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The Chiaroscuro Stem Cell
Posted on: December 14, 2003

Owen first recognized stem cells experimentally in 1945, when he found lifelong blood chimerism between twin cows. He postulated that an interchange of cells between bovine twin embryos occurred as a result of conjoined blood vessels and that the interchanging cell had to be "ancestral" to the terminal erythrocyte. More than 15 years later, investigators formally tested for these ancestral blood cells by preventing radiation death in mice with bone marrow transplantation. These stem cells were noted for their ability to give rise to clonal colonies of differentiated blood cells in the recipient spleen and for their ability to rescue subsequent generations of lethally irradiated mice. This multilineage reconstitution by a self-renewing cell is a cardinal feature of stem cells. To this day, transplantation experiments like those performed in the 1960s that showed clonal, robust, and functional differentiation by a cell transplantable over many generations remain the gold standard in testing stem cells.

Some stem cells have a greater capacity of self-renewal and multilineage differentiation than others (Table 1). At the time of conception, the fertilized egg (zygote) contains dividing cells (blastomeres) that form an embryo and placenta (Figure 1). These blastomeres are totipotent; they have the potential to form an entire living organism. After about 4 days, these totipotent cells begin to specialize and form into a hollow ball, the blastocyst, containing an outer shell (trophoectoderm) and a cluster of cells called the inner cell mass (ICM), from which the embryo develops. Human embryonic stem (hES) cells are derived by removing the trophoectoderm, which would normally become the placenta, and culturing cells from the ICM. These hES cells of the ICM are pluripotent; they are able to differentiate into tissues of all 3 germ layers but cannot produce another embryo because they are unable to give rise to the placenta and supporting tissues. Transplanting hES cells into a woman's uterus would not produce a fetus. Blastocysts harvested for hES derivation are usually acquired from unused in vitro fertilizations (IVFs). The hES cells express specific surface antigens, as well as OCT-4 and human telomerase, proteins associated with a pluripotent and immortal phenotype. A second source of pluripotent cells is human embryonic germ cells, derived from the fetal gonadal ridge, which normally gives rise to either sperm or egg. It remains to be tested whether human embryonic germ cells have the same capacities that make hES cells so important. A hallmark of hES cells is their long-term self-renewal capability in culture dishes. Whereas adult stem cells divide asynchronously and eventually lose their ability to self-renew, hES cells have been cultured over many years, maintaining developmental potential, proliferative capacity, and karyotypic stability.

Table 1. Cell Characteristics in Stem Cell Biology

Term Definition Example
Totipotent Able to produce an entire being. Blastomeres
Pluripotent Able to produce all tissues and self-renew indefinitely. Embryonic stem cells
Multipotent Able to produce many cell types and self-renew over the lifetime of the being and over many subsequent generations if transplanted. Hematopoietic stem cells
Progenitor Able to produce restricted number of cell types and with limited to no capacity of self-renewal Neutral stem cells

Because of this unlimited self-renewal and boundless developmental potential, hES cells may serve as powerful tools to unlock important medical challenges. As a basic science tool, these cells help to identify molecular mechanisms in pluripotent cell differentiation, affording investigators a much better understanding of fetal development. Ultimately, this understanding aims to reduce infertility, pregnancy loss, and birth defects-major health care challenges in the modern world. Also, hES cells are helpful in drug development, identifying potentially embryotoxic teratogens. Moreover, normal cell lines derived from these pluripotent cells may serve as representative tissues for in vitro toxicity testing of medicinal compounds in development. Finally, organ regeneration from hES cells would not only halt disease progression but also could help to remodel damaged organs. Investigators are already using embryonic stem cells to create heart muscle, brain, pancreatic islet cells, and blood vessels. Techniques used for tissue engineering include transplanting a patient's somatic cell nucleus into an enucleated oocyte, activating the cell to mimic fertilization, culturing the totipotent cells in a dish, and then differentiating the cells into tissue of need. The resulting tissue, whether cardiac, pancreatic, hematopoietic, neural, or hepatic, is genetically identical to the patient's tissue, would not be rejected because of identical HLA antigen expression, and could be used for tissue repair.

Figure 1. Embryonic stem cell. At the time of conception, a single cell (zygote) is formed and subsequently grows into a cluster of totipotent blastomeres. Further cell division produces a hollow ball with an outer trophoectoderm, producing placenta and supporting tissues as well as an inner cell mass. Embryonic stem cells are derived from plating inner cell masses on a plastic dish and culturing them in incubation ovens. With further culturing, these pluripotent embryonic stem cells can form tissues of all 3 germ layers. CNS = central nervous system. (...) = other organs.

