|Vol. 7, No. 1 / February 2009
When will there be a cure for…?
The future of embryonic stem cell research
Federally funded research using embryonic stem cells is limited to the 22 stem cell lines created before August 9, 2001.
Fetal and adult stem cells are more abundant and less controversial; therefore they are the most widely used type.
Clinicians are a direct conduit to the patients who donate embryos and eggs to research.
Embryonic stem cells hold promise for treatment of neurodegenerative disorders, spinal injuries, and metabolic and blood diseases, among others.
President, California Institute for Regenerative Medicine, San Francisco, California
Having struggled with infertility for several years, Mr and Mrs D are about to begin in vitro fertilization (IVF). Of the 7 eggs that are successfully harvested and fertilized in vitro, 2 will be transferred at the blastocyst stage and the rest will be frozen. Anticipating that there may be frozen blastocysts remaining after IVF if the treatment is successful, you begin a discussion with the couple about their options. After asking Mr and Mrs D how many children they hope to have, you present them with the possibilities: discard any remaining blastocysts, donate them to other couples, or donate them for research. They are curious about how the blastocysts could contribute to research and ask you for more information.Use of embryonic stem cells (ESCs) will undoubtedly have a profound effect on medicine. Clinical trials in spinal cord repair to treat paraplegia are about to begin, and promising developments are occurring in retinal epithelial repair, regeneration of pancreatic beta cells, and treatment of myocardial infarction, among others. Considerable funding and efforts are going into worldwide research in order to bring the discoveries from the lab into the clinic.Clinicians and patients have an important role in advancing regenerative medicine. Donations from a couple that has successfully undergone assisted reproduction and completed childbearing contribute to research that could have a major impact in the treatment of more than 70 diseases and injuries. The state of the science is reviewed here, along with suggestions for clinicians and patients who wish to contribute to the field.
DISCLOSURE: Dr Trounson reports that he is a shareholder and scientific director of Monash IVF and a shareholder of Sydney IVF.
Stem cell science
Stem cells vary in their ability to develop into specialized cell types. ESCs are the most plastic, and various techniques are used to manipulate them into any of the cell types found in adults. However, federally funded research using ESCs is limited to the 22 stem cell lines created before August 9, 2001.
Fetal and adult stem cells are more restricted in their ability to specialize but are also more abundant and less controversial; therefore, they are the most widely used type of stem cells.
Not all stem cells are embryonic
ESCs are pluripotent, that is, they are in the most basic state and, by nature, can form all the cells of the body. This category of stem cells encompasses a single-cell egg, cells from a blastocyst, and embryonic germ cells taken from an embryo at 8 to 12 weeks. With single-cell eggs, it is possible to induce parthenogenesis in the human
and, via nuclear transfer, create pluripotent stem cells in an animal model.
The pluripotentiality of ESCs is demonstrated by placing them by transplantation in vivo, such as under the kidney or testes capsules, and observing the formation of a teratoma containing a variety of mixed tissue types, including glandular epithelium, neurons, bone, muscle, or even lung tissue.
In contrast, adult stem cells are multipotent and cannot develop into as many different cell types. Fetal stem cells are considered adult stem cells in this regard because they also lack the flexibility of ESCs. Hematopoietic cells, the best known stem cells, are an adult type that are currently being used in bone marrow transplantation because of their ability to form the full gamut of blood cells. Neural stem cells are derived from adult stem cells and have been used in the treatment of Batten disease, a genetic lysosomal storage disease that causes neurologic damage in children.
Cord blood and placental stem cells, also part of this category, are used widely for immune and blood disorders.
Recently, researchers have caused specialized adult skin cells to differentiate by exposing them to specific transcription factors, forming induced pluripotent stem (iPS) cells.
The ability of cells to drive specialized cells to a pluripotent or multipotent state or even to another committed cell state holds great promise in the future of regenerative medicine.
Embryonic stem cell growth and differentiation
Embryonic stem cells are usually formed by isolating the inner mass of cells in a blastocyst and cultivating this into a colony of undifferentiated cells; alternatively, cells can be derived from morulae or intact blastocysts from which the zona pellucida has been chemically removed.
A 50% success rate for establishing a stem cell line from embryos is considered very high but has been achieved.
Production of ESCs is favored by selecting euploid embryos free of chromosomal abnormalities, as determined by preimplantation genetic diagnosis. There is little difference in the ability to form ESC lines based on the stage of preimplantation embryos.
