In recent years, data from numerous experimental studies has suggested that the potential uses of stem cells in medicine may reach far beyond bone marrow transplantation. How applicable is recent research to modern medicine, and how soon might we expect to see stem cell therapies applied to tissue engineering problems? These and other questions are explored in this special issue of Bone Marrow Transplantation. It is altogether fitting that a discussion of the therapeutic potentials of stem cell therapy be grounded in our field, being the first to apply stem cell therapy to the clinical management of acquired and inherited diseases. But what is a stem cell? In the context of bone marrow transplantation, we understand the answer to this question in a concrete and functional sense because of decades of research and clinical applications that grew out of the need to understand the effects of ionizing radiation on biological systems. In the years following the Second World War, a considerable amount of scientific effort was focused on the prevention and treatment of radiation sickness. From these studies came the observation that transplants of spleen or bone marrow cells contribute to cellular recovery following lethal radiation.1 Almost 50 years after this dramatic insight, we now understand that the ability of such transplants to reconstitute hematopoiesis following radiation depends upon the presence of extremely rare stem cells found predominantly in the bone marrow but capable of mobilization into peripheral tissues via the blood vascular system.2
After many years shrouded in mystery and controversy, the characteristics of blood stem cells were gradually revealed through novel assays3,4,5 and methods for isolation of these rare cells.6,7 We now understand that the definition of a stem cell must include the two essential characteristics of self-renewal (cellular division maintains stem cell potential) and multipotency (differentiation into functionally distinct lineages). To complicate matters, it is clear that progenitor cells, which are multipotent but lack self-renewal potential, are often difficult to distinguish from true stem cells.8 Finally, at least some confusion persists in the tissue stem cell field, where unipotent precursor cells which maintain a tissue through a self-renewing process are often considered stem cells.
The general field of stem cell biology has been the subject of intense public interest in recent years for several reasons. First, the demonstration that recipients of bone marrow transplants harbor donor-derived cells in a variety of tissues has radically changed our expectations for the applications of this type of therapy,9 even though many questions have been raised by these interesting findings.10 Second, the derivation of totipotent human stem cells from both embryonic and fetal sources has introduced a potential new source of tissue for engineering applications. Equally importantly, this new technology marks the genesis of a new level of conflict between science and religion that surpasses that raised by older questions of creationism vs evolution. The potential use of stem cells derived from adult tissues introduces yet another side to this complex story. How are we to define when a stem cell is a stem cell? It is in this vein that I examine a few of the historical aspects of stem cell biology in order to better understand where we have come from at this stage in the development of the stem cell field.
Embryonic stem cells: a timeline
Lewis11 has correctly identified the origins of the stem cell biology field in the work of Leroy Stevens, a developmental biologist who identified frequent testicular tumors arising spontaneously in strain 129 mice at the Jackson Laboratories. This work was published to little fanfare beginning in 1958.12 However, the curiosity of Mintz and Illmensee led to a startling observation. When malignant teratocarcinoma cells were mixed into developing mouse embryos, the environment of the embryo harnessed the unregulated growth of the tumor and directed these cells to proper channels of proliferation and differentiation.13 The result was chimeric mice in which a significant portion of the body mass was derived from the teratocarcinoma. This startling discovery was viewed at the time as evidence for environmental regulation of malignant growth, but the potential of these cells was certainly not overlooked by developmental biologists. Embryonic stem cell lines were derived from the inner cell mass of mouse blastocysts in 1981,14,15 as shown in Figure 1. These cells were adapted for growth in culture without differentiation, but could differentiate into mesoderm, endoderm, and ectoderm in vitro and in vivo. The derivation of embryonic stem cell lines was rapidly exploited to give birth to the field of targeted mutagenesis,16,17 an entirely new approach to the investigation of complex mammalian biological systems. Today, it is difficult to imagine a world in which the genome could not be mutated in a specific manner. The true power of stem cell biology was revealed to the world at large with the announcement that the transfer of nuclei derived from adult somatic cells into enucleated oocytes produced, at a low frequency, viable offspring clonally derived from the donor of the nuclei.18
Figure 1.
From the initial descriptions of the ability of testicular carcinoma cells to produce pluripotent embryonic stem cells, these cells have since been derived from blastocysts as well as the primordial germ cells in the developing genital ridge in both mouse and man. Figure courtesy of Terese Winslow, used with permission of the artist.
Full figure and legend (54K)Mouse to man
The successful application of targeted mutagenesis in the mouse was not the only useful application of embryonic stem cell lines. A variety of investigators utilized these cell lines to model the development of the early embryo in culture systems, and successfully recapitulated several aspects of embryogenesis. When the application of in vitro fertilization to the clinical problem of infertility resulted in the birth of the first test-tube baby in 1978, the stage was set for the eventual derivation of human embryonic stem cells from embryos fertilized in vitro but not implanted into a womb.19 Since these early embryos are frozen in quantities that exceed clinical need, large banks of fertilized embryos destined for destruction now exist around the world as a consequence of the widespread application of in vitro fertilization. Some of these embryos have been cultured to derive embryonic stem cell lines: however, the derivation of cell lines in addition to those already in existence has been deemed unnecessary and will not be supported by federal funding agencies in the United States.
