey, UK), supplemented with 20% KO-Serum Replacement (Gibco), 1% non-essential amino acids (Biological Industries), 1 mM L-glutamine (Biological Industries), 0.5% insulin-transferrin-selenium (Invitrogen, Paisley, UK), 50 U/ml penicillin and 50 mg/ml streptomycin (Biological Industries), 0.1 mM betamercaptoethanol (Sigma-Aldrich), and 30 ng/ml bFGF (R&D Systems, Europe Ltd, Abingdon, UK). hESCs were manually propagated during the first passages. Following 5 – 7 passages, the newly established lines were further propagated by collagenase IV 1 mg/ml (Invitrogen). Established cell colonies were grown in hESC medium supplemented with 4 ng/ml bFGF as previously described (Frumkin et al., 2010).

Fluorescent in situ hybridization For FISH analysis, hESCs were fixed with methanol and acetic acid (3:1), and the analysis were carried out using the same probes panel for chromosomes

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11 and 22 and the same method used for the single-cell PGD analysis as previously described (Barbash-Hazan et al., 2009).

Karyotyping hESCs were incubated with 100 ng/ml colcemide (Biological Industries) for 30 min at 378C. Following trypsinization, the cells were incubated with 0.075 M potassium chloride for 10 min at 378C, fixed with methanol and acetic acid (3:1) and dropped onto glass slides. Karyotype analysis of chromosome spreads were determined by Giemsa staining of at least 20 different metaphase-stage cells.

FACS analysis FACS analysis of undifferentiated hESCs was performed using AF488 anti-human SSEA-3 antibodies (BioLegend) and their respective isotype control. Samples were analyzed using a BD FACSCanto flow cytometer (BD Biosciences). Briefly, the cells were grown on matrigel-coated plates until confluence. A total of 1 × 106 cells were incubated in the dark for 1 h with 5 ml anti-human SSEA-3 antibody (Santa Cruz cat330305) or 5 ml normal rat IgM antibodies (for control). After incubation, the cells were washed with PBS and subjected to FACS analysis.

In vivo hESC differentiation In vivo differentiation into teratomas was performed as described previously (Amit et al., 2004). Briefly, hESCs were injected into the rear leg muscle of three 4-week-old male SCID-beige mice, and the mice were sacrificed 10 weeks later for teratoma isolation. Experiments were performed with the approval of the Committee for Animal Care and Use of the Rappaport Faculty of Medicine, at the Israel Institute of Technology (Technion, Haifa). Histological sections were prepared by paraffin embedding and stained for H&E and for b-hCG in the Institute of Pathology at the Tel-Aviv Sourasky Medical Center.

Directed differentiation into trophoblasts Undifferentiated hESCs were passaged with collagenase type IV 1 mg/ml and plated on 12-well plates coated with matrigel (BD Biosciences) (diluted 1:100 in cold DMEM) in a density of 80 – 100 000 cells per well. The cells were cultured in MEF-conditioned medium supplemented with 8 ng/ml bFGF for 2 days and then treated with feeder-free conditioned medium supplemented with 100 ng/ml human BMP4 (R&D).

b-hCG secretion measurements Fluorimetric immunoassays were performed to assess the secreted b-hCG concentration (mIU/ml) in medium samples taken at different time points following induction of differentiation of the hESCs. Fresh hESCs were used as a negative control.

Gene expression by quantitative real-time PCR Total RNA was extracted on Days 0, 2, 4, 6 and 8, following induction of differentiation, using an RNeasy Mini Kit (Qiagen Crawley, UK) including the DNase step. RNA was reversed transcribed using the SuperScript III First-Strand Synthesis System for RT– PCR (Invitrogen). Reverse transcriptase PCR (RT – PCR) was performed using ReddyMix (Thermo-Scientific, Surrey, UK). Quantitative Real-Time PCR (qRT – PCR) was performed using SYBR Green (Thermo-Scientific). Relative transcript levels were calculated relative to GAPDH. All primers used in these processes are listed in Supplementary Table SI.

Statistical analysis Relative gene expression is presented as mean + standard error. Values were compared by one-tailed Student’s t-test using SPSS version 19. P , 0.05 was considered statistically significant.

