Frances A. Brook

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.

Embryonic stem cell-derived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance

Although human embryonic stem (ES) cells may one day provide a renewable source of tissues for cell replacement therapy (CRT), histoincompatibility remains a significant barrier to their clinical application. Current estimates suggest that surprisingly few cell lines may be required to facilitate rudimentary tissue matching. Nevertheless, the degree of disparity between donor and recipient that may prove acceptable, and the extent of matching that is therefore required, remain unknown. To address this issue using a mouse model of CRT, we have derived a panel of ES cell lines that differ from CBA/Ca recipients at defined genetic loci. Here, we show that even expression of minor histocompatibility (mH) antigens is sufficient to provoke acute rejection of tissues differentiated from ES cells. Nevertheless, despite their immunogenicity in vivo, transplantation tolerance may be readily established by using minimal host conditioning with nondepleting monoclonal antibodies specific for the T cell coreceptors, CD4 and CD8. This propensity for tolerance could be attributed to the paucity of professional antigen-presenting cells and the expression of transforming growth factor (TGF)-β2. Together, these factors contribute to a state of acquired immune privilege that favors the polarization of infiltrating T cells toward a regulatory phenotype. Although the natural privileged status of ES cell-derived tissues is, therefore, insufficient to overcome even mH barriers, our findings suggest it may be harnessed effectively for the induction of dominant tolerance with minimal therapeutic intervention.

Directed differentiation of dendritic cells from mouse embryonic stem cells

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

A structurally defined mini-chromosome vector for the mouse germ line

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Does lumbosacral spina bifida arise by failure of neural folding or by defective canalisation

The aim of this study was to determine whether open lumbosacral spina bifida results from an abnormality of neural folding (primary neurulation) or medullary cord canalisation (secondary neurulation). Homozygous curly tail (ct) mouse embryos were studied as a model system for human neural tube defects. The rostral end of the spina bifida was found to lie at the level of somites 27 to 32 in over 90% of affected ct/ct embryos. Indian ink marking experiments using non-mutant embryos showed that the posterior neuropore closes, and primary neurulation is completed, at the level of somites 32 to 34. Since neurulation in mammals progresses in a craniocaudal sequence, without overlap between regions of primary and secondary neurulation, we conclude that spina bifida in ct/ct embryos arises initially as a defect of primary neurulation. The position of posterior neuropore closure in human embryos is estimated to lie at the level of the future second sacral segment indicating that in humans, as in the ct mouse, lumbosacral spina bifida usually arises as a defect of posterior neuropore closure. Cranial NTD affect females predominantly, whereas lower spinal NTD are more common in males, both in humans and ct mice. We offer an explanation for this phenomenon based on (a) differences in the effect of embryonic growth retardation on the likelihood that an embryo will develop either cranial or lower spinal NTD and (b) differences in the rate of growth and development of male and female embryos at the time of neurulation.

Female predisposition to cranial neural tube defects is not because of a difference between the sexes in the rate of embryonic growth or development during neurulation.

The susceptibility of females to anencephaly is well established and has been suggested to result from a slower rate of growth and development of female embryos during cranial neurulation. We have tested this hypothesis by measuring the rates of growth and development, both in utero and in vitro, of male and female embryos of the curly tail (ct) mutant mouse strain, in which cranial neural tube defects occur primarily in females. Embryonic growth was assessed by increase in protein content, while development progression was judged from increase in somite number and morphological score. Embryos were sexed by use of the polymerase chain reaction to amplify a DNA sequence specific to the Y chromosome, and by sex chromatin analysis. We find that, during neurulation (between 8.5 and 10.5 days of gestation), males are advanced in growth and development relative to their female litter mates, but that the rates of growth and development do not differ between the sexes during this period. We conclude that rate of embryonic growth and development is unlikely to determine susceptibility to cranial neural tube defects. It seems more likely that male and female embryos differ in some specific aspect(s) of the neurulation process that increases the susceptibility of females to development of anencephaly.

Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro.

We have screened neurulation-stage mouse embryos for regional differences in protein distribution, by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The screen has revealed an 83-kD protein (pI 6.8) that is present in embryo regions where neurulation is in progress but not in regions where neurulation is complete. The 83-kD protein is not synthesized in the neurulation-stage embryo or in the yolk sac, but is taken up from the culture serum in vitro and, probably, from the maternal serum in utero. The 83-kD protein has been identified as transferrin on the basis of its electrophoretic migration and recognition on Western blots by an antitransferrin antibody. Culture of embryos in serum containing 125I-transferrin, followed by autoradiography of embryo sections, shows that transferrin is taken up and localized in the gut beneath the closing neural folds at several levels of the body axis in 8.5- and 9.5-day embryos. In situ hybridization studies show that the transferrin receptor mRNA is expressed in all cells of the 9.5-day embryo, including the gut endoderm. These findings are consistent with a role for transferrin in development of the gut and perhaps, indirectly, in completion of neurulation during early mouse embryogenesis.

The Maestro (Mro) Gene Is Dispensable for Normal Sexual Development and Fertility in Mice

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.

Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

Direct measurement of myofilament calcium in living cardiomyocytes

Visualising when and where calcium appears and disappears in cardiomyocytes is a major goal of cardiovascular research. Surprisingly we find that the chemical dyes widely used for this purpose disrupt cell contractility, due at least in part due to direct inhibition of the acto-myosin ATPase required to generate force. In order to improve calcium detection methods, we have developed a genetically encoded indicator that sits within the myofilament to directly visualise the changes occurring at the sarcomere. This tool improves on established chemical dyes and untargeted genetically encoded indicators for analysing small molecule modulators of myofilament-based calcium signalling. Importantly this is achieved without any measurable change in contractile function.Subcellular calcium indicators applied to cardiomyocytes reveal an orchestra of intracellular sources interacting during every heartbeat. Here we describe a myofilament-restricted calcium indicator which enables visualisation of transients in their target, the sarcomere, where calcium regulates contraction. This tool improves on established chemical dyes and untargeted genetically encoded indicators for analysing modulators of contractility and myofilament-based calcium signalling.