Charles E. Murry, ... Lior Gepstein, in Heart Development and Regeneration, 2010

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. Mouse embryonic stem cells (mESCs) have been studied for several decades, and have provided major advances in our understanding of developmental biology and gene function in the adult organism. The single greatest application of mouse embryonic stem cells has been in studies of gene function through homologous recombination (knockout or knockin strategies). These studies were made possible by the remarkable ability of genetically-modified embryonic stem cells to incorporate into all tissues of a developing mouse after injection into a blastocyst, followed by the ability of resulting chimeric mice to pass the genetic modification via the germline. Embryonic stem cells have also been useful tools for understanding molecular events controlling differentiation into the early germ layers and more distal branches of the developmental tree. Over the last 15 years an increasing number of groups have become interested in the use of mouse embryonic stem cells as a cell source to treat murine models of cell deficiency.

Research in this area gained worldwide prominence, extending far beyond the usual scientific community, when Jamie Thomsons group at the University of Wisconsin reported developing the first lines of human embryonic stem cells (hESCs) in 1998 (Thomson et al., 1998b). A veterinary pathologist with an interest in early human development, Thomson had honed his skills by first deriving lines of embryonic stem cells from nonhuman primates (marmosets and rhesus monkeys) (Thomson et al., 1995, 1996). To derive human embryonic stem cells, Thomsons group worked with blastocysts donated by fertility clinic patients, who no longer intended to use these spare embryos for reproductive purposes (these blastocyts are commonly discarded if they are not to be used for reproductive purposes). The embryos were 5 days post-in vitro fertilization, and were at the blastocyst stage, a hollow ball of 150 cells surrounded by a carbohydrate-rich zona pellucida. Blastocysts contain a rim of trophoectoderm cells, which gives rise to the placenta and amniotic membranes, and an inner cell mass, which gives rise to the embryo proper. (By way of comparison, a 5-day-old embryo derived from traditional fertilization is at a preimplantation stage, still residing in the fallopian tube). To derive the human embryonic stem cells, Thomsons group enzymatically digested the zona pellucida and removed the trophoectoderm using antibodies and complement (immunosurgery) (Fig. 1), leaving the inner cell mass intact. The inner cell mass was placed into a culture system, using feeder layers of mouse embryonic fibroblasts to provide a still-unknown set of factors that had maintained other primate embryonic stem cells in the undifferentiated state. The human cells thrived in this environment, growing for hundreds of population doublings while still expressing molecular markers of pluripotency and retaining the ability to differentiate into a wide variety of cell types in vitro. Importantly, after implantation into immuno-tolerant mice, human embryonic stem cells formed teratomas, tumors comprised of cells from endoderm, mesoderm and ectoderm. At present, teratoma formation represents the most definitive evidence for human embryonic stem cell potency, since human blastocyst injection is widely-considered to be unethical. Since this original publication, over 100 lines of human embryonic stem cells have been derived worldwide by similar techniques (Cowan et al., 2004; Musri et al., 2006).

Figure 1. Embryonic stem cell derivation. Cells in the inner cell mass (ICM) of pre-implantation embryos are isolated by the removal of the trophectoderm by immunosurgery (antibody and complement-mediated lysis). To maintain cells in the undifferentiated state, inner cell mass cells are plated on a mouse embryonic fibroblasts feeder layer. These undifferentiated cells can be induced to differentiate into cells from the different germ layers.

It is important to consider the scientific context in which this advancement came. The late-1990s and early-2000s had yielded a number of other major scientific advancements, including sequencing of the human genome (Lander et al., 2001) and cloning of the first mammal, Dolly the sheep (Campbell et al., 1996). Thus, within a few short years, science had delivered the genetic blueprint of humanity, techniques to completely dedifferentiate a cell and grow a new mammal from it, and early human cells that could develop into any tissue. Understandably, this triggered a response that extended beyond the scientific community and into the lay press, public coffeehouses, churches and political forums. Most countries are still debating the extent to which human embryonic stem cell research should be regulated, and public policies range widely, from governmental encouragement, to legal restrictions, to outright bans. While not the topic of this chapter, we would encourage all readers to explore the ethics and policy implications of human embryonic stem cell research, and we refer those interested to references (Green, 2001; Daley et al., 2007; Sugarman, 2007) for in-depth analyses.

Human embryonic stem cells share many similarities with their murine counterparts, but they also have several important differences. Like mouse embryonic stem cells, human embryonic stem cells can divide extensively without telomere shortening and by this criterion appear to be immortal. Although there is not complete overlap with mouse embryonic stem cells, human embryonic stem cells express surface markers characteristic of pluripotent cells. Additionally, both embryonic stem cell types express transcription factors required for pluripotency, including Oct4 and Nanog. In mouse embryonic stem cells, the cytokine leukemia inhibitory factor (LIF) is necessary to maintain cells in their pluripotent state (Williams et al., 1988; Pease and Williams, 1990). In contrast, human embryonic stem cells will differentiate in the presence of LIF (Zaehres et al., 2005) and require FGF for pluripotency. Bone morphogenetic proteins (BMPs) contribute to the maintenance of pluripotency of mouse embryonic stem cells (Ying et al., 2003), whereas they induce trophoblast differentiation in human embryonic stem cells (Xu et al., 2002). The optimal conditions to maintain human embryonic stem cells in the pluripotent state are still being worked out. For this reason, most investigators currently use either mouse embryonic fibroblast feeder layers, or medium conditioned by these cells (supplemented with bFGF) (Xu et al., 2001) for growth and maintenance of undifferentiated human embryonic stem cells.

Mouse embryonic stem cells typically grow as tight clusters and show a high plating efficiency after dissociation to single cells. These characteristics facilitate their low-density plating and subsequent isolation of subclones. In contrast, undifferentiated human embryonic stem cells typically grow as flat two-dimensional colonies, which are passaged by forming smaller clumps (either through partial enzymatic digestion or mechanical dissociation) and allowing them to expand. Furthermore, the establishment of clonal lines is more difficult with human embryonic stem cells, because they do not tolerate single cell dispersion as well as mouse embryonic stem cells. Human embryonic stem cells must be karyotyped regularly to screen for chromosomal abnormalities, such as trisomies 12 and 22, as well as several translocation variants (reviewed in Baker et al., 2007) that can accumulate with time in culture. Time in culture can also affect the differentiation efficiency of some cell types, including cardiomyocytes. It is likely that these difficulties reflect our still-imperfect ability to culture the cells, which may improve as the community gains experience.

The difference between mouse embryonic stem cells and human embryonic stem cells has been assumed to relate to species differences in signaling requirements for pluripotency. Recently, however, two groups isolated pluripotent cells from postimplantation stage mouse epiblasts (Brons et al., 2007; Tesar et al., 2007). These epiSCs do not use the LIF/STAT3 pathway for maintaining pluripotency, but instead use pathways initiated by activin/nodal and FGF, similar to human embryonic stem cells. Interestingly, while epiSCs formed teratomas after injection into host mice, they did not generate chimeric embryos after blastocyst injection. The similarity of mouse epiSCs to human embryonic stem cells has raised the possibility that signaling pathways between species are actually conserved, with the difference being that human embryonic stem cells represent a later developmental stage than mouse embryonic stem cells.

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