Results PGD for translocation t(11;22) and derivation of a translocated hESC line A 26-year-old woman carrier of the balanced translocation 46, XX, t(11;22)(q23;q12), who had a history of six early (first trimester) miscarriages and two previous failed IVF cycles, underwent PGD-FISH for translocation t(11;22) with specific fluorescent probes located to chromosomes 11 and 22 which are informative for this specific translocation (Fig. 1). When examined on metaphase spreads extracted from the peripheral blood of the parental carrier, this set of probes demonstrated a signal pattern of a balanced carrier of chromosomal translocation t(11;22)(q;q) (Fig. 1A). These FISH results confirmed the carrier’s karyotype and further validated the PGD-FISH protocol developed specifically for this couple. PGD-FISH analysis on single blastomeres aspirated from seven embryos identified two embryos with balanced translocation t(11;22): they were transferred back to the uterus, resulting in the implantation of one embryo and the birth of a healthy child. Five embryos were found to carry unbalanced translocation t(11;22) and were therefore donated for hESC line derivation. Three of these five embryos developed into expanded blastocysts by Day 6 post-fertilization, and one hESC line with der(11)t(11;22)(q23;q12) was established and labeled Lis05_t (11;22). FISH analysis of .30 Lis05_t(11;22) cells performed at passages 7 and 34 confirmed the single-cell PGD results of der(11)t(11;22), demonstrating the existence of one maternal derivate of chromosome 11 and normal paternal chromosomes 11 and 22. The missing maternal derivate 22 led to an unbalanced translocation (Fig. 1B I–II). Full karyotyping performed at passages 13, 18 and 33 confirmed partial monosomies of chromosomes 11 and 22, with no other chromosomal aberrations (Fig. 1B III). The haploid genomic loss in Lis05_t(11;22) included 11q23.1 to 11qter and 22p13 to 22q12.3. This haploid genomic loss comprised a total of 363 genes from chromosome 11 and 530 genes from chromosome 22 (NCBI Map Viewer).

Characterization of the pluripotent Lis05_t(11;22) hESC line The Lis05_t(11;22) hESC line harbored a partial monosomy which had been shown to be lethal (Jobanputra et al., 2005). Interestingly, we now showed that the translocation t(11;22) hESC line derived from the unbalanced embryo was able to self-renew in vitro while remaining undifferentiated (currently at passage .60), demonstrating a typical hESC line morphology (Fig. 2A). In addition, Lis05_t(11;22) colonies expressed pluripotent markers, such as Oct4 and Nanog (Fig. 2B), and .90% of the cells were SSEA-3 positive (Fig. 2C). In vitro spontaneous differentiation of Lis05_t(11;22) hESCs resulted in the formation of embryoid bodies (EBs), similar to control hESCs (Fig. 3A I). Histological sections of these EBs revealed the presence of cells derived from the three embryonic germ layers (Fig. 3A II). FISH analysis demonstrated the unbalanced translocation t(11;22) also in the differentiated cells within the EB (Fig. 3A III). Moreover, in vivo differentiation of

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(neural rosettes) (Fig. 3B II) and the mesoderm (blood vessels and cartilage) (Fig. 3B III). Taken together, the results displayed in Figs 2 and 3 demonstrate that the translocation t(11;22) cells are indeed undifferentiated pluripotent hESCs with a wide differentiation potential.

Trophoblastic differentiation of Lis05_t(11;22)

Figure 1 Diagnosis and characterization of t(11;22). (A) Balanced t(11;22) in the parental genome. (I) Fluorescent image of the metaphase spreads following FISH analysis of a balanced t(11;22) carrier in white blood cells in preparation for PGD. The aqua probe is located to the chromosome 11 a-satellite region; the orange probe is located to chromosome 22 at the q11.2 region; the green probe located to chromosome 22 at the q13.3 region. The green signals at der11 indicate the 22q region that had translocated with the 11q region (which was not probed at der22). (II) Schematic depiction of the FISH probes used in (A). (B) The unbalanced t(11;22) in the Lis05_t(11;22) hESC line. (I) FISH analysis of unbalanced t(11;22) hESCs performed at passage 34. (II) Schematic depiction of the FISH probes used in (B-I). (III) Karyotype analysis performed at passage 33. The arrow indicates derivate 11 with part of chromosome 22 attached to the q arm of chromosome 11. Note the single chromosome 22 present in this unbalanced partial monosomy line and that der22 is missing.

