This article is a US Government work and, as such, is in the public domain in the United States of America.
Stem cells have two remarkable properties. They can either renew themselves or they can differentiate into one or more adult cell types. Stem cells derived from a human embryo appear to have an unlimited capacity to self-renew in cell culture, and they are also able to differentiate into hundreds of adult cell types. Human embryonic stem cell lines offer a platform technology that has the potential to elucidate the molecular mechanisms that determine adult cell fate, generate cellular models for discovery of new drugs, and create populations of differentiated cells for novel transplantation therapies. The National Institutes of Health (NIH) has identified some of the rate-limiting steps toward realizing this potential, and has forged funding initiatives to accelerate research progress. Given the remarkable potential, NIH support for research using stem cells is an important priority for the foreseeable future. Developmental Dynamics 236:3193–3198, 2007. Published 2007 Wiley-Liss, Inc.
Stem cells have two defining properties: (1) they have the capacity to renew themselves under appropriate conditions, either in vitro or in vivo; and (2) they are pluripotent, which means they are able to differentiate into most, if not all, mature cell types. Human stem cells derived from an early stage blastocyst (an embryo approximately 5 days after fertilization) have the capacity to renew indefinitely in the laboratory and can provide a theoretically unlimited source of cells. Stem cells and progenitor cells—early offspring of stem cells that can differentiate but not replicate indefinitely—can also be found in many different organs, including the amniotic fluid, placenta, umbilical cord, brain, gut, bone marrow, and liver. Sometimes referred to as “adult” stem cells, these stem cells are typically rare in the organ of origin, consisting of as few as 1 cell in 10,000. Stem cells from organs typically are more limited in their capacity to self-renew in the laboratory, making it more difficult to isolate a large number of stem cells for a specific experimental or therapeutic application. In addition, because their principal function is to restore damaged cells, they are typically limited to grow into the cell types found in the organ from which they originate. So far, the evidence for transdifferentiation, whereby a stem cell from one organ differentiates into a mature cell type of a different organ, is not compelling if the goal is to demonstrate appropriate mature cell function and not simply gene expression properties. Any or all of these limitations of stem cells derived from organs may some day be reduced or eliminated by new discoveries, and, in fact, such research is currently under way in several laboratories. In the future, it may be possible to reverse the differentiation process, thereby generating pluripotent cells from differentiated cells. This is an important area of research because this mode of generation would not require the destruction of human embryos.
WHAT IS THE MEDICAL PROMISE OF STEM CELL RESEARCH?
The availability of human pluripotent, self-renewing cell cultures affords scientists an exciting platform technology whose impact on biomedical research is believed by many in the research community to be significant. There is no question that these cells will be an enabling resource for scientists whose goal is to understand the molecular mechanisms that determine how a pluripotent, self-renewing cell differentiates into a specialized cell type (Fig. 1). These mechanisms include epigenetic changes in the chromatin structure, developmental changes in gene expression, exposure to growth factors, and interactions between adjacent cells. This knowledge could, in the future, lead to new approaches to mobilize and differentiate endogenous populations of pluripotent cells to re-populate a cell type ravaged by injury or one of many cellular degenerative diseases, including Type 1 diabetes mellitus, Parkinson's disease, myocardial infarction, and spinal cord injury, to name a few prominent examples. Alternatively, scientists may some day be able to coax human pluripotent cells grown in the laboratory to become a specific type of specialized cell, which physicians might be able to subsequently transplant into a patient to replace cells damaged by these same disease processes.
Once it is possible to direct the differentiation of pluripotent cell cultures into a specific type of cell, scientists can develop cellular models of human disease that may be useful in drug discovery or toxicity studies. In the case of a neurodegenerative disorder such as Huntington's disease (an autosomal dominant genetic disease in which neuronal degeneration leads to death), one could imagine that pluripotent cells differentiated into neurons in culture could be used to test drugs that may ultimately delay or prevent the metabolic processes that result in degeneration in these cells. It is impossible to predict the many ways by which a heightened understanding of how pluripotent cells differentiate into specialized cell types may ultimately lead to new approaches for treating patients with cellular degenerative diseases.
WHAT ARE THE SOURCES OF HUMAN PLURIPOTENT CELL LINES?
Using existing technology and protocols, scientists are able to generate human pluripotent cell lines using three methods. The first method involves the development of human embryos to help an infertile couple have a baby through in vitro fertilization (IVF). During the IVF process, more embryos are usually generated than may actually be needed to assist the couple in their efforts to have a family. These extra, or spare, embryos are typically stored in a freezer in the event that the couple attempts to try to have additional children in the future. It is estimated that there are approximately 400,000 such spare embryos worldwide. If these embryos are never used by the couple, they will either remain in storage or be discarded as medical waste. Alternatively, these embryos could potentially be used to generate a human embryonic stem cell line.
