No abstract

Cultured human embryonic stem (hES) cells have a known predisposition to aneuploidy of chromosomes 12, 17 and X. We studied 17 hES cell lines by array-based comparative genomic hybridization (aCGH) and found that the cells accumulate other recurrent chromosomal abnormalities, including amplification at 20q11.21 and a derivative chromosome 18. These genomic changes have a variable impact at the transcriptional level.

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).

The contribution of the POU domain, class 5, transcription factor-1 (POU5F1) in maintaining totipotency in human embryonic stem cells (hESCs) has been repeatedly proven. In humans, two isoforms are encoded: POU5F1_iA and POU5F1_iB. So far, no discrimination has been made between the isoforms in POU5F1 studies, and it is unknown which isoform contributes to the undifferentiated phenotype.Using immunocytochemistry, expression of POU5F1_iA and POU5F1_iB was examined in hESCs and all stages of human preimplantation development to look for differences in expression, biological activity, and relation to totipotency. POU5F1_iA and POU5F1_iB displayed different temporal and spatial expression patterns. During human preimplantation development, a significant POU5F1_iA expression was seen in all nuclei of compacted embryos and blastocysts and a clear POU5F1_iB expression was detected from the four-cell stage onwards in the cytoplasm of all cells. The cytoplasmic localization might imply no or other biological functions beyond transcription activation for POU5F1_iB. The stemness properties of POU5F1 can be assigned to POU5F1_iA because hESCs expressed POU5F1_iA but not POU5F1_iB. However, POU5F1_iA is not the appropriate marker to identify totipotent cells, because POU5F1_iA was also expressed in the nontotipotent trophectoderm and was not expressed in zygotes and early cleavage stage embryos, which are assumed to be totipotent. The expression pattern of POU5F1_iA may suggest that POU5F1_iA alone cannot sustain totipotency and that coexpression with other stemness factors might be the key to totipotency.

No abstract

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT-PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.