the translocated hESCs resulted in the formation of teratomas containing a variety of structures representing cells from the three germ layers, including the endoderm (intestine-like structures) (Fig. 3B I), the ectoderm

We then studied the potential of the translocated cells to differentiate into trophoblast cells, in order to determine whether the translocation t(11;22) results in an impairment in the development of extra-embryonic lineages rather than embryonic lineages. For this purpose, we performed two different experiments: in vivo spontaneous differentiation by injection of the hESCs into nude mice to obtain teratomas, and in vitro directed differentiation of hESCs into trophoblasts. In vivo trophoblastic development was demonstrated in the control hESCs by positive b-hCG staining of paraffin-embedded teratoma sections (Fig. 4A, Supplementary Fig. S1(D –F)). In contrast, teratoma sections obtained from Lis05_t(11;22) hESCs showed no b-hCG-secreting cells (Fig. 4B. Supplementary Fig. S1A –C). Human placenta served as positive controls for in situ b-hCG staining (Supplementary Fig. S2). It is important to note that the b-hCG-positive cells in the control teratomas are random cells in a gland-like structure rather than large areas of trophoblastic cells characterizing the placenta. However, it is well accepted that extra-embryonic cells, including trophoblasts, are rarely found in teratomas generated from hESCs. Therefore, we conclude that Lis05_t(11;22) hESCs, unlike the control line, cannot generate trophoblasts following in vivo differentiation into teratomas, probably due to the translocation. In vitro directed differentiation into trophoblast was performed by treating the hESCs with bone morphogenetic protein 4 (BMP4) which is considered to be an efficient protocol for differentiating hESCs into trophoblasts (Xu et al., 2002). Differentiation into trophoblasts was evidenced by their ability to secrete b-hCG into the medium. The control cells began to secrete b-hCG already at 4 days after BMP4 treatment, and b-hCG secretion increased dramatically with culture time, reaching extreme levels of 55 000 mIU/ml at 8 days following differentiation induction (Fig. 4C). In contrast, b-hCG secretion from translocation t(11;22) cells was significantly delayed and increased considerably more slowly, reaching a maximum of only 10 000 mIU/ml, which was much lower than the controls. In order to rule out the possibility that the difference in proliferation rate between the Lis05_t(11;22) cells and the controls might affect the results, and that the lower amount of b-hCG secretion could have been due to lower amounts of differentiated cells, we first counted the number cells at every time point and normalized the b-hCG levels to the number of cells (Supplementary Fig. S3). The results of the normalized data showed that the proliferation rate was similar in translocation and control cells, supporting our conclusion that the significant differences in b-hCG secretion shown in Fig. 4C are due to the impaired trophoblast differentiation in the translocated lines compared with controls. In order to explore the mechanism responsible for the impaired trophoblastic differentiation in translocation t(11;22), we analyzed the expression levels of several trophoblastic genes in RNA samples extracted from Lis05_t(11;22) hESCs at the same time points during in vitro trophoblastic differentiation in which b-hCG was measured. We specifically examined the expression of CDX2, KRT7, PPARG, ERVW1, KLF4, TP63, CGA and GCM1, all of which were recently

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Figure 2 Characterization of the Lis05_t(11;22) hESC line. (A) Phase image of two classical colonies of undifferentiated Lis05_t(11;22) hESCs (passage 39). (B) Expression of the pluripotent markers Oct4 and Nanog by PCR analysis in control (HUES-6, HUES-16 and HUES64) and Lis05_t(11;22) hESCs. The housekeeping gene GAPDH was used as a control. (C) Quantitative FACS analysis of SSEA-3 expression in Lis05_t(11;22) hESCs at passage 39 revealing 95% positive-stained cells.

