Abstract
Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.
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.
Stem cell hierarchy. Zygote and early cell division stages (blastomeres) to the morula stage are defined as totipotent, because they can generate a complex organism. At the blastocyst stage, only the cells of the inner cell mass (ICM) retain the capacity to build up all three primary germ layers, the endoderm, mesoderm, and ectoderm as well as the primordial germ cells (PGC), the founder cells of male and female gametes. In adult tissues, multipotent stem and progenitor cells exist in tissues and organs to replace lost or injured cells. At present, it is not known to what extent adult stem cells may also develop (transdifferentiate) into cells of other lineages or what factors could enhance their differentiation capability (dashed lines). Embryonic stem (ES) cells, derived from the ICM, have the developmental capacity to differentiate in vitro into cells of all somatic cell lineages as well as into male and female germ cells.
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).
Developmental origin of pluripotent embryonic stem cell lines of the mouse. The scheme demonstrates the derivation of embryonic stem cells (ESC), embryonic carcinoma cells (ECC), and embryonic germ cells (EGC) from different embryonic stages of the mouse. ECC are derived from malignant teratocarcinomas that originate from embryos (blastocysts or egg cylinder stages) transplanted to extrauterine sites. EGC are cultured from primordial germ cells (PGC) isolated from the genital ridges between embryonic day 9 to 12.5. Bar = 100 m. [From Boheler et al. (40).]
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.
Human pluripotent embryonic stem (ES) and embryonic germ (EG) cells have been derived from in vitro cultured ICM cells of blastocysts (after in vitro fertilization) and from primordial germ cells (PGC) isolated from aborted fetuses, respectively.
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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Embryonic Stem Cells: Prospects for Developmental Biology ...