ESCs are maintained on feeder cells, such as murine or human fetal fibroblasts, or human endometrium, foreskin fibroblasts, bone marrow cells, or differentiated ESCs. This environment provides the growth factors necessary for ESC renewal; however, it also has the potential to induce differentiation or alter the chromosomal karyotype. Currently, the goal is to optimize culturing procedures that use sterilized extracellular matrices and serum-free media and that do not involve animal reagents.
Barring changes in the chromosomal karyotype, ESCs can be maintained continuously in the laboratory for at least 10 years, even though staining for surface markers indicates that within 1 week of culturing, a mass of ESCs is nonhomogeneous.
For differentiation, ESCs can be cultured as flat (single-cell layer attached to the culture dish) or aggregated embryoid bodies as unattached spherical structures, which are grown in hanging drops or forced aggregation.
Embryoid bodies contain a wider variety of cell types and can be driven toward specific lineages by activating endogenous transcription factors or virally introducing new transcription factors; exposing the ESCs to particular growth factors or antagonists; or co-culturing ESCs with cell types that induce a specific lineage.
The mass of differentiated cells may be filtered by flow cytometry and the cells of interest purified. Neurons have been produced by exposing ESCs to growth factors and antagonists.
Co-culturing has also been used to produce ESC-derived neurons, as well as cardiomyocytes, pneumocytes, keratinocytes, and hematopoietic cells.
Such approaches have potential for generating tissue that will be transplanted into patients to cure degenerative diseases.
ESC research: What to expect in the clinic
The translation of stem cell research into clinical practice will be driven by biotechnology companies that usher the science through the development pipeline to obtain regulatory approval for clinical trials, and by clinicians who are able to gain hospital ethics committee support for early clinical trials of these cells to prove their usefulness for patients. In addition to their use in treatment of disease and tissue repair (
), ESCs have the potential to accelerate basic medical research and drug development.
Spinal cord repair
The FDA has received a proposal for a phase 1 clinical trial of the use of human ESC-derived neural progenitors for spinal cord repair. These investigators are seeking to use oligodendrocytes derived from ESCs to remyelinate damaged neurons in patients with low lumbar injuries. This approach is based on data from an animal model that show enhanced remyelination and improved mobility when ESC-derived oligodendrocytes are transplanted shortly after a spinal cord injury.
Although cervical injuries are more common, investigators believe it is safer to begin in the lower region of the spinal column for the first safety study.
In the area of diabetes, a partnership between a biotechnology firm and a number of academic institutions has produced pancreatic beta islet cells by deriving endoderm-type cells.
These investigators pushed the embryoid body into a endoderm lineage. They selected for the cells under careful conditions, producing glucose-responsive endocrine cells that function in hyperglycemic mice. Initial problems with teratoma formation have been resolved by filtering out undifferentiated cells. Currently, this group is investigating ways to overcome the autoimmune dysfunction that destroys insulin-producing cells in type 1 diabetes, so that the ESC-derived beta cells can survive.
All of the cells needed for cardiac repair after a myocardial infarction have been produced from ESCs, including pacemaker cells, fiber atrial cells, sinus cells, and left ventricular cells.
Placed in the heart of rat models, ESC-derived cardiomyocytes connect to the existing cells, forming tight junctions. When an infarction is induced in the rat, there is an improvement in the animal; however, the improvement is small and the benefits are transient.
Most recently, engineers have begun to develop fiber scaffolds that encourage the growth of functional myocytes. Early attempts have successfully grown muscle tissue that could be transplanted as a patch.
Researchers hope to be able to restore sight by generating ESC-derived pigmented retinal epithelial cells. Initially, cells grown in culture were injected into the eye in a rat model. The rats became responsive to light but remained unable to see structure.
Using matrix and membrane culture techniques similar to that used for cardiomyocytes, researchers have developed single-layer patches of retinal epithelial cells in the rat model that may be able restore the ability to see structure.
Respiratory diseases frequently cause death when patients essentially drown due to the amount of fluid in their lungs. If it were possible to stop the inflammatory processes that produce this excess of fluid, many lives could be saved. This is an opportunity to use the bone marrow mesenchymal stem cells, cord blood cells, and placental amniocytes to prevent the anti-inflammatory conditions for respiratory disease progression.