Figure 3 Differentiation potential of Lis05_t(11;22) hESCs. (A) In vitro differentiation into EBs. (I) Phase image of Lis05_t(11;22) embryoid bodies (EBs) at Day 25 following spontaneous in vitro differentiation. (II) Hematoxylin and eosin (H&E) staining of a paraffin-embedded Lis05_t(11;22) EB section showing different cell types derived from the three germ layers. (III) Confirmation of the unbalanced t(11;22) hESCs by FISH analysis. (B) In vivo differentiation into a teratoma. H&E staining of paraffin-embedded sections of a Lis05_t(11;22) teratoma developed in mice reveals structures derived from all three germ layers: (I) gut-like structure (endoderm derivative), (II) neural rosette-like structure (ectoderm derivative) and (III) cartilage-like structure (mesoderm derivative).

shown to mediate BMP4-induced differentiation of human and mouse pluripotent stem cells into trophoblast lineages (Marchand et al., 2011; Li et al., 2013). We first show that in vitro trophoblastic differentiation induced down-regulation of the pluripotent markers Nanog and Oct4 already at Day 4 following induction in both the control and Lis05_t(11;22) hESCs (Fig. 5A and B), demonstrating that Lis05_t(11;22) cells effectively

undergo differentiation. More importantly, the expression of the trophoblastic genes CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG was increased in the control cells as expected, indicating that trophoblast cells were indeed generated in vitro and they are the ones secreting b-hCG (as seen in Fig. 4). In contrast, applying the same protocol of directed trophoblastic differentiation by BMP4 on the translocated hESCs Lis05_t(11;22)

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Figure 4 Impaired trophoblastic differentiation of Lis05_t(11;22) hESCs. (A and B) Immunostaining of b-hCG in teratoma sections developed from control hESCs (A, black arrows), and in Lis05_t(11;22) hESCs (B, no specific staining). (C) b-hCG secretion (mIU/ml) following in vitro trophoblastic differentiation of control hESCs (blue lines) and Lis05_t(11;22) hESCs (red line); (n ¼ 3).

resulted in reduced or delayed expression of the trophoblastic genes CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG (Fig 5C–H).

Discussion We report the establishment of a novel hESC line that carries an unbalanced translocation and has partial monosomies in chromosomes 11 and 22. Unbalanced translocations are known to be lethal during early embryonic development, and they are probably the reason for recurrent miscarriages in carriers of translocations in general and in carriers of translocation t(11;22) in particular. In spite of the extensive genomic alteration that is observed in unbalanced translocation t(11;22)(q23;q12) cells, the ICMs isolated from such a blastocyst successfully generated a stable pluripotent hESC line. Compared with monogenic disorders, the efficiency of deriving translocated hESCs following PGD is very low. We are aware of only a few reports on the derivation of hESC lines with unbalanced translocations (Sun et al., 2008; Frydman et al., 2009; Narwani et al., 2010; Stephenson and Braude 2010). Frydman et al. plated 22 unbalanced blastocysts for derivation, but only one was successfully derived into an established hESC line carrying a partial chromosome 21 monosomy and a partial chromosome 1 trisomy. In contrast, the derivation efficiency of hESC lines following PGD for monogenic mutations was reportedly not

different from that of WT-hESC lines (Frumkin et al., 2010; Tropel et al., 2010). Indeed, over 200 PGD-hESC lines have been derived thus far worldwide (summarized in http://www.umassmed.edu/iscr/ GeneticDisorders.aspx). The Lis05_t(11;22) line described here has typical hESCs morphology and expresses transcription factors and surface markers associated with bona-fide undifferentiated hESCs. In addition it displayed a stable karyotype, even at high passages. These results are in agreement with Sun’s observations that hESC lines with normal and abnormal karyotypes present similar biological characteristics (Sun et al., 2008). We demonstrated that our unbalanced translocation t(11;22) cells can differentiate both in vitro and in vivo into cells from the three germ layers. However, our results show that the translocation t(11;22) line is specifically linked to impaired differentiation into trophoblastic/placental lineages, as shown by the reduced and delayed b-hCG secretion from trophoblast cells differentiated from these translocated hESCs. In addition, activation of trophoblastic genes, which are needed for trophoblast development and functions (Niwa et al., 2005; Strumpf et al., 2005), are dramatically altered in trophoblastic cells differentiated from the translocated hESC line compared with control cells. The first lineage decision during mammalian embryo development is the differentiation of the totipotent cleavage stage embryo into the ICM and trophectoderm that comprises the blastocyst. The ICM cells