The current procedure for generating a human embryonic stem cell line involves embryos at the blastocyst stage, which occurs approximately 5 days after the human oocyte, or egg, is fertilized (Fig. 2). During the normal reproductive process, the 5-day-old blastocyst is still in the fallopian tube, en route to the uterus where it may implant within approximately 7 to 8 days after fertilization. The blastocyst consists of approximately 150–200 cells that form a hollow sphere of cells, the outer layer of which is called the trophectoderm. During normal development, the trophoblast will become the placenta and umbilical cord, structures essential for supporting continued development of the embryo. At one pole of this hollow sphere, 30 to 50 cells form a cluster that is called the inner cell mass, which will progress to form the developing fetus and child. Cells in the inner cell mass are pluripotent, possessing the capacity to become any of the several hundred specialized cell types found in a developed human, with the exception of the placenta and umbilical cord.
To generate a human embryonic stem cell line, scientists remove the inner cell mass from a blastocyst no longer needed for reproductive purposes and place these cells into a specialized culture medium. In approximately one of five attempts, a human embryonic stem cell line begins to grow. The conditions for culturing the cells are critical for maintenance of the self-renewing and pluripotent properties of these remarkable cells.
Success in generating human embryonic stem cell lines was first reported by the laboratory of James Thomson, PhD, VMD, in 1998 at the University of Wisconsin. These ground-breaking studies built on roughly 20 years of experience using mouse embryonic stem cells in research studies. In the same year, the second method for generating human pluripotent stem cell lines was discovered in the laboratory of John Gearhart, PhD, at The Johns Hopkins Medical School. They reported the generation of human pluripotent cell lines by isolating specialized cells known as primordial germ cells from a 5- to 7-week-old embryo and placing these cells into culture (Fig. 2). Primordial germ cells are destined to become either oocytes or sperm cells, depending on the sex of the developing embryo. The resulting cell lines are called embryonic germ cell lines, and they share many of the same properties as embryonic stem cells. Defining the similarities and differences between these two types of human pluripotent cells is an active area of research.
The third potential method to generate human embryonic stem cell lines is a process called somatic cell nuclear transfer, or SCNT (Fig. 2). This procedure is also referred to as therapeutic cloning, a term that causes confusion given the multiple uses of the word “cloning.” Cloning refers to making an identical copy of anything—a molecule, cell, or animal. In this procedure, scientists begin by harvesting human oocytes from a female volunteer donor—typically a young woman. This harvesting procedure is not completely benign and carries risks to the donor because drugs that stimulate the production of more than one oocyte during a woman's menstrual cycle are usually administered before the harvest procedure. The cell nucleus from the donated oocyte is gently removed and replaced by a nucleus from another somatic cell, an adult cell from elsewhere in the body, growing in culture—hence the name somatic cell nuclear transfer. The oocyte with the newly transferred nucleus is stimulated to develop through a process called parthenogenesis—which is defined as development without the joining of an egg and sperm. Development can only progress if the transplanted nucleus—which came from a differentiated cell—is returned to the pluripotent state by factors found in the oocyte cytoplasm. This alteration in the state of the chromatin in the nucleus is called nuclear reprogramming; understanding the molecular mechanisms that facilitate this process is another active area of research.
When parthenogenesis progresses to the blastocyst stage, the inner cell mass is removed and placed into culture in an attempt to establish a pluripotent stem cell line. Embryos generated by SCNT have successfully produced pluripotent cell lines in mice that appear to behave exactly the same as pluripotent cell lines generated from mouse blastocysts created by IVF. At this time, there are no published reports in which human embryos generated by SCNT have been used to generate a pluripotent cell line, although scientists worldwide are actively pursuing this area of research.
Why are scientists interested in using embryos generated by SCNT to create pluripotent cell lines? The nuclear genes of such a pluripotent cell line will be identical to the genes in the donor nucleus. If such a nucleus came from a cell that carries a gene mutation underlying a human genetic disease such as Huntington's disease, then all cells derived from the pluripotent cell line would carry this mutation. Cellular models of human genetic disease could be developed with this procedure, both to explore the underlying biology of disease and to develop drugs to slow or halt disease progression. Alternatively, if the cell providing the donor nucleus comes from a specific patient, all cells derived from the resulting pluripotent cell line would be a genetic match to the patient with respect to the nuclear genome. If these cells were used in transplantation therapy, the likelihood that the patient's immune system would recognize the transplanted cells as foreign and initiate tissue rejection would be reduced. However, because mitochondria also contain DNA, the donor oocyte will be the source of the mitochondrial genome, which is likely to carry mitochondrial gene differences from the patient, which may still lead to tissue rejection.
WHAT IS THE STATE OF THE SCIENCE OF HUMAN PLURIPOTENT CELLS?
