Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Embryonic Stem Cell Lines Derived from Human Blastocysts

High-order chromatin folding in topologically associating domains has a critical role in proper long-range transcriptional control around the Xist locus, and presumably throughout the genome. The spatial organization of the genome is linked to biological function, and advances in genomic technologies are allowing the conformation of chromosomes to be assessed genome wide. Two groups present complementary papers on the subject. Bing Ren and colleagues use Hi-C, an adaption of the chromosome conformation capture (3C) technique, to investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types. Large, megabase-sized chromatin interaction domains, termed topological domains, are found to be a pervasive and conserved feature of genome organization. Edith Heard and colleagues use chromosome conformation capture carbon-copy (5C) technology and high-resolution microscopy to obtain a high-resolution map of the chromosomal interactions over a large region of the mouse X chromosome, including the X-inactivation centre. A series of discrete topologically associating domains is revealed, as is a previously unknown long intergenic RNA with a potential regulatory role. In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment1. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C)2 and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.

Spatial partitioning of the regulatory landscape of the X-inactivation centre

https://www.cell.com/cell/pdf/0092-8674(87)90646-5.pdf

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.

Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

https://science.sciencemag.org/content/sci/244/4910/1288.full.pdf

Altering the genome by homologous recombination.

https://www.cell.com/cell/pdf/S0092-8674(03)00847-X.pdf

BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3

The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK CP1 , EK CC1 1 and EKCC1 2) which produce a very high proportion (greater than 50%) of live-born animals that are overtly chimaeric Seven chimaeric male mice, derived from these three lines, have, so far, proved to be functional germ-line chimaeras

Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

Embryonic stem cells isolated directly from mouse embryos1 can be cultured for long periods in vitro and subsequently repopulate the germ line in chimaeric mice2,3. During the culture period these embryonic cells are accessible for experimental genetic manipulation4–6. Here we report the use of retroviral vectors to introduce exogenous DNA sequences into a stem-cell line and show that these modified cells contribute extensively to the somatic and germ-cell lineages in chimaeric mice. Compared with current methods for manipulation of the mouse genome, this approach has the advantage that powerful somatic-cell genetic techniques can be used to modify and to select cells with germ-line potential, allowing the derivation of transgenic strains with pre-determined genetic changes. We have by this means inserted many proviral vector sequences that provide new chromosomal molecular markers for linkage studies in the mouse and that also may cause insertional mutations.

Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector

The human Lesch–Nyhan syndrome is a rare neurological and behavioural disorder, affecting only males, which is caused by an inherited deficiency in the level of activity of the purine salvage enzyme hypoxanthine-guanosine phosphoribosyl transferase (HPRT)1–3. How the resulting alterations in purine metabolism lead to the severe symptoms characteristic of Lesch–Nyhan patients is still not understood3,4. No mutations at the Hprt locus leading to loss of activity have been described in laboratory animals. To derive an animal model for the Lesch–Nyhan syndrome, we have used cultured mouse embryonic stem cells5, mutagenized by retroviral insertion and selected for loss of HPRT activity, to construct chimaeric mice6,7. Two clonal lines carrying different mutant Hprt alleles have given rise to germ cells in chimaeras, allowing the derivation of strains of mutant mice having the same biochemical defect as Lesch–Nyhan patients. Male mice carrying the mutant alleles are viable and analysis of their cells shows a total lack of HPRT activity.

A potential animal model for Lesch–Nyhan syndrome through introduction of HPRT mutations into mice

The predictions of a model for the initiation of X-chromosome inactivation based on a single inactivation centre were tested in a cytogenetic study using six different embryo-derived (EK) stem cell lines, each with a different-sized deletion of the distal part of one of the X-chromosomes. Metaphase chromosomes were prepared by the Kanda method from each cell line in the undifferentiated state and after induction of differentiation, and cytogenetic evidence sought for a dark-staining inactive X-chromosome. The results confirm the predictions of the model in that when the inactivation centre is deleted from one of the X-chromosomes neither X present in a diploid cell can be inactivated, and in addition considerably further localize the position of the inactivation centre on the X-chromosome.

X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation

Eggs from 129 SvE and (C57BL × CBA)F1 hybrid female mice were activated parthenogene tically following their exposure to a 7% solution of ethanol in PBS. Only the haploid cla ss which developed a single pronucleus following second polar body extrusion was examined further. These eggs were transferred to suitable recipients and ‘delayed’ blastocysts subsequently recovered. The ‘delayed’ blastocysts were explanted into tissue culture and a total of four haploid-derived pluripotent cell lines established from individual embryos. Chromosome analysis of morulae revealed that over 80% contained only haploid mitoses. However, chromosome analysis of early passage cell lines revealed that all were diploid with a modal number of 40 chromosomes. When transplanted into syngeneic hosts, all lines formed well-differentiated teratocarcinomas. This technique provides a source of homozygous diploid cell lines of parthenogenetic origin.

/pdf/establishment-of-pluripotential-cell-lines-from-haploid-10gnhkp07b.pdf

Elizabeth J. Robertson

Papers

Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector

A potential animal model for Lesch–Nyhan syndrome through introduction of HPRT mutations into mice

X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation

Establishment of pluripotential cell lines from haploid mouse embryos