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Figure 5 Gene expression of trophoblastic and stemness markers in control and Lis05_t(11;22) hESCs differentiated into trophoblasts. qRT –PCR analysis of (A) NANOG, (B) Oct4, (C) CDX2, (D) TP63, (E) KRT7, (F) ERVW1, (G) CGA, (H) GCM1, (I) KLF4 and (J) PPARG in control (blue bar) and Lis05_t(11;22) (red bar) hESCs. Relative transcript levels of each gene were analyzed in undifferentiated hESCs (Day 0) and in samples taken every 2 days during in vitro trophoblastic differentiation. Data are presented as mean + standard error and represents two to three experiments performed on the translocated line and on the three different control lines. *P , 0.05, **P , 0.01.

278 will give rise to the three germ layers of the developing embryo, and the trophectoderm will give rise to the trophoblast lineage that will eventually contribute to the placenta. It has been previously shown that the presence of unbalanced chromosomal translocations does not affect the embryo’s ability to create a blastocyst (Evsikov et al., 2000), however, their development is impaired in most cases. Most unbalanced translocations are lethal and lead to RMs. Indeed, for translocation t(11;22), there is only one report on a pregnancy of an embryo carrying an unbalanced karyotype with similar variant to that of Lis05_t(11;22) (Jobanputra et al., 2005). The ultrasound examination of this pregnancy had revealed a viable embryo with a beating heart at 6 weeks of gestation, which spontaneously aborted shortly thereafter. RMs associated with unbalanced translocation t(11;22) in humans can be caused by the improper development of either of the two cell lineages: the ICM that creates embryonic tissues or the trophectoderm that creates the extra-embryonic tissues. We studied the differentiation potential of our Lis05_t(11;22) cells in order to explore the basis for the occurrence of RMs in unbalanced translocation t(11;22) embryos. As mentioned above, our Lis05_t(11;22) cells were fully pluripotent and capable of developing into derivatives of the three embryonic germ layers. This led us to hypothesize that the RMs of embryos carrying unbalanced translocation t(11;22) cells could be due to a specific impairment in the development of extra-embryonic lineages rather than the embryonic lineages. However, since we performed direct differentiation to only the trophoblast lineage, we cannot rule out the possibility that abnormal differentiation into other lineages could also contribute to recurrent miscarriages in these embryos. During early human placenta development, a mononuclear cytotrophoblast differentiates into either syncytiotrophoblasts or extra-villous trophoblasts. The former are responsible primarily for gas and nutrient exchange, whereas the latter invade the uterine stroma and spiral arterioles in order to access maternal blood and establish the maternal– fetal interface (Roberts and Fisher 2011). Although hESCs are derived from the ICM, following removal of the trophectoderm, previous studies have demonstrated that hESCs are also able to differentiate into trophoblasts (Xu et al., 2002; Gerami-Naini et al., 2004; Ezashi et al., 2012). The most common methodology for differentiating hESCs into trophoblasts is by treating them with BMP4. BMP4-treated hESCs have been shown to secrete b-hCG, the definitive functional test for syncytiotrophoblasts development (Hay and Lopata 1988; Staun-Ram and Shalev 2005; Cole 2012). We demonstrated that unlike control hESC lines, the Lis05_t(11;22) cells displayed reduced and delayed secretion of bhCG, indicating abnormal trophoblast differentiation. b-hCG secretion of control hESCs treated with BMP4 was reportedly accompanied by the expression of cytotrophoblast genes (CDX2, KRT7, TP63) and syncytiotrophoblasts genes (ERVW1, GCM1, PPARG, KLF4, CGA). Indeed, as expected, all of these genes were up-regulated in our control hESCs following treatment with BMP4, resembling the expression pattern characterizing proper in vivo development, with genes important for cytotrophoblast differentiation being up-regulated before genes of the syncytiotrophoblasts. In contrast, the translocated Lis05_t(11;22) hESCs showed an impaired expression of almost all of these genes, concomitantly with delayed and reduced secretion of b-hCG. CDX2 and KRT7 are markers of mouse trophectoderm and epithelial cells (Adjaye et al., 2005; Bernardo et al., 2011), p63 is expressed in proliferative cytotrophoblast (Lee et al., 2007; Li et al., 2013) and ERVW1, also known as syncytin-1, mediates the fusion of cytotrophoblasts into syncytiotrophoblasts as well as