It is generally accepted that each organ of our body is in balance between degradation and repair. The liver that we were born with is not the same liver that we have when we die. Throughout life, toxic insults wound our organs, bringing about the question of what keeps the balance between destruction and construction. In adults, stem cells have been found in many tissues, such as liver, bone marrow, pancreas, and brain, maintaining this homeostasis. Moreover, some of these adult stem cells, once thought to mend only local property, also help in disaster relief of other more distant organs.

To test cell plasticity, investigators use a variety of cell transplantation models. The basic procedure includes injecting donor cells of interest into a recipient and subsequently analyzing the recipient's organs for donor contribution. Modifications have been made to this basic procedure, sometimes resulting in conflicting reports in stem cell plasticity. These differences in findings may be explained by disparities in donor stem cell separation, cell cycle of transplanted cells, time from transplantation to evaluation of end-organ chimerism, type of injury eliciting plasticity, and ability of target niche to support stem cell transdifferentiation.

Thus, before plasticity can be confirmed, several criteria must be addressed. First, clonal repopulation should be demonstrated from the transplanted stem cell. Second, a self-renewing cell should be responsible for observed plasticity. Although cells capable of organ regeneration have been identified in various organs, if the cell does not exhibit self-renewal, it is not considered a stem cell; rather, it is termed a progenitor cell. Third, multiple markers and proper morphology should be used in tissue analyses. Recently, stem cells thought to transdifferentiate into pancreatic islet cells were found to have simply absorbed insulin from the tissue culture media. Fourth, the transdifferentiated cells should be functional. Fifth, robust repopulation is preferred. Sixth, analyses for cell fusion between stem cell and differentiated cell should be performed. In culture dishes, stem cells can acquire the markers of differentiated cells by fusing with them spontaneously. Some investigators regard cell fusion as an artifact confounding plasticity; however, many organs in the body, including the liver, heart, skeletal muscle, and brain, have functional multinucleated cells. Until proved otherwise, fusion may be a natural and useful process.

One of the first discoveries in end-organ repair and cell plasticity occurred in the liver. When hepatocytes are prevented from proliferating in response to liver damage, hepatic oval cells come to the rescue and produce more hepatocytes and bile duct cells. Interestingly, these oval cells, viewed as resident liver progenitor cells, share similar protein production with the HSC. Human studies have also found that the human liver may accommodate remote repair, although some scrutinizing investigators have found that repairs are short fixes and are not long-lived. To clarify these mixed results, further studies in liver transplant recipients have found that liver remodeling by a remote stem cell is more robust in situations of active hepatitis; however, bile duct repair happens early after transplantation and at unwavering levels. Studies of liver plasticity describe the possibility of remote repair, variable intraorgan accommodation to remote restoration, and injury as an important nidus for plasticity.

The liver and pancreas have close ties, going back to development in which both emerge from the same general area of the ventral foregut endoderm. The pancreatic progenitor cell, located within the ductal epithelia of the pancreas, has the capacity to differentiate into endocrine islets of Langerhans, including the glucagon-producing α cells, the insulin-producing β cells, the pancreatic polypeptide-producing γ cells, and the somatostatin-producing δ cells. Given the close relationship between the liver and pancreas, it is not surprising that these same pancreatic progenitor cells can differentiate into liver cells, and, conversely, hepatic oval cells can produce insulin-producing cells. With the obvious benefit to diabetes mellitus research, future studies will seek to identify the best stem cell sources and methods of transplantation for effective pancreatic islet neogenesis.

Unlike the liver and pancreas, the kidney progenitor cell has yet to be discovered. During development, the human kidney results from collision of the ureteric bud (lower urinary tract) and the metanephric mesenchyme (upper urinary tract). Precursors for the collecting tubules reside within the ureteric bud, whereas precursors for the rest of the kidney come from the metanephric mesenchyme. After development, in situations of injury, the kidney undergoes repair, with myofibroblast cells observed at sites of parenchymal remodeling. However, the origin of these myofibroblast cells is debatable. They may be remnants from the primitive metanephric mesenchyme or from bone marrow. Experimentally, bone marrow transplantation in mice has led to marrow-derived renal tubular epithelial cells. In humans, kidney transplant studies have shown renal tubular repair from an extrarenal source. Several human studies have also addressed the issue of renal blood vessel repair, finding endothelial cell repair by a source outside the kidney, particularly in conditions of graft rejection and vascular inflammation. The remaining important issue is the true identification and location of this kidney colonizing progenitor cell.

