many that the development of human ES cell lines was not far off. The wait ended in November last year when John Gearhart's group at the Johns Hopkins Hospital reported on the development of what seemed to be a human EG line and James Thomson's group at the University of Wisconsin described the isolation of human cells with the properties of ES cells. When discussing these studies, the first critical question that comes to mind is: do these cell lines possess the characteristics of true EG and ES cells? The fact that these lines were generated from appropriate cell populations, that they both have extensive proliferative potential and display a capacity for multilineage differentiation suggests that they do indeed represent EG and ES cells. In these studies, the EG cell lines demonstrated some potential to undergo spontaneous differentiation in culture although the efficiency and extent of this differentiation remains to be fully defined. The pluripotential nature of the human ES cells was demonstrated in teratomas generated following their injection into immunocompromised mice. But further characterization of the developmental potential of both types of cells in culture is necessary.
To appreciate the potential importance of human ES/EG cell lines, it is worth examining recent advances made with mouse ES cells. Although mouse ES cells are most noted for their ability to produce new strains of animals with specific genetic alterations, it is their capacity to differentiate and generate multiple lineages in culture that is most relevant to human cell lines. When removed from conditions that maintain them in an undifferentiated state, ES cells will spontaneously differentiate to form multicellular structures known as embryoid bodies (EBs) that contain elements of all three embryonic germ layers: ectoderm, mesoderm and endoderm. As differentiation continues, a wide range of cell types, including hematopoietic, endothelial, muscle, and neuronal develop within the EBs in a defined and reproducible temporal order. When analyzed in detail, the developmental programs associated with lineage commitment in EBs show remarkable similarities to those found in the normal embryo. Thus, EBs provide a rich source of normal developing cell populations with both embryonic and adult phenotypes. Given this potential, what opportunities do human ES and EG cell lines offer?
The most obvious application of human ES/EG cells and the one that receives the most attention is in cell–replacement therapies: to replace diseased or degenerating tissues, or cell populations (such as those of the hematopoietic system) that have been destroyed by chemotherapy. In theory, EBs could provide an unlimited supply of specific cell types for transplantation. To date, ES cell–derived cardiomyocytes, neural precursors and hematopoietic precursors have been transplanted into recipient animals. Although the analyses of the long–term outcome of such experiments are limited, the findings suggest that the transplanted cells are able to function in the host animal.
These preliminary studies are encouraging and suggest that ES cell–based therapies for certain neuronal, hematopoietic and cardiovascular diseases will be possible. However, before this goal can be reached a number of obstacles need to be overcome, the most significant of which is donor/recipient compatibility and graft rejection. Possible solutions to this problem include the banking of large numbers of ES/EG cell lines encompassing a significant fraction of the histocompatibility types in the population, and/or the genetic modification of the stem cells to make the graft more acceptable to the recipient. In addition, recent cloning studies suggest a third solution in which nuclear transfer could be used to produce starting material that would allow the isolation of individualized ES cell lines. In this situation, the cells used for transplantation would be genetically identical to those of the patient.
Another application of ES/EG cells is for the study of development in both human and animal model systems. Embryoid bodies provide an unprecedented opportunity to study the events regulating lineage commitment, and tissue growth and maturation during embryonic development. This approach includes the identification and isolation of novel precursor cells and of medically important genes. Such genes might encode proteins that have direct therapeutic applications, such as growth factors, or that are important targets for drug development. The ability to experimentally manipulate this system opens many new avenues of research as access to normal embryo–derived precursors at comparable stages of development is often very difficult in animal models, and is almost impossible in humans because of ethical considerations.
The ES cell differentiation system can also be used to develop a rapid assay to study the function of genes. In the mouse, most gene function studies require the generation of transgenic and/or gene knockout animals, which can be considered long–term assays. The ES cell differentiation system offers a rapid complementary in vitro approach that is not subject to the limitations of in vivo studies. The system is well suited to understanding the role of genes through 'loss–of–function' studies by developing ES cell lines that lack a specific gene. Understanding how the absence of a gene influences the normal growth and development of specific tissues is an important first step towards elucidating its function. The ES cell differentiation system is also ideal for 'gain–of–function' studies, as it is possible to analyze the consequences of overexpression of specific genes on the development of different cell lineages. With the development of human ES/EG cells, these same approaches can now be applied to the study of human genes using a human in vitro developmental system.
