Richard L. Gardner

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.

The Relationship Between Cell Lineage and Differentiation in the Early Mouse Embryo

The notion that particular types of terminally differentiated cells originate from specific ancestral cells during ontogeny is one of wide currency and long standing. It arose principally from studies on lower chordate and invertebrate embryos carried out in the latter part of the last century and the beginning of the present one (see Davidson, 1976, for a recent review). The concept of stem cells, which may persist into adulthood in a variety of tissues, is clearly related to that of specific cell lineages (Cairnie et al., 1976). Thus neural crest cells, haematocytoblasts, and neuroblasts are believed to give rise to distinct constellations of specific mature cell types.

Contributions of blastocyst micromanipulation to the study of mammalian development

This is a personal account of why the author chose to focus on devising techniques for micromanipulating the blastocyst stage conceptus as a way of investigating early development in mammals. Its aim is to provide insight into what such technical innovations entailed and how they have contributed to present understanding of both embryology and the analysis of gene function in mammals. The ability to dissect and reconstitute mouse blastocysts, and to inject cells or tissue into them, enabled genes to be harnessed as markers for elucidating the lineage of cells and interactions between tissues from the stage when differentiation is first evident. Most importantly, it made it possible to apply clonal analysis to the study of cell fate in mammals. The scope of blastocyst micromanipulation was further enhanced when embryonal carcinoma (EC) cells and, particularly, embryonic stem (ES) cells were found to be able to participate in normal development and contribute to the germ line following injection into the blastocyst.

Scrambled or bisected mouse eggs and the basis of patterning in mammals.

Several findings challenge the notion that specification of cell types and embryonic axes in mammals are rooted entirely in the temporal and spatial relations between cleaving blastomeres. They raise the question as to whether, as in most non-mammalian species, these processes depend on information already present in the egg. However, experiments designed to investigate this possibility directly by perturbing the organization of the zygote or, very recently, by deleting one or other of its polar regions [M. Zernicka-Goetz. Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles. Development 1998;125:4803-4808 (Ref. 1)], have been interpreted to mean that such a role for the egg can be discounted. This conclusion seems premature in view of continuing uncertainty regarding the developmental potential of individual blastomeres in mammals.

Experimental analysis of the transdifferentiation of visceral to parietal endoderm in the mouse.

The visceral endoderm (VE) of isolated extraembryonic regions (ExEmbs) of 7 days postcoitum (dpc) prestreak mouse conceptuses have been shown to convert readily to parietal endoderm (PE). The present study addresses the following three unanswered questions. On what does conversion depend, how rapidly does it occur, and is it an enduring general property of a residual small population of relatively immature cells? In situ hybridization reveals that change in cell state occurs within 2 days of culture. Deprivation of the mesoderm also promotes it in later ExEmbs. Conversely, the conversion to PE in isolated 7 dpc ExEmbs is suppressed by grafting 8 dpc or 9 dpc mesoderm. Hence, the conversion provides an example of transdifferentiation that is promoted by the absence of extraembryonic mesoderm. The presence of mesoderm seems to be necessary to enable the VE to grow rather than convert to PE, as occurs if it retains contact with the extraembryonic ectoderm. Developmental Dynamics 233:837–846, 2005.

Delayed and disturbed morphogenesis of the umbilical blood vessels in insulin-like growth factor-II deficient conceptuses (Igf2m+/p−)

Insulin‐like growth factor‐II (IGF‐II) deficiency occurs when a conceptus inherits an inactive gene from the father (Igf2m+/p−): fetal wet weight is reduced to 60% of wild‐type, with the decline starting at E11. The umbilical cord vessels of mutant and wild‐type were compared. At E8.0–E8.5, the timing of somite formation and chorioallantoic fusion was not altered. At E14.5–E16.5, the left umbilical artery degenerated approximately 1 day later in Igf2m+/p− conceptuses when compared with the wild‐type. In the common umbilical artery at E15.5, muscle volume was reduced by one third in IGF‐II deficiency. Treating the umbilical arteries as ideal tubes, the values of radius4/length suggest that blood flow through the placenta may be reduced by more than half in the Igf2m+/p− conceptuses. Developmental Dynamics 233:88–94, 2005.