Current cancer drugs target rapidly dividing cells, but the apparent rare quiescent, drug-resistant cells are just as dangerous. Quiescent cells are thought to cause cancer recurrence and metastases, and they represent a new target for cancer treatment. Current ESC research is focused on understanding how a normal cell maintains its proliferative ability and loses its capacity for apoptosis, senescence, and differentiation. Researchers hope that by creating quiescent cancer cells from ESCs, they will understand more about the cellular process of moving from quiescence to proliferation and thus how to target these cancer cells.
understanding genetic disorders
ESCs hold great promise for understanding many aspects of genetic disorders. Researchers have generated lines of ESCs with the mutations for cystic fibrosis and Huntington’s disease, among others. Neurons and glia have been derived from ESCs with Huntington’s disease trinucleotide expansion. Using these cells, it is possible to produce the polyQ-expanded huntingtin protein that is toxic to neurons, especially the medium spiny neurons of the striatum, and potentially drive the accumulation of aggregating proteins characteristic of Huntington’s disease in a laboratory dish. The capacity to recreate these conditions facilitates the search for drugs that may slow the disease progression. Such drugs might significantly prolong the healthy years of life for an individual with a Huntington’s disease mutation.
Another example of the use of stem cells to elucidate the mechanisms and treatment of a genetic disorder is the case of polycythemia vera, a myeloproliferative disorder that causes the overproduction of red blood cells and acute leukemia in most patients.
Drug screening and development
Use of ESCs will improve understanding of the causes and cures of a range of diseases. Furthermore, the ability to screen drugs using ESCs will reduce costs and the need for animals as test subjects. In neurologic diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and glioblastoma, unaffected neurons developed from normal ESCs can be exposed to candidate drugs and observed for the disease phenotype. If the disease phenotype occurs, it provides information about the development of these disorders. This type of experimentation also creates an opportunity to find molecules that inhibit the disease. The pharmaceutical industry is already using ESCs to identify the chemical composition of the small molecules needed for treatments, reducing the need for animal testing. Environmental toxicology tests conducted in rodents can be replaced with tests of toxicity in ESC-derived human hepatocytes or cardiac cells.
The clinic and the lab: A critical partnership
Clinicians are a direct conduit to the patients who donate embryos and eggs to research. By talking with patients about donating to stem cell research, they can become aware of their potential role in contributing to important developments in regenerative medicine.
The possibility of donating to research can be mentioned in the context of a discussion about how many children the couple desires and what they wish to do if there are excess embryos or eggs. It is most practical for patients to choose among the 3 options—destruction, donation to another couple, or donation to research—before the eggs or embryos are frozen. Patients’ questions about these options can also be addressed at this time. While they are undergoing fertility treatments, people are generally thoughtful about the process and the possibilities. However, once a couple has left the clinic, it is often difficult to locate them and obtain the necessary consent forms.
The majority of patients are positive about the possibility of contributing to research. It can be useful to discuss the state of the research, particularly if the patient has a family member who is affected by diseases or injuries currently under investigation.
Stem cell research is being conducted across the country, and particularly in California. Proposition 71, passed in California in 2005, approved the use of state funds distributed by the California Institute of Regenerative Medicine for ESC research at California institutions. Twelve major research institutes are under constructions, and millions of grant dollars have been awarded to date. Other hot spots for stem cell research include Massachusetts, New York, and New Jersey, where it is especially easy for clinicians to collaborate with researchers at nearby institutions.
ESCs hold promise for treatment of neurodegenerative disorders, spinal injuries, and metabolic and blood diseases, among others. Further down the road, it may be possible to treat sterility by repairing the hypothalamus and pituitary gland with ESCs and replenishing the germ cells in the testes and ovary with the patient’s own cells reprogrammed by transduction with transcription factors used to form iPS cells. The use of a clinician’s time to talk to patients about donation and consent can be a huge contribution to the field of regenerative medicine.
CASE STUDY CONCLUSION
You explain the importance of ESCs in regenerative medicine and describe the potential applications in the treatment of a variety of diseases and injuries. Mr D’s nephew was recently diagnosed with type 1 diabetes, and he is particularly excited to learn about research developments in that area. Mr and Mrs D decide to donate any remaining embryos after they have completed IVF. You present them with the paperwork that they need to complete to provide their informed consent.
A year later, Mr and Mrs D, the new parents of beautiful twin girls, send you the paperwork to release the remaining embryos for ESC research.
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