Human ES/EG cells will be valuable as a test system for evaluating the toxicity and efficacy of new medicines or chemicals. The wide range of cell types and tissues that develop in EBs represent a biological system that mimics many of the complex interactions of the cells and tissues of the body, and as such provides an attractive screening tool. This type of assay could have wide applications in the pharmaceutical, cosmeceutical and agrochemical industries. It has the potential to reduce the need for animal testing and to increase the efficiency and reduce the costs of developing safe and effective drugs and chemicals.
Given the progress with mouse ES cells, it is important to ask how close we are to similar experiments with human cells? Before one can fully answer this question, a number of basic issues need to be addressed. First, we need a more thorough understanding of the efficiency and regulation of human ES/EG cell differentiation in culture. There are also basic technical issues relating to the growth and propagation of these cells in culture that need to be overcome. Specifically, the reported difficulty in dissociating the human ES/EG cell clusters into viable single cells is problematic, particularly for gene–targeting experiments. In addition, we need more data on the reproducibility and efficiency of human ES/EG cell isolation. Will it be possible for other investigators to establish comparable cell lines, and if so, at what frequency? With respect to cell–replacement therapy, we will need to develop methods for the purification of large numbers of lineage–specific precursors from EBs. In addition, certain safety issues need to be addressed before this type of therapy can be moved to the clinic. For instance, are genetic mutations introduced during cell differentiation and expansion in EBs? What are the potential problems if the precursor cell population isolated for transplantation contains contaminating cell types? These concerns will be addressed as more investigators develop their own ES/EG lines and/or gain access to existing ones.
It is clear that ES cell technology has revolutionized modern biology and provides us with unique opportunities to understand the mechanisms that control basic biological processes. The development of human ES/EG cells is a significant milestone towards applying the potential of this technology to the direct treatment of human disease, an application anticipated by many. Significant additional research will be necessary to capitalize on the full therapeutic potential of these cells, but the resulting novel therapies should more than justify the effort.
The production of blood cells, or hematopoiesis, takes place in the bone marrow. Among the billions of cells in the bone marrow, there is a very small subpopulation that has a pivotal role in the maintenance of hematopoiesis. This subpopulation is composed of hematopoietic stem cells (HSC) that, with their distinctive capabilities of self-renewal and differentiation, furnish a constant supply of blood cells of all hematopoietic lineages throughout life. Thus, the stem cell can either replicate and remain a stem cell or differentiate into myeloid or lymphoid stem cells, which in turn can further proliferate and mature, ultimately giving rise to all the circulating blood cells
Currently, allogeneic bone marrow transplants are recognized as a treatment of choice for chronic myelogenous leukemia, acute leukemias failing initial treatment, aplastic anemia, and several lethal disorders of the immune system and of hematopoiesis. Allogeneic bone marrow transplantation has become increasingly used as a cure for a variety of genetic defects of the hematopoietic and immune systems, and for lipid storage diseases. Genetic diseases that have been successfully cured by bone marrow transplantation include Cooley's anemia, sickle cell anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi anemia, Blackfan-Diamond anemia, ataxia telangiectasia, infantile agranulocytosis, Chediak-Higashi disease, chronic mucocutaneous candidiasis, mucopolysaccharidosis, cartilage-hair hypoplasia, Gaucher's and other lipid storage diseases. Some of these diseases, such as Cooley's anemia (beta-thalassemia) and sickle cell anemia, are major worldwide public health problems. Others are devastating orphan diseases that are extremely costly to treat. Collectively, these genetic diseases occur in tens-of-thousands of births per year.
It is also recognized that several malignant disorders are sensitive to agents which have, as their dose-limiting toxicity, myelo-ablation. This knowledge, along with the initial success of marrow and peripheral blood-derived autografts administered after myelo-ablative therapy, have clearly defined the rationale for the use of hematopoietic stem and progenitor cells in the treatment of several non-hematopoietic malignancies, including breast cancer, which occurs with alarming frequency.
