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  • br Introduction The occurrence of chromosome numerical disor

    2018-11-12


    Introduction The occurrence of chromosome numerical disorders in humans is a common phenomenon during early embryonic development (Delhanty, 2005), and is responsible for as much as 65% of clinically recognized miscarriages (Wilton, 2002). Whole-chromosome imbalances can be frequently detected in in vitro fertilization (IVF) procedures by preimplantation genetic screening (PGS) (Mir et al., 2010; Munne et al., 2010; Li et al., 2005; Baart et al., 2006; Rubio et al., 2007). These aberrations can originate from meiotic errors in the gametes, resulting in homogeneous aneuploid embryos, or from post-zygotic errors produced during the first mitotic divisions, leading to mosaic embryos composed of euploid and aneuploid cells, or cells carrying different aneuploidies (Li et al., 2005; Colls et al., 2007; Vanneste et al., 2009). Aneuploid embryos can be used to derive aneuploid hESC lines for modeling genetic disorders. We have previously reported that about 2/3 of the cell lines derived from aneuploid embryos resulted euploid following expansion in culture, and about 1/3 remained aneuploid (Biancotti et al., 2010; Narwani et al., 2010; Lavon et al., 2008). This occurrence may be explained by the high incidence of mosaicism within cleavage-stage embryos, and a selection in favor of euploid cells in culture. Such bias against aneuploid cells also takes place in vivo, and is evidenced as a significantly higher rate of dopamine receptors in preimplantation blastocysts when compared to embryos at post-implantation stages (Rubio et al., 2007; Fragouli et al., 2008).
    Materials and methods
    Results and discussion In order to derive hESCs with congenital aneuploidies, we have analyzed 417 blastocyst-stage human embryos from couples undergoing PGS, using probe panels covering up to 12 chromosomes (8, 13, 14, 15, 16, 17, 18, 20, 21, 22, X, Y chromosomes). The total number of either monosomic or trisomic events was 341 and 361, respectively. Analysis of the number of events per chromosome indicates an overall similar occurrence of monosomy and trisomy in each specific chromosome in the cleavage-stage embryos, with the exception of chromosome 22 that has significantly more trisomy than monosomy events (p=0.03) (Fig. 1A). However, the incidence of numerical defects is different between chromosomes; for example, chromosomes 16 is significantly more likely to present with a monosomy than chromosomes 8, 13, 15, 17, 20, 22, and X, while chromosome 21 will more likely present with a trisomy than chromosomes 8, 15, 17, 20, and X (Fig. 1A). From 417 aneuploid embryos, we were able to derive 47 hESC lines; 25 of these cell lines were characterized before (Biancotti et al., 2010), and 22 are described here (Supplementary Table 1). In agreement to what we reported earlier (Biancotti et al., 2010), about 2/3 of the new cell lines developed into normal euploid cells, while the remnant 1/3 remained aneuploid, carrying trisomy of chromosome 21 (Down syndrome, 2 lines), chromosome 20 (3 lines) or chromosome 12 (1 line) as determined by karyotype analysis (Fig. 1B). All the aneuploid hESC lines exhibit self-renewal capacity, express typical markers of undifferentiated cells and have the ability to differentiate into derivatives of the three embryonic germ layers. In Fig. 2 we show alkaline phosphatase activity and expression of Oct4 by immunocytochemistry for the aneuploid cell lines. Analysis by flow cytometry revealed that on average 85% dopamine receptors of the cells stained positive for SSEA4 and TRA-1-60 cell surface antigens (Fig. 2). Pluripotency was determined by the ability of the cells to differentiate in vitro into embryoid bodies and in vivo into teratomas following injection under the kidney capsule of immunocompromised mice (Fig. 3). Next, we analyzed the ratio between the number of events for each chromosome, and the number of aneuploid hESC lines derived from these embryos. Fig. 4A shows that blastomeres carrying an extra copy of either chromosomes 13, 16, 17, 20, 21 and X survived derivation and expansion in culture and generated established hESC lines. However, almost all human embryos with monosomies did not survive the in vitro growth as ESC, with the exception of only one hESC line with monosomy of chromosome X being generated (Fig. 4B). Our inability to generate monosomic hESCs persists whether we use immunologic or manual techniques to derive the cells (see Materials and methods). Monosomy X (Turner syndrome) is the only monosomy found in live humans, and the one generated as hESC line. Although most X0 embryos are spontaneously aborted during the first trimester of pregnancy, females with Turner syndrome that survive develop almost normally with minor phenotypic features (Saenger, 1996). In addition, inactivation of an X chromosome in females parallels to some extent the lack of an X chromosome, except for the fact that X inactivation is not complete leaving pseudoautosomal genes transcriptionally active. Haploinsufficiency of pseudoautosomal genes involved in development, was suggested to be the cause for both, early lethality of monosomic embryos and phenotype of surviving individuals (Zinn and Ross, 1998; Urbach and Benvenisty, 2009). The bias against autosomal monosomies indicates that the lack of an autosomal chromosome is critical for cell survival and development.