The research community is currently in the stage of fundamental discovery, or the basic science phase of understanding the properties of human pluripotent cells. Researchers are investigating how to direct these cells to differentiate into specialized cell types of interest for research, and are also making attempts at drug discovery and transplantation therapy (Fig. 3). A more complete understanding of the molecular mechanisms that determine the differentiated state of cells derived from pluripotent cells will be necessary before clinical applications are pursued. Scientists will need to pilot experimental transplantation therapies in animal model systems to assess the safety and long-term stable functioning of transplanted cells. In particular, care needs to be taken to ensure that any transplanted cells do not continue to self-renew in an unregulated manner after transplantation, which may result in a teratoma, or stem cell tumor. In addition, scientists will need to make sure that cells transplanted into a patient are not recognized as foreign by the patient's immune system and rejected. Currently, no clinical trials are being conducted in patients using cells generated by differentiating human embryonic stem cells, although scientists are hopeful that such trials will commence in the not-too-distant future. Human pluripotent cells derived from other sources, such as the umbilical cord and bone marrow are currently being used to treat patients with a variety of disorders that require replacement of cells made by the bone marrow, including Fanconi's anemia and chemotherapy-induced bone marrow failure after cancer treatment.
WHAT FEDERAL LAWS AND POLICIES AFFECT HUMAN EMBRYONIC STEM CELL RESEARCH IN THE UNITED STATES?
There is no Federal law that limits the scope of research involving human embryos and embryonic stem cell research. For example, no law at the Federal level prohibits attempts at cloning humans for reproductive purposes using SCNT—an activity that many individuals believe is morally repugnant because SCNT in other mammals often leads to a failure of the embryos to develop into a normal fetus. Several states have adopted laws that limit the scope of research within their borders, and several states have passed laws that provide state-based support for stem cell research.
However, limits have been placed on research activities that can be funded by the Federal Government. On August 9, 2001, in his first televised nationally televised address, President Bush set forth his policy placing limits on the use of Federal funds for human embryonic stem cell research. The President announced that he would, for the first time, allow the use of Federal funds for study of embryonic stem cell line so long as before his announcement: (1) the derivation process (which commences with the removal of the inner cell mass from the blastocyst) had already been initiated; and (2) the embryo from which the stem cell line was derived no longer had the possibility of development as a human being. In addition, the President indicated that these additional conditions must be met: (1) the stem cells must have been derived from an embryo that was created for reproductive purposes; (2) the embryo was no longer needed for these purposes; (3) informed consent was obtained for the donation of the embryo; and (4) no financial inducements were provided for donation of the embryo. The President's policy does not pertain to human embryonic germ cell lines, whereby the pluripotent cells are derived from the primordial germ cells from a 5- to 7-week fetus rather than a human embryo.
To facilitate research eligible to receive Federal funding, the NIH created the Human Embryonic Stem Cell Registry, which lists all human embryonic stem cell lines—at varying stages of development—that meet the eligibility criteria. Seventy-eight such derivations were subsequently located at private institutions around the world, seven of which are duplicates located at Geron, a biotechnology company, and WiCell, a nonprofit research subsidiary of the Wisconsin Alumni Research Fund. Because all 78 derivations are owned by private entities, the owners are under no obligation to provide human embryonic stem cells to the research community and NIH has no authority to insist that this limited resource be shared. In addition, because some of the derivations are little more than a frozen inner cell mass, there is no guarantee that the cells will propagate as a cell line when thawed.
The scale-up and characterization effort required to distribute cell lines is both time- and resource-intensive (Fig. 4). In an effort to maximize the availability of cell lines eligible for Federal funding to the research community, NIH provided support through Infrastructure Grant Awards to allow private institutions with derivations on the Registry to prepare, expand, and characterize cell lines for responsible distribution to the community. Of the 15 private entities that own the 78 derivations eligible for Federal funding, 9 have applied for and received NIH Infrastructure Awards with the intent of generating distribution-ready human embryonic stem cell lines from their derivations. These nine private entities control 40 of the 78 derivations eligible for Federal funding. Of these 40 derivations, 16 have failed to expand into pluripotent, self-renewing human embryonic stem cell lines, and one derivation was withdrawn by the donors. At the present time, 21 cell lines have been scaled up and characterized to the point at which they can be distributed to the research community for human embryonic stem cell research supported by Federal funding.
A second limitation was placed on the use of NIH budget authority by the Legislative Branch of the Federal Government for research that involves human embryos. Beginning in 1996 and every year thereafter, the Human Embryo Research Ban (also called the Dickey Amendment) to the Department of Health and Human Services (DHHS) annual Appropriation Act prohibits the use of funds appropriated to DHHS to support the creation of a human embryo for research purposes or research in which a human embryo is destroyed, discarded, or subjected to risk of injury or death greater than that allowed under Federal requirements for fetuses in utero. For the purposes of this prohibition, the definition of a human embryo is very broad, including embryos generated by parthenogenesis. Because NIH budget authority falls under the DHHS appropriation, NIH funds cannot be used to create a new human embryonic stem cell line, because a human embryo created by either IVF or SCNT would be destroyed in the process.
