I. INTRODUCTION
Several seminal discoveries during the past 25 years can be regarded not only as major breakthroughs for cell and developmental biology, but also as pivotal events that have substantially influenced our view of life: 1) the establishment of embryonic stem (ES) cell lines derived from mouse (108, 221) and human (362) embryos, 2) the creation of genetic mouse models of disease through homologous recombination in ES cells (360), 3) the reprogramming of somatic cells after nuclear transfer into enucleated eggs (392), and 4) the demonstration of germ-line development of ES cells in vitro (136, 164, 365). Because of these breakthroughs, cell therapies based on an unlimited, renewable source of cells have become an attractive concept of regenerative medicine.
Many of these advances are based on developmental studies of mouse embryogenesis. The first entity of life, the fertilized egg, has the ability to generate an entire organism. This capacity, defined as totipotency, is retained by early progeny of the zygote up to the eight-cell stage of the morula. Subsequently, cell differentiation results in the formation of a blastocyst composed of outer trophoblast cells and undifferentiated inner cells, commonly referred to as the "inner cell mass" (ICM). Cells of the ICM are no longer totipotent but retain the ability to develop into all cell types of the embryo proper (pluripotency; Fig. 1). The embryonic origin of mouse and human ES cells is the major reason that research in this field is a topic of great scientific interest and vigorous public debate, influenced by both ethical and legal positions.
ES cell research dates back to the early 1970s, when embryonic carcinoma (EC) cells, the stem cells of germ line tumors called teratocarcinomas (344), were established as cell lines (135, 173, 180; see Fig. 2). After transplantation to extrauterine sites of appropriate mouse strains, these "funny little tumors" produced benign teratomas or malignant teratocarcinomas (107, 345). Clonally isolated EC cells retained the capacity for differentiation and could produce derivatives of all three primary germ layers: ectoderm, mesoderm, and endoderm. More importantly, EC cells demonstrated an ability to participate in embryonic development, when introduced into the ICM of early embryos to generate chimeric mice (232). EC cells, however, showed chromosomal aberrations (261), lost their ability to differentiate (29), or differentiated in vitro only under specialized conditions (248) and with chemical inducers (224). Maintenance of the undifferentiated state relied on cultivation with feeder cells (222), and after transfer into early blastocysts, EC cells only sporadically colonized the germ line (232). These data suggested that the EC cells did not retain the pluripotent capacities of early embryonic cells and had undergone cellular changes during the transient tumorigenic state in vivo (for review, see Ref. 7).
To avoid potential alterations connected with the growth of teratocarcinomas, a logical step was the direct in vitro culture of embryonic cells of the mouse. In 1981, two groups succeeded in cultivating pluripotent cell lines from mouse blastocysts. Evans and Kaufman employed a feeder layer of mouse embryonic fibroblasts (108), while Martin used EC cell-conditioned medium (221). These cell lines, termed ES cells, originate from the ICM or epiblast and could be maintained in vitro (Fig. 2) without any apparent loss of differentiation potential. The "pluripotency" of these cells was demonstrated in vivo by the introduction of ES cells into blastocysts. The resulting mouse chimeras demonstrated that ES cells could contribute to all cell lineages including the germ line (46). In vitro, mouse ES cells showed the capacity to reproduce the various somatic cell types (98, 108, 396) and, only recently, were found to develop into cells of the germ line (136, 164, 365). The establishment of human ES cell lines from in vitro fertilized embryos (362) (Fig. 3) and the demonstration of their developmental potential in vitro (322, 362) have evoked widespread discussions concerning future applications of human ES cells in regenerative medicine.
Primordial germ (PG) cells, which form normally within the developing genital ridges, represent a third embryonic cell type with pluripotent capabilities. Isolation and cultivation of mouse PG cells on feeder cells led to the establishment of mouse embryonic germ (EG) cell lines (198, 291, 347; Fig. 2). In most respects, these cells are indistinguishable from blastocyst-derived ES cells and are characterized by high proliferative and differentiation capacities in vitro (310), and the presence of stem cell markers typical of other embryonic stem cell lines (see sect. II). Once transferred into blastocysts, EG cells can contribute to somatic and germ cell lineages in chimeric animals (197, 223, 347); however, EG cells, unlike ES cells, retain the capacity to erase gene imprints. The in vitro culture of PG cells from 5- to 7-wk-old human fetuses led to the establishment of human EG cell lines (326) (Fig. 3). These cell lines showed multilineage development in vitro but have a limited proliferation capacity, and currently can only be propagated as embryoid body (EB) derivatives (325). Following transplantation into an animal model for neurorepair, human EG cell derivatives, however, show some regenerative capacity, suggesting that these cells could be useful therapeutically (190). Although pluripotent EG and EC cells represent important in vitro models for cell and developmental biology, this review focuses mainly on fundamental properties and potential applications of mouse and human ES cells for stem cell research.
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