Adult cardiac muscle regeneration is modest and largely restricted to viable myocardium. Several investigators have sought to enhance the heart regeneration challenge by injecting different cell sources into damaged myocardium and observing for engraftment. Cell sources such as cell lines, skeletal muscle myoblasts, and bone marrow cells have been used. Interestingly, these cellular cardiomyoplasty approaches have identified the bone marrow as one of the richest founts of cardiac repair. In murine models, bone marrow cells can differentiate into cardiomyocytes and coronary arterioles. However, within the bone marrow, the operative cell (mesenchymal stem cell vs. HSC) is debatable. Furthermore, when these same experimental models are taken from rodent to primate experimental systems, the previously observed robust engraftment appears modest at best. This brings into question the clinical relevance of stem cell therapy for heart disease. Unfortunately, studies of human cardiac allografts have not clarified the picture, with some investigators finding cardiomyocyte chimerism and others finding only coronary artery chimerism. Future controlled trials will be important to clarify the role of cell transplantation in myocardial repair.

Animal studies of the brain have found that bone marrow can differentiate into neurons and glial cells. Whether these transplanted cells are functional and impact cognitive function has yet to be determined; however, environmental influences such as ischemia, seizure, learning, and exercise can substantially stimulate hippocampal neuropoiesis. Human studies have found that bone marrow can make brain cells. Female patients undergoing therapeutic bone marrow transplantation were found to have donor neurons, astrocytes, and microglia in the hippocampus years after transplantation and without evidence of cell fusion. Interestingly, whereas some investigators found no cell fusion in the hippocampus region of the adult brain, the others found evidence of cell fusion in the cerebellum. In a series of bone marrow transplant recipients, these investigators found recipient cerebellar Purkinje cells containing extra donor DNA. These cerebellar cells are postulated to have been rescued from radiation and chemotherapy toxicity. The functionality of Purkinje cell fusion will certainly be a focus for future research. Furthermore, the mechanisms of how blood can make brain cells are unknown; however, between the hematopoietic and central nervous systems, there are similarities in homing and migration.

The close ties between blood and blood vessels have always been suspected given the recognition of a hemangioblast in the developing embryo. However, whether an adult hemangioblast worked double-duty, replenishing blood and repairing vessels, was debatable until 2002, when certain investigators formally proved that the adult HSC could function as a hemangioblast. Furthermore, these investigators were the first to show functional plasticity, with active perfusion of donor-derived vessels. Follow-up studies also seem to indicate that it is in the setting of injury, whether acute or chronic, that remotely derived repair by the HSC is most robust, a similar lesson learned from liver plasticity studies.

In addition to their role in responding to physiologic repair, stem cells are implicated in certain cancers. This conviction is still being deliberated; however, lines of evidence seem compelling. Cancer cells have the ability to self-renew much like stem cells. In human acute myelogenous leukemia studies, investigators have shown that the acute myelogenous leukemia stem cell could be purified from the CD34+CD38- fraction-the same fraction as the HSC. Solid tumors like lung cancer also have a tumorigenic subset amid a crowd of differentiated cells. Furthermore, many tumors like colon cancer, small cell lung cancer, and melanoma express proteins restricted to embryonic growth and development. Signaling pathways such as Notch, Wnt, and Sonic hedgehog (Shh) supporting stem cell self-renewal and multipotency are also found in cancers of the blood, colon, and breast. Finally, stem cells and cancer cells share similar homing and migration pathways such as the stromal cell derived factor 1/CXCR4 axis in tumor spread and metastasis of breast cancer, prostate cancer, and lung cancer. The importance of discovering and characterizing a cancer stem cell is to better target the carcinogenic subset, bringing about a quicker remission and preventing future relapse.

New discoveries in stem cell biology will soon bring revolutionary changes in the way physicians approach degenerative diseases, wound repair, autoimmune conditions, cancer, and reproductive medicine. Stem cells are self-renewing cells capable of producing many different cell types. Adult stem cells do well in repairing their organ of origin but have limited capabilities in self-renewal and distant organ repair under normal physiologic conditions. The degree of plasticity potential of the adult stem cell has yet to be determined. Embryonic stem cells have tremendous therapeutic and research potential to produce any tissue of the body and to grow unperturbed in plastic culture dishes for many years. Stem cells currently are used in transplantation regimens to repair wounded organs. They are also used experimentally in toxicity studies to test drug safety, cancer investigations to pinpoint methods of unregulated growth, and reproduction protocols to identify critical steps in fertility and pregnancy. However, along with these remarkable abilities, use of stem cells carries many ethical challenges.

Certainly, stem cells are not the first human discovery to stretch the boundaries of medical knowledge and create waves of ethical debate. Our society has grappled with permission to perform autopsies for crucial understanding of human anatomy and consent to produce recombinant DNA for lifesaving medications. In all instances of stretching knowledge boundaries, a societal consciousness was at play, in some ways encumbering progress and questioning techniques of intervention.

