Human Reproduction Vol.23, No.8 pp.

1742– 1747, 2008 Advance Access publication on May 24, 2008

doi:10.1093/humrep/den190

The four blastomeres of a 4-cell stage human embryo are able to develop individually into blastocysts with inner cell mass and trophectoderm
Hilde Van de Velde1,2,4, Greet Cauffman1, Herman Tournaye1,2, Paul Devroey1,2 and Inge Liebaers1,3
1 2 4

Research Centre Reproduction and Genetics, Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium; Centre for Reproductive Medicine, UZ Brussel, Belgium; 3Centre for Medical Genetics, UZ Brussel, Belgium Correspondence address. Tel: þ 32-24776696; Fax: þ32-24776692; E-mail: hilde.vandevelde@uzbrussel.be

BACKGROUND: Early mammalian blastomeres are thought to be flexible and totipotent allowing the embryo to overcome perturbations in its organization during preimplantation development. In the past, experiments using single blastomeres from 2-, 4- and 8-cell stage mammalian embryos have provided evidence that at least some of the isolated cells can develop into healthy fertile animals and therefore are totipotent. We investigated whether isolated blastomeres of human 4-cell stage embryos could develop in vitro into blastocysts with trophectoderm (TE) and inner cell mass (ICM). METHODS: Six 4-cell stage human embryos were split and the four blastomeres were cultured individually. The expression of NANOG, a marker for ICM cells, was analysed by immunocytochemistry. RESULTS: The majority of the blastomere-derived embryos followed the normal pattern of development with compaction on Day 4 and cavitation on Day 5 and developed into small blastocysts with TE and ICM on Day 6 (n 5 12). The four cells of one embryo were individually capable of developing into blastocysts with TE and ICM, and NANOG was expressed in the ICM. CONCLUSIONS: Although based on a small number of embryos, we conclude that the blastomeres of a 4-cell stage human embryo are flexible and able to develop into blastocysts with ICM and TE.
Keywords: blastomere; totipotent; inner cell mass; NANOG; blastocyst

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Introduction Early blastomeres are assumed to be totipotent allowing the embryo to regulate its development in order to overcome perturbations in its organization such as cell loss. Direct evidence for totipotency is provided when an isolated blastomere is able to develop into a normal fertile offspring (reviewed in Edwards and Beard, 1997). In mice, splitting experiments have shown that a single blastomere of a 2-cell stage embryo is totipotent, but a single blastomere of a 4-cell stage embryo is not. A blastomere of a 4-cell stage mouse embryo can develop into a blastocyst and implant, but will die soon because of its small size and the insufficient cell number of its inner cell mass (ICM). Indirect evidence that at least some of the blastomeres of a 4-cell stage mouse embryo are totipotent was provided in chimeric models using carrier blastomeres of a different genotype to keep the size and cell number of the embryos normal (Tarkowski et al., 2001). Some isolated cells of an 8-cell stage mouse embryo form only small trophoblasts (Edwards and Beard, 1997). Both blastomeres of 2-cell stage ovine embryos can develop into lambs (Willadsen and Godke, 1984), and all the cells of a 4-cell stage bovine embryo are totipotent (Johnson et al., 1995). 1742

The aim of our study was to investigate whether isolated blastomeres of human 4-cell stage embryos could develop in vitro into blastocysts with trophectoderm (TE) and ICM. To our knowledge, this is the first time such an experiment has been carried out. Materials and Methods
Oocytes and embryos According to Belgian legislation, embryos can be generated for research purposes as long as the study is filed to the Federal Ethical Committee on Embryos. The Ethical Committee of UZ Brussel gave its approval for this project because the embryos would not be transferred into a uterus and would therefore be destroyed by the experimental procedure. Therefore, reproductive cloning, which is not allowed in Belgium, was not an issue here. Mature oocytes, empty zona pellucida (ZP) from immature oocytes and blastocysts were donated for research by couples treated at our IVF centre, who had given their informed consent. Embryos were obtained after microinjection with the spermatozoa of a consenting donor. In vitro culture, micromanipulation and inverted microscopy Oocytes were denuded and injected with spermatozoa and the resulting zygotes were cultured (Medicult series, Lucron Bioproducts,