A second approach to the application of stem cell technology in humans utilizes tissue derived from the genital ridge of aborted first trimester fetuses (Figure 1).20 These cells, which normally develop into mature gametes, can be cultured under specific conditions to produce cell lines with all known characteristics of blastocyst-derived embryonic stem cells but lacking apparent tumorigenic potential. This combination of multipotential differentiation in the absence of tumor formation has lead to the proposed use of these cells in clinical trials to treat spinal cord injury, Parkinson's disease, and other cell-based therapies. With the specter of the cloning of human beings looming before us, the National Academy of Sciences initiated a comprehensive analysis of this brave new world.21 The current state of federal funding will support the utilization of fetal-derived embryonic germ cells in clinical applications, most likely because the derivation and use of these cells avoids some of the concerns raised by the concept of frozen embryos as sources of embryonic stem cells. Embryonic germ cells are unable to be implanted into a surrogate mother to produce a genetically normal human, unlike the embryos formed during in vitro fertilization. As such, the only embryos that might be formed by embryonic germ cells would be genetic mosaics of the germ cell and a blastocyst in which such cells might be introduced, or would be the product of a somatic cell nuclear transfer. Since the latter process can be performed using a wide variety of cell types, the embryonic germ cell provides no special advantage in this sense.
Adult stem cells
Undifferentiated cells that are found in a differentiated adult tissue are considered adult stem cells, particularly when these cells contribute to ongoing tissue maintenance or repair. These cells may be capable of self-renewal, but do not replicate indefinitely in culture. Adult stem cells may differentiate to produce progenitor, precursor, and mature cells, but these activities are usually limited to the cells contained in the tissue of origin. Adult stem cells usually comprise a small minority of the total tissue mass, and as such are usually quite difficult to identify and isolate. Adult stem cells have been described in regenerating tissues such as the liver, epithelium, and muscle, as well as in tissues like the brain, which previously was thought not to possess extensive regenerative properties. By far, the most well-characterized example of adult stem cells is that of the hematopoietic system.
Hematopoietic stem cells: paradigms for stem cell biology
The limited lifespan of most blood cells demands that a continual source of these cells be assured throughout life. It is likely for this reason that the hematopoietic system has so readily lent itself to applications involving clinical transplantation. Indeed, the challenges faced by cells utilized in bone marrow transplantation are not so different than the normal physiologic role played by these cells during maintenance of hematopoiesis over the lifetime of the normal mammal. Compared to the hematopoietic system, other tissues of the adult mammal display relatively limited potential for replacement from endogenous stem cells in response to tissue injury. Furthermore, no other tissue is characterized by such a wide variety of different cell lineages, which all arise from a common stem cell in a developmental process that continues throughout life. The ability to model many of these differentiation pathways under controlled conditions in vitro, and the availability of recombinant proteins that select or direct differentiation along specific lineages makes the hematopoietic system the premier paradigm for the field of stem cell biology.
Plasticity
The concept of stem cell plasticity refers to the phenomenon of trans-differentiation, which is the ability of an adult stem cell from one tissue to differentiate as a specialized cell type of another tissue. A recent study showed that neural stem cells were capable of regenerating blood lineages in transplant recipients,22 and the field rapidly advanced as examples of muscle, epithelium, liver, and other tissues derived from heterologous stem cells (usually bone marrow derived) were reported.23 With few exceptions, these studies involved transplantation of large numbers of cells, leaving open the possibility that distinct classes of stem cells were responsible for regeneration of the different tissues. Furthermore, the magnitude of tissue replacement has often been minor, suggesting that this approach to tissue engineering will require extensive optimization in order to be clinically useful. Finally, technical artefacts24 and difficulty in reproducing some of the reported findings25 suggest that caution is indicated in interpreting many of the experiments. The concept that stem cells derived from adult tissues will provide a viable alternative to the embryo as a source of material for tissue engineering is far from validated.
Stem cells as therapeutic agents
The ability of stem cells to provide a self-renewing source of normal differentiated cells has been extensively exploited in the bone marrow transplantation field. Applications include the treatment of marrow failure syndromes, leukemia and lymphoma, certain inherited blood disorders and autoimmune diseases.
Recent advances in vector design have made gene correction a feasible approach to the treatment of a number of genetic diseases, including X-linked severe combined immunodeficiency disease, hemophilia, and a number of autoimmune disorders. Clinical application of stem cell therapy depends on robust self-renewal and differentiation of the transplanted cells, and in this sense trans-differentiation must be sufficiently frequent and robust to achieve enough tissue replacement to be clinically useful. However, if harnessed and properly regulated, it is not difficult to imagine the application of stem cell therapy in diverse settings such as diabetes (generation of islet cells from stem cells), repair of damaged heart muscle, and rebuilding the nervous system after injury or age-related decline.26
Regulatory and funding issues
The United States government now provides some funding for human stem cell research. While NIH funds cannot be used for derivation of human ES lines, this type of research can be performed using private sources of funding if proper informed consent is obtained under a protocol approved by an institutional review board according to NIH guidelines. NIH funds can be used for research that utilizes existing embryonic stem cell lines, as well as for derivation and use of embryonic germ cells from fetal tissues. This apparent discrepancy in policy arises because of the consideration that established stem cell lines and aborted fetal tissues are not embryos and cannot, by themselves, develop into human beings. The NIH guidelines and the FDA regulate experimental and clinical use of human pluripotent stem cells and fetal tissues. As of September 25, 2002, five NIH grants have been approved and funded for a total of $4.2 million, and administrative supplements for embryonic stem cell research have been awarded to 30 additional investigators.27
The future of stem cell therapy?
A number of challenges remain before the promise of stem cell therapy can be translated into application. First and foremost, the political and ethical conflicts that surround the use of human embryonic and fetal tissue for medical applications must be resolved. The concept that stem cells derived from adult tissues will substitute for those obtained from fetal or embryonic sources is simply too premature to be used as a basis for legislation and regulation. While the combination of gene therapy with stem cell therapy has proven to be effective for certain diseases, methods of gene delivery must be improved to prevent unpredictable adverse events. Animal models must be refined to allow comprehensive analysis of potential risks and benefits prior to clinical application. These and other barriers stand before us, marking the path toward new applications in clinical medicine.
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