Additionally, shared concerns among all instances of testing medical boundaries, as well as using stem cell technology, include issues of safety, efficacy, and resource allocation. For decades, patients have undergone adult HSC transplantation in the treatment of immune deficiencies and cancer. Although graft-vs.-host disease and post transplantation infections are major risks of allogeneic transplantation, investigators have worked to minimize these consequences, and many patients accept these risks in the hope of the lifesaving benefit of disease eradication. In contrast, reproduction by SCNT into embryonic stem cells has been inefficient and carries the concerns of developmental abnormalities and early aging. However, the field of stem cell therapy is still in its infancy, with researchers incrementally improving safety, efficacy, and applicability to a wider spectrum of disease.

Stem cell therapy differs from previous technologies in how these founts of plasticity are tapped. Adult stem cells are typically acquired by harvesting adult tissues. Patients give informed consent and usually undergo little risk at donation. In contrast, hES cells are obtained by culturing cells from the ICM of a blastocyst, usually acquired from an unused human embryo produced by IVF or from an already aborted fetus. The harvesting process requires dissolving the blastocyst, bringing into question the moral and legal status of the human embryo.

Many religious perspectives consider the human fetus to constitute an individualized human entity. However, there is substantial debate regarding at which specific stage dignity is conferred in development (conception, primitive streak development, implantation, "quickening," or birth). Recently, a less specific "developmental view" of moral status surfaced, meriting moral rights to the individual as consciousness and relationships develop. Taking into account the many perspectives on the moral status of the human embryo and the scientific promises of a healthier tomorrow through stem cell technology, our society has attempted to define the legal status of the human embryo.

The embryonic stem cell also has tremendous potential in producing replicate tissues and genetically identical offspring. Cloning is accomplished by removing the chromosomes of an oocyte, inserting the nucleus of a donor adult (somatic) cell, and then stimulating the hybrid cell to divide as if it were a totipotent zygote. The cell is then prepared to produce any tissue of the body (therapeutic cloning) or if implanted in a female uterus to produce offspring with the same genetic material as the donor adult cell (reproductive cloning). Resultant tissues from this totipotent cell have the same major histocompatibility complexes and enzyme machinery as the adult donor, allowing for drug testing on pure tissues and organ transplantation without rejection. Many find cloning a misleading and politically charged term; thus, it is best to refer to this technology as cell replacement through nuclear transfer or somatic cell nuclear transfer (SCNT).

Aside from the important issues of safety, efficacy, and resource allocation, opponents of SCNT for reproductive purposes raise concerns over potential problems of identity for cloned offspring, troubled family relationships, and cloning of malevolent people. Whereas these points must be discussed, they fall prey to the overly simplistic viewpoint of genetic reductionism. We are more than our DNA. All children, not just identical twins, already experience living up to the expectations of siblings, parents, and grandparents. Furthermore, parent and child relationships are not defined by haplotype DNA matching but by supportive relationships developed in the home. In addition, although some people may have genetic susceptibility toward hostility or malevolence, the environment nurtures or quells these behaviors. Moreover, conventional sexual reproduction passes on the same genetic susceptibilities to behavior traits that would be true of reproductive cloning. Another concern includes the potential for eugenics, the selection of healthier genetic traits, potentially leading to the commercialization of human procreation. Although this concern argues on a slippery slope, further debate on this topic will include the true importance of asexual reproduction, the issue of why human reproduction should be left to chance, whether humans should have greater control over reproduction, and whether safeguards will be effective.

Probably the most unshakable consequence of SCNT for reproductive purposes is that it impacts not only the partakers but also our entire society indirectly. The cost to society of foregoing use of this technology, either by failure to correct genetic abnormalities or by improving the success of lifesaving organ transplantations, may be equal to or greater than the perceived costs to the dignity of life as held by those with the most inviolable concept of the moral status of embryos.

The climate in which stem cells are explored can be nurturing or profoundly limiting. As medical scientists, we must not make judgments or ethical decisions on our own; rather, we must ensure full informed consent of the population as a whole. This approach may limit quick progress and may disqualify avenues of research and therapy, but as responsible researchers, we must use the resources of society in a worthy manner to explore fully the tremendous potential of stem cells.

Source: An Overview of Stem Cell Research and Regulatory Issues. Christopher R. Cogle, Steven M. Guthrie, Ronald C. Sanders, William L. Allen, Edward W. Scott and Bryon E. Petersen Mayo Clinic Proceedings; Aug 2003; 78; 8: 993-1003.
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