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embryo quality was evaluated on Day 2 prior to biopsy. Their size could thus be compared with the size of regular in vitro developing human embryos. of which 12 had an ICM. UK) were used as spacers. Diaphot 300. three of four blastomeres developed into blastocysts on Day 5 (in total 15). 6. Peterborough. They were cultured individually for 6 days in sequential medium. aspirated after ZP drilling as in the case of embryo biopsy for preimplantation genetic diagnosis (PGD) (Van de Velde et al. Sigma-Aldrich) at a concentration of 2 mg/ml. consisting of a small number of TE cells. 6. regular IVF embryos were stained for NANOG. ND. not done because lost during fixation. Merelbeke. Result of splitting six 4-cell stage human embryos. Abcam. Nikon. 10% fragmentation and regular sized blastomeres were split. 1743 . Olympus. Between every step. Blastomeres were removed by aspiration using a pipette with an inner diameter of 50 mm. Belgium) was performed to record the fluorescent images. Embryo 1 2 3 4 5 6 Total Day 2 Survived 4 4 3 4 4 4 23 Day 3 2 cell 4 4 3 4 3 1 19 Day 4 Compaction 3 3 3 4 3 3 19 Day 5 Cavity 3 3 3 4 3 3 19 Day 6 Full-expanded blastocyst (ICM/NANOG) 2 (1/ND) 3 (2/2) 3 (1/1) 4 (4/4) 3 (3/2) 1 (1/0) 16 (12/9) The number of blastomeres that survived biopsy on Day 2. a transcription factor exclusively expressed by ICM cells in human embryos (Hyslop et al. Indirect immunocytochemistry Embryos were washed in phosphate-buffered saline (PBS.. Positive and negative control experiments on regular preimplantation embryos. On Day 5.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 min at room temperature. 2005). respectively. In the ICM of the two Bl4AA and two Bl4BA blastocysts. Culturing in a ZP enabled size comparison between the small blastomere-derived blastocysts and regular human embryos in the IVF laboratory. In vitro development was followed by an experienced clinical embryologist using an inverted microscope (  100 or  200 magnification.org/ by guest on January 1. the number of blastocysts with inner cell mass (ICM)/the number of ICM expressing NANOG is also presented.. After immunocytochemistry and confocal microscopy. Japan) and photographs were taken daily (OCTAX Eyeware MX). an ICM was visible. Next. In positive controls. In 12 of these blastocysts. After staining. 19 blastomeres divided into two (Table I). One day later. 1). Confocal scanning microscopy with a HeNe laser (633 nm) (IX71 Fluoview 300. as used in the IVF programme (Van Landuyt et al. Sigma-Aldrich. One blastomere did not survive the aspiration. 2000). 2a). according to the scoring system of Gardner and Schoolcraft. Sigma-Aldrich). the four blastomeres were each capable of developing individually into a good quality expanded blastocyst with cohesive TE and tightly packed ICM (two type Bl4AA with many ICM cells and two type Bl4BA with fewer cells. Leuven. Tokyo. we looked for the presence of the stemness marker NANOG. showed signs of compaction on Day 4. which were donated for research after informed consent. 3 and 2 cells.No allocation in the 4-cell stage human embryo Belgium) as described previously (Van de Velde et al. fixed with 3.7% formaldehyde (Merck. Aartselaar.. All blastomeres were put one by one into an empty ZP (ZP of immature oocytes had been emptied by aspiration). Bornem. VWR International. In the sixth embryo. some ICM cells expressed NANOG. Switserland) as described before (Van de Velde et al. had a cavity on Day 5 and developed into full/expanded blastocysts on Day 6 are represented in the respective columns. the majority of the blastomeres followed the normal pattern of preimplantation development in accordance with the biological clock of compaction on Day 4 and cavitation on Day 5. 2000).. 2012 Table I. In five out of six embryos. They were then incubated with 2 mg/ml anti-NANOG rabbit polyclonal antibody (ab21624. one by one. samples were incubated with 10 mg/ml Alexa Fluor 647 donkey anti-rabbit immunoglobulin (Ig) G (A-31573. They were evaluated daily until Day 6 of preimplantation development. 16 blastomeres developed into full/expanded blastocysts on Day 6. In the last column. embryos with . A hole was made in the ZP using laser pulses (Fertilase. At the moment of evaluation on Day 3 (72 h after injection). Four blastomeres degenerated during the in vitro culture. Belgium) supplemented with 2% bovine serum albumin (BSA. Fertilization was assessed 16–22 h after microinjection. UK) in PBS with 2% BSA at 48C overnight. Belgium) in PBS for 10 min at room temperature and permeabilized with 0. In negative controls. were included in each experiment (data not shown). We therefore conclude that the cells of a 4-cell stage human embryo can individually develop into small blastocysts with ICM and TE cells. 23 single blastomeres inside a ZP were cultured individually in vitro. we found that in 9 of 11 blastocysts (one was lost during fixation) with ICM and TE tested. 2000) and put into an empty ZP (Fig. MTM Medical Technologies Montreux. Downloaded from http://humrep. Finally. round glass cover slips (10 mm diameter) fixed with acrytol mounting medium (Surgipath. Some blastocysts were artificially hatching through the hole previously made in the ZP. 19 embryos were compacting/compacted (made up of 2 – 4 – 8 cells).oxfordjournals. these 19 blastomere-derived embryos developed into blastocysts. embryos were put between two glass cover slips (24  60 mm) in 2 ml SlowFadew Light Antifade reagent (Invitrogen). were found to express NANOG (Fig. In order to support the microscopical observation of ICM cells. with a cavity. Results Twenty-four blastomeres of six human embryos (Table I) were. 1999) (Fig. divided on Day 3. Belgium) in PBS with 2% BSA for 2 h at 48C in the dark. 2b). 2005). Invitrogen. Cambridge. To prevent squeezing of the embryos. the NANOG antibody was replaced with rabbit IgGs (R-9133. samples were washed three times for 5 min in PBS with 2% BSA on a shaking plate at room temperature.