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trophoblast proliferation (Huang et al., 2013). GCM1 and its upstream regulator PPARG are required for syncytiotrophoblasts development and the proper branching of trophoblast villi (Anson-Cartwright et al., 2000; Tarrade et al., 2001; Baczyk et al., 2009). KLF4 is a nuclear marker of syncytiotrophoblasts (Sen et al., 2012), and CGA encodes the alpha subunit of the hormone human chorionic gonadotrophin (hCG) (Hay and Lopata 1988; Staun-Ram and Shalev 2005). The impaired expression of trophoblastic genes is probably the reason for the inability of Lis05_t(11;22) cells to differentiate into trophoblast cells. In conclusion, our results provide valuable information regarding the molecular mechanism responsible for human implantation failure in a recurrent unbalanced translocation t(11;22). It is well established that monosomy embryos will not develop into advanced fetuses of ongoing pregnancies. Autosomal monosomies are rarely found in spontaneous abortions and are thought to be responsible mainly for preclinical miscarriages. Moreover, deficiencies involving any part of the chromosome (i.e. partial monosomies) lead, in most cases, to lethality at very early stages of human development (Stephenson et al., 2002; Biancotti et al., 2012). Whether these monosomy embryos fail to implant or succeed in implanting and only subsequently result in a spontaneous miscarriage is not always understood since it is impossible to study human embryos in vivo. However, some variants of unbalanced chromosomal translocations, including partial monosomy, can develop into fetuses and lead to the birth of a child with severe malformations (Yao and Jenkins 1994). The live birth of an affected offspring indicates that unbalanced translocations do not necessarily affect prenatal embryo viability, and that the outcome of each pregnancy depends on the specific karyotype of the embryo. Our translocated hESC line Lis05_t(11;22) carries a partial monosomy, and this particular unbalanced translocation has been previously shown to be lethal and leads to pregnancy loss (Jobanputra et al., 2005). We are aware that this work was done on only one variant of only one translocation due to the scarcity of translocated hESC lines. At the same time, the translocation t(11;22) is the most frequent balanced translocation in the human population. Further studies on hESC lines with different variants of different translocations are needed to validate our conclusion that the correlation between impaired trophoblast differentiation and implantation failure also applies to other translocations.

Supplementary data Supplementary data are available at http://molehr.oxfordjournals.org/.

Acknowledgements The authors thank Tamar Schwartz, Tanya Cohen Nava Mei-Raz and Shiri Asaf, the embryologists of the IVF lab, for their skillful processing of all the materials and the Institute of Pathology at the Tel-Aviv Sourasky Medical Center for b-hCG staining. Sigalit Siso is thanked for the graphics and Esther Eshkol for editorial assistance.

Authors’ roles A.S.: acquisition of data; analysis and interpretation of data; manuscript writing. Y.K.: conception and design analysis and interpretation of data; manuscript writing. T.F.: acquisition of data; analysis and interpretation of data; manuscript writing. M.T. acquisition of data; analysis and interpretation of data; manuscript writing. A.C.: clinical treatment of patients.

hESCs to study implantation failure in unbalanced t(11;22)

A.A.: acquisition of data; clinical treatment of patients. D.B.-Y.: conception and design analysis and interpretation of data; manuscript writing; final approval of manuscript.

Funding This work was supported by a grant from the Recanaty Foundation (Tel Aviv University), the joint Weizmann-TASMC Research Grant, and the Ministry of Health (Israel).

Conflict of interest None declared.

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