Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

BACKGROUND: Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. RESULTS: As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. CONCLUSION: We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).

New cell lines from mouse epiblast share defining features with human embryonic stem cells.

The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development.

The derivation of highly germline-competent embryonic stem cells containing NOD-derived genome.

It would be extremely advantageous to the analysis of disease mechanisms in the spontaneous mouse model of type 1 diabetes, the nonobese diabetic (NOD) strain, if genes in this strain could be modified in vivo using embryonic stem (ES) cells and homologous recombination. However, a NOD ES cell line with adequate germline transmission has not yet been reported. We report the development of highly germline-competent ES cell lines from the F1 hybrid of NOD and 129 for use in NOD gene targeting. Consequently, we developed ES cell lines derived from (NOD x 129)F1 x 129 backcross 1 mice, which were intercrossed to select for homozygosity of particular regions of NOD genome known to contain disease loci.

Sex-specific differences in meiotic chromosome segregation revealed by dicentric bridge resolution in mice.

The meiotic properties of paracentric inversion heterozygotes have been well studied in insects and plants, but not in mammalian species. In essence, a single meiotic recombination event within the inverted region results in the formation of a dicentric chromatid, which usually breaks or is stretched between the two daughter nuclei during the first meiotic anaphase. Here, we provide evidence that this is not the predominant mode of exchange resolution in female mice. In sharp contrast to previous observations in other organisms, we find that attempts to segregate the dicentric chromatid frequently result not in breakage, stretching, or loss, but instead in precocious separation of the sister centromeres of at least one homolog. This often further results in intact segregation of the dicentric into one of the meiotic products, where it can persist into the first few embryonic divisions. These novel observations point to an unusual mechanism for the processing of dicentric chromosomes in mammalian oogenesis. Furthermore, this mechanism is rare or nonexistent in mammalian spermatogenesis. Thus, our results provide additional evidence of sexual dimorphism in mammalian meiotic chromosome behavior; in "stressful" situations, meiotic sister chromatid cohesion is apparently handled differently in males than in females.

Derivation and characterisation of the human embryonic stem cell line, OxF1.

OxF1 is a human embryonic stem cell line derived from a surplus embryo donated through the Oxford IVF clinic. The cells have a stable 46 XX karyotype and show expression of Oct 4, Nanog and TRA-1-60. Embryoid bodies differentiate into cells that represent all three germ layers as demonstrated by immunohistochemical localisation of beta III tubulin, nestin, desmin, smooth muscle actin, Gata 6 and cytokeratin 18. Directed differentiation through haematopoiesis has been demonstrated.

Changing potency by spontaneous fusion.

Recent reports have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs 1-9. Here we define a mechanism by which progenitor cells of the central nervous system can give rise to non-neural derivatives. Cells taken from mouse brain were co-cultured with pluripotent embryonic stem cells. Following selection for a transgenic marker carried only by the brain cells, undifferentiated stem cells are recovered in which the brain cell genome has undergone epigenetic reprogramming. However, these cells also carry a transgenic marker and chromosomes derived from the embryonic stem cells. Therefore the altered phenotype does not arise by direct conversion of brain to embryonic stem cell but rather through spontaneous generation of hybrid cells. The tetraploid hybrids exhibit full pluripotent character, including multilineage contribution to chimaeras. We propose that transdetermination consequent to cell fusion 10 could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells 9.