. but individually they are equivalent and may not yet be irreversibly allocated. We postulate that allocation to ICM or TE occurs after the maternal to embryonic genome switch on Day 3. Hiiragi et al.. the blastomeres communicate and behave differently. 2005). Gardner’s group initially found that the embryonic (ICM) – abembryonic (cavity) axis in the blastocyst is approximately orthogonal to the plane of the first cleavage. one cell will become TE and one cell will contribute to the germline (Edwards and Beard.. 2005. Torres-Padilla et al. Moreover. Others argue that this pre-patterning model has only been established in particular mouse strains under certain experimental 1744 conditions (manipulation. blastomeres are thought to be totipotent at least up to the 8-cell stage. (a) The cells are aspirated one by one and (b) gently put into an empty zona pellucida (ZP) and (c) the four blastomeres are put into four separate ZP.. cells are allocated based on order and/or orientation of cleavage divisions. 2006). 2012 . In non-mammals. Polarity and pre-patterning in the mammalian embryo are currently under debate (Vogel. who supports the pre-patterning model. 2001. We conclude that the cells are flexible and may not yet be irreversibly allocated to either cell lineage.and 8-cell stage (Braude et al. 2005). they showed that the cells have distinct fates (PiotrowskaNitsche et al. 2006). Edwards. Discussion The results of the experiments performed in this study provide evidence that the isolated blastomeres of a human 4-cell stage embryo can individually develop in vitro into blastocysts with TE and ICM. We cannot exclude that within the structure of the embryo. indicating that polarity in the mouse blastocyst might find its origin already in the fertilized egg (Gardner. 1988. and only the former plays a role in totipotency (Cauffman et al.. 2004. two cells are determined to become ICM.. Gray et al. This idea is supported by Zernicka-Goetz’ group.oxfordjournals.to 5-cell stage embryos. whereas the late-dividing cell tends to form the abembryonic region. Edwards.. The expression of b-HCG and POU5F1 was negatively correlated in these embryos. who reported that it is already possible at the 2-cell stage to predict the fate of the sister cells (Piotrowska et al. Lee et al. 1967). invasive techniques and mechanical pressure) that might have induced tendencies/patterns that are biologically irrelevant (Vogel. 2005. Lately. The human embryonic genome is switched on at the 4. which will form the fetus as well as extraembryonic tissue and the outer cells (TE). The first morphological sign of polarity appears in the blastocyst when at least two cell types can be distinguished: the inner cells (ICM). Cauffman et al.. (2004) after expression analysis of b-HCG (potential marker of TE). experiments with POU5F1 should be reinterpreted because the isoforms POU5F1_iA and POU5F1_iB are often disregarded. 2004): the early-dividing cell tends to form the embryonic region. however. Nevertheless. caution should be taken when interpreting these data since the result in a number of blastomeres was not considered because no transcripts of b-actin were found. 2007).2001). 1988) explains the flexibility of the cells at the 4-cell stage. because at the 4-cell stage all cells still have the individual capacity to develop into the two lineages ICM and TE. but embryonic expression is not necessarily synchronized and therefore variability in expression should be taken into account when drawing conclusions at the early cleavage-stage (Braude et al.. Having an up– down or left – right axis seems to be clearly defined in frog and fly oocytes where morphogens in varying concentrations regulate differentiation after fertilization. Our splitting results contradict Edwards’ human pre-patterning model. The formation of outer and inner cells during the fourth cleavage is the result of differential division into polar/outer cells and apolar/inner cells (inside – outside hypothesis) (Tarkowski and Wroblewska. 2001. 1997). Hiiragi et al. The timing of human embryonic genome activation between the 4.. they cross the zygote and early embryo and dictate the neighbouring cells to differentiate. By creating chimeric embryos from the individual cells from a 4-cell stage mouse embryo.. The embryonic genome becomes activated earlier in the mouse Downloaded from http://humrep. POU5F1 (potential marker of totipotent cells. 1997. in mice an isolated blastomere of a 2-cell stage embryo is totipotent (Edwards and Beard. 1997. although data showing some pre-patterning have been reported. In this regulative or cleavage-driven model. gradients of mRNA and morphogens are established by the cytoskeleton.Van de Velde et al. formerly called OCT-4) and b-ACTIN (positive control) in single blastomeres of five human 2.to 8-cell stage. however. 2006). several research groups found evidence in mice that the fertilized oocyte is already polarized (morphological and/or molecular asymmetry) and that the early blastomeres are predestined to become either ICM or TE (pre-patterning model). Data in support of this model were provided by Hansis et al. 2006). which will develop into part of the placenta. Monk and Holding. 2006. Expression analysis of POU5F1_iA throughout human preimplantation development showed that POU5F1_iA is expressed in TE and ICM cells of the human blastocyst and does not induce a specific allocation of the dividing blastomeres towards a certain lineage (Cauffman et al. In mammals. has introduced a model—extrapolated from the non-mammalian situation—where in the 4-cell stage embryo.org/ by guest on January 1. Figure 1: Biopsy of a 4-cell stage human embryo.