Policies and laws in other countries are sometimes more permissive and other times more restrictive than in the United States, depending on the country in question. This complex issue is beyond the scope of this discussion, although it is fair to note that the United States is unique in having a policy that restricts activities funded by the Federal government but places no restrictions on research funded by other sources.
WHAT ARE RATE-LIMITING STEPS FOR MAKING PROGRESS IN HUMAN EMBRYONIC STEM CELL RESEARCH?
It is extremely difficult to grow human embryonic stem cell lines in culture. The cells require highly specialized growth media. In addition, most human embryonic stem cell lines are grown in the presence of a feeder cell line, a layer of cells from a mouse or human source on which stem cells can grow and obtain nutrients but which has been treated so the feeder cells cannot divide. Proper preparation of the feeder cells is essential for successful culture conditions. Human embryonic stem cell lines used as source of cells for transplantation therapies should be propagated on a human feeder cell layer to reduce the risk of harmful mouse viruses present in mouse feeder cell are transferred to the human stem cells. Human embryonic stem cell cultures must be expanded using an exacting protocol, or the cells will either die or begin to differentiate spontaneously and lose pluripotency and self-renewal properties. Because only a few laboratories in the United States are growing these cells, there is a shortage of people well-versed in the art and science of successful human embryonic stem cell culture. To expand this rate-limiting human resource, NIH offers training grants for institutions to provide hands-on training in the techniques needed to culture human embryonic stem cells. In 2003, five such courses were established, and approximately 200 scientists were trained. In 2006, the number of courses was increased to seven, and NIH plans to continue to support this activity as long as the demand is evident. In addition, the NIH is supporting training in many independent laboratories funded to perform investigator-initiated human embryonic stem cell research.
There is a compelling need for simplified, cost-effective, and uniform cell culture conditions that will support the growth and pluripotency of most if not all human embryonic stem cell cultures. Optimally, these conditions would eliminate the need for the feeder cell layer and in its place, provide purified stocks of necessary growth factors. Feeder cells add more steps to cell culture protocols, and may be problematic if an undesirable biological agent or molecule is unwittingly transmitted from the feeder layer to the cultured human embryonic stem cells. This issue could result in additional safety concerns on the part of the U.S. Food and Drug Administration when the first clinical trials involving transplantation of cells differentiated from human embryonic stem cells are proposed. The NIH is supporting efforts at several different institutions to establish culture conditions using only well-defined components, and progress toward eliminating the need for feeder cells either to establish or propagate human embryonic stem cell lines has been reported at the University of Wisconsin. It will be essential to determine whether the protocols for culture developed at Wisconsin can be simplified further or rendered less costly, because it requires the addition of purified growth factors that are very expensive. In addition, the cells must be monitored for genetic stability, sustained pluripotency, and continuous self-renewal over many passages in the new culture conditions.
Availability of human embryonic stem cell lines is another potential impediment to research progress. NIH-supported Infrastructure Grant Awards have resulted in the generation of 21 human embryonic stem cell lines that are eligible for Federal funding and are ready to be shipped to investigators. However, these cell lines are scattered among a variety of different providers, each specifying different requirements to be satisfied before shipment. In addition, a $5,000 licensing fee must be paid by all not-for-profit entities who request a human embryonic stem cell line, because the intellectual property for derivation of human embryonic stem cell lines is currently held by WiCell, a biotechnology spin-off company started by the Wisconsin Alumni Research Fund. In an effort to consolidate as many of the 21 available lines as possible to one location and standardize quality control, NIH initiated a research and development contract to fund a National Stem Cell Bank at WiCell, after the completion of a national competition. The hope is that this step will simplify efforts on the part of the research community to obtain human embryonic stem cells for research.
In the short-term, some of the challenges include the development of more robust culture conditions and protocols, understanding the molecular mechanisms that direct differentiation into specific cell types, and developing the human infrastructure to advance this exciting new scientific opportunity. Once these challenges have been met, transplantation studies in animal models (rodent and nonhuman primates) that are shown to be effective and of long-term benefit will need to be performed before the research challenge moves into clinical trials. The risks and benefits of transplantation therapies will need to be very carefully considered, as these interventions represent a lifelong experiment with unknown consequences. Although it is clear that transplantation-based therapies using human embryonic stem cells are far from imminent, we can never know the full potential of these remarkable cells unless we begin the journey.
Research opportunities and advances, as well as links to other information about stem cell research can be found on the NIH Stem Cell Information Web site: stemcells.nih.gov.