is a freelance medical writer in Mamaroneck, NY.
Embryonic stem cells are isolated from developing organisms between the time of fertilization and 8 weeks of development. As primitive, undifferentiated cells, they are pluripotent, having the potential to develop into most, if not all, cell types found in the adult. Michael Rudnicki Professor, University of Ottawa, and Scientific Director of the Canadian Stem Cell Network, Ottawa, Canada, point out that embryonic stem cell lines derived from embryonic stem cells have a finite life in culture. Other problems with many existing lines include abnormal karyotypes, resulting from accumulating chromosomal abnormalities, and the presence of animal proteins in those grown with feeder layers. “World-wide, there are fewer than 100 lines that have been developed, and the broad human diversity has not been explored. It's naïve and defies logic why grandfathered lines are ok, and new ones are not. Morally and ethically, we have an obligation to use embryos created by in vitro fertilization (IVF) that would be discarded. It's like organ donation,” he concludes.
Stem cells can also be created by transfer of the nucleus from a differentiated somatic cell into an enucleated ovum. While this has been demonstrated for some animal species, it still seems to be theoretical for humans. Stem cells from bone marrow and peripheral blood have been used for decades to treat hematologic malignancies. Adult stem cells are now known to exist in many other tissues, although most are multipotent, rather than pluripotent, and therefore capable of giving rise to a limited number of cell types related to their tissue of origin. Umbilical cord blood also contains multipotent stem cells.Gene Therapy
Cynthia Dunbar, Head, Molecular Hematopoiesis Section, National Heart Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD, studies hematopoiesis and gene transfer into primary hematopoietic cells. “For all types of gene therapy, it's the best of times and the worst of times,” she observes. “The good news is that proof-of-principle has been achieved that it is possible to ameliorate or cure genetically based immunodeficiencies and that vectors for gene transfer, as well as conditioning regimens for engraftment, have improved.” Unfortunately, integrating vectors can turn on oncogenes and cause cancer, most likely by insertional mutagenesis.
Dunbar believes that her group's use of nonhuman primate models of viral vector-based gene therapy, unlike murine models or in vitro human hematopoietic assays, is absolutely predictive of what happens in humans. They identify insertion sites of retroviral and lentiviral vectors using linear amplification-mediated PCR (LAM-PCR). However, the cancer-causing potential of many genes at insertion sites remains to be determined, nor does this technique identify genes at distant sites that might be differentially promoted by an insertion. Although lentiviral vectors appear to confer less of a risk for tumors than retroviral vectors, other gene transfer systems may prove more useful. These include bacteriophage-based vectors that target pseudobacterial sites in the human genome or the use of modified zinc finger DNA-binding proteins to target specific integration sites. “It's hard to get genes into hematopoietic stem cells without a viral vector,” Dunbar says. “We need to be able to get them into primitive cells without killing them. Electroporation and chemical methods that look good for cell lines don't work as well with stem cells.” Dunbar predicts that within the next 2 to 3 years, gene therapy trails to treat sickle cell anemia and other globinchain related diseases (e.g., thalassemia) will begin. It will be necessary for some of these diseases not only to provide normal hemoglobin chains, but also to knock down production of abnormal chains, perhaps with small interfering RNA (siRNA). Therapy based on recombination of homologous gene sequences is probably 10 to 15 years away, Dunbar predicts. “I have tried to think of myself all along as someone studying hematopoiesis. You need to know the target population to cure the disease,” Dunbar says.Image 1.
Elaine Fuchs, Professor, Laboratory of Mammalian Cell Biology and Development, Rockefeller University, New York, NY, is trying to understand how the multipotent stem cells in mammalian skin develop into epidermis, hair follicles, and sebaceous and sweat glands. Skin is an ideal model system, because it and its associated structures are readily accessible, and because human and murine skin stem cells are easily grown in culture. Her group is addressing the relationship between adult and embryonic skin stem cells and trying to identify the unique genes in embryonic skin stem cells that keep them in a pluripotent state. These genes seem not to be expressed in adult skin stem cells, yet the adult cells retain some ability to regenerate skin and skin structures, as can be seen when skin heals after injury and in the normal cycling of hair follicles. Fuchs hopes to answer “where the cells come from, where they are sequestered, how the animal knows later in life that it will need cells to repair and undergo the normal homeostasis of skin.” If it were possible to expand the repertoire of the adult stem cells more complete repair of skin after cancer surgery or severe burns might be possible, she speculates. Currently, the skin resulting after burn operations does not make hair or sweat glands because the stem cells in grafted epidermis are unipotent, Fuch's group has shown that hair follicles contain a reservoir of multipotent cells that can not only replenish skin but also make sweat glands and hair, “In the future, those cells could generate better skin in burns. Although other uses include hair replacement, I didn't go into science to solve that problem.” she says. There are lots of similarities among adult stem cells in different tissues, including skin, bone marrow, and intestine. “The parallels are quite striking,” notes Fuchs, pointing out that these stem cells can give rise to a variety of cell types, but that these remain tissue-specific. “Everyone agrees that signaling pathways play an important role in activation and proliferation. These pathways specify whether you get follicles of sweat glands, T or B cells, or intestinal epithelium.” “However, what is not known is how those signals are orchestrated to stimulated the proper number of the appropriate cell types in a correctly organized structure and the mechanisms underlying how things go wrong and result in aberrant growth or malignancy.Image 2.
"Without question, the hype in the early years has attenuated down to the serious business of describing the cellular molecular biological mechanisms in stem cells,” Rudnicki says. His work focuses on embryonic myogenesis and the function of stem cells in adult skeletal muscle, including identification of transcription and signaling factors. “We're very much interested in regulatory networks and are beginning to understand extrinsic factors and the role of epigenetic factors,” he says. “We have the molecular genetic tools to address how satellite cells in muscle, which may be either stem cells or progenitors, daughters of stem cells that are committed in some way, make of repair muscle.” It's conceivable, he says, to design drugs to stimulate intrinsic repair, for example in muscular dystrophy, and eventually turn a lethal disease into a chronic one. Regenerative medicine will really arrive, Rudnicki believes, when there is a pharmacopoeia of small molecular drugs to target cells, and that this will be achieved by studying all cell types. “It's a really exciting frontier between genomics and genetics. Technologies, including high-throughput mass spectroscopy and new sequencing platforms, will help move human biology forward in a huge way,” he says. Having the ability to sequence an entire genome for $1000 will do away with expression profiling on chips, Rudnicki believes. He looks forward to a map of stem cell regulatory networks that will allow visualization of the cell as whole system. “We will all become systems biologists,” he predicts.Ongoing Issues
Increased knowledge about how to isolate and propagate pure populations of various types of stem cells has been a major advance. However, Fuchs thinks that increased funding of basic science is required to advance the field. “The U.S was the leader in biomedical research, and we have lost the lead in the recent past,” she observes. “All basic science is hurting, and stem cell biology, like every other field of medicine, depends basic science.”
John A, Kessler, Professor and Chair, Interdepartmental Neuroscience Program, Northwestern University, Chicago, IL, believes that stem cell research will “ultimately revolutionize how we practice medicine. In the past, we treated symptoms of disease. If an organ was damaged, there was little we could do to repair it. The excitement,” he says, “is that we are just learning how to make cells in the pancreas, heart, and nervous system. Soon we will be able to think about ways to develop therapies.” Kessler frequently gives talks about stem cell research. “It's absolutely essential for people to be educated. The anti-intellectuals won't stop me or my colleagues. I would rather be doing science than talking, but if we don't who will?”