2006). Moreover.. 2006) with a low success rate (respectively. to provide evidence that the four cells are pluripotent. NANOG is considered to be one of the key markers of totipotency/stemness (Hyslop et al. we investigated the expression of NANOG. Therefore. the majority developed into full/expanded blastocysts with clearly visible ICM and TE.oxfordjournals. From the six split embryos. 2005). Bolton et al. Nevertheless. it is the only marker that is specifically expressed in the ICM cells of expanded human blastocysts (Hyslop et al. (b) immunocytochemistry and confocal microscopy showed NANOG expression in cells of the inner cell mass on Day 6. cavitated on Day 5 and developed into expanded blastocysts on Day 6 (the same scale applies to all photographs). compacted on Day 4. the four sister cells did not always develop in vitro into blastocysts. 2003). The finding that in one embryo the four cells can equally develop into blastocysts has implications on the current knowledge of preimplantation development and cell loss by fragmentation. (ii) lack of developmental potential or (iii) normal in vitro preimplantation development.. However. which is practically almost impossible.. 1984). In order to confirm the presence of ICM cells within the blastomere-derived blastocysts. Totipotency or rather pluripotency could have been proven by deriving embryonic stem cells (ESCs) from the ICM. Although 1745 .. they followed the biological clock of compaction on Day 4 and cavitation on Day 5..org/ by guest on January 1. NANOG was chosen because. For ethical and legal reasons. The expression of POU5F1_iA was not investigated because it is not restricted to ICM cells in human blastocysts (Cauffman et al.. but the chance to succeed was a priori low.No allocation in the 4-cell stage human embryo Downloaded from http://humrep. (a) Single blastomeres divided on Day 3. This could be due to: (i) damage after manipulation. biopsy for PGD or cryodamage. NANOG null murine embryos have been shown to have normal ICM. we could not transfer the blastocysts into a uterus to determine their implantation capacity and provide evidence that they are totipotent. but they fail to produce epiblast and die shortly after implantation (Mitsui et al. 4 and 2%). The critical step seemed to be the production of cellular mass (expansion) between Day 5 and Day 6. to our knowledge.. 2006) and human embryos (Klimanskaya et al. ESC lines have been derived from single blastomeres of 8-cell stage mouse (Chung et al. 2012 Figure 2: In vitro development of four single blastomeres derived from one 4-cell stage human embryo. 1982. 2005). an ESC line should have been derived from each of the four blastomeres. The presence of trophoblast cells was obvious under the inverted microscope. indicating that some of these blastomeres are pluripotent. which could explain the differences between the two species. we conclude that the blastomere-derived blastocysts are potentially totipotent. (Flach et al. The size of the blastocysts derived from the individually cultured blastomeres was smaller when compared with the size of regular in vitro cultured human embryos (actually four times smaller from Day 3 until Day 5). On Day 6.

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