Embryonic stem Cells

SCIENTIFIC SUMMARY

Ground-breaking research, particularly in the biomedical fields, is often associated with controversy, both from within and without the scientific community; at times, scientists face outright opposition to their work. A topic that has recently raised public concerns—among religious leaders, politicians, celebrity spokespersons, and many others—is research on embryonic stem cells (ESC), primitive cells found in the early embryo that can differentiate into any adult tissue. Although the great promise of ESC therapy is widely accepted, vociferous debate continues as to the ethical validity of ESC harvesting, which presently cannot be done without destroying a potentially viable human embryo. This summary will not attempt to address directly the important ethical issues surrounding ESC research, but rather will outline current understanding of ESC development and progress towards ESC therapy.

Definition

All types of stem cells are capable of asymmetric division, in which a parent cell divides into one daughter cell identical to the parent and a second daughter cell capable of further differentiation. The identical daughter cell contributes to the self-renewing population of stem cells, while the second daughter differentiates along a given lineage to form cells of a particular tissue. These differentiated cells lose proliferation ability and cannot regenerate the tissue in absence of the self-renewing stem cells. An example of such terminally-differentiated cells is a neuron, which has minimal capability of self-repair and often dies if damaged. Most organs in the adult body contain a population of stem cells that serve as sources of cell replacement throughout life. Red blood cells, for example, are constantly replenished from hematopoietic stem cells residing in the adult bone marrow. 

Adult stem cells are constrained to produce cells along given lineages—such as keratinocytes for epidermal stem cells and gametocytes for germ stem cells—within just one of the three embryonic germ layers. Embryonic stem cells, however, can differentiate into cells of any lineage within any germ layer, with the sole exception of the trophoblast-derived placenta. Adult stem cells are termed multipotent for their ability to differentiate along several lineages, whereas the more primitive ESC are called pluripotent for being able to recreate tissues from all three germ layers. Pluripotency is what causes such great enthusiasm for ESC; if properly manipulated, these cells should be able to grow or regenerate any organ in the adult body.

Identification

After fertilization with a sperm, the oocyte begins dividing rapidly. The cells formed during these initial divisions are totipotent, because they will give rise to the placenta as well as the three germ layers. At day five post-fertilization, the embryo is a blastocyst, which consists of an outer layer of cells called the trophoblast surrounding a hollow cavity (blastocoel) and an inner cell mass.

The trophoblast will develop into the placenta, while the inner cell mass will eventually form the three germ layers—ectoderm, mesoderm, and endoderm—and therefore all the organs of the adult body. Researchers studying ESC obtain them by harvesting the inner cell mass of the blastocyst, a technique first performed for human ESC by Dr. James Thompson at University of Wisconsin (Thomson, Itskovitz-Eldor et al. 1998).

A powerful way to demonstrate that a population contains ESC is to show that the cells can differentiate into any other cells. One technique is transplantation of putative ESC into severe-combined immunodeficient (SCID) mice, which have no ability to reject the foreign cells. In a SCID mouse, ESC grow a teratoma—a tumor containing tissues of all three germ layers—as a result of a Ras-like gene expressed in these primitive cells (Takahashi, Mitsui et al. 2003).

Differentiation

The pluripotency of ESC was first studied in the mouse. ESC were isolated and then injected into the blastocyst of a genetically distinct mouse (Edwards 2004). The resulting chimera had donor cells in all tissues of the adult mouse, in tissues from all three germ layers. Although murine ESC and human ESC share functional properties, recent studies have shown that ESC from the two species express different markers and use different pathways to regulate differentiation (Ginis, Luo et al. 2004). Therefore, caution must be taken in applying knowledge learned from mice to humans. Mice are still essential to ESC research, however, as SCID mice can be used as hosts for xenogeneic transplants.

One focus of ESC research is elucidation of the signals and pathways involved in differentiation to different lineages. Inducing differentiation in ESC is not difficult; rather, controlling differentiation towards a therapeutically desired target is a challenge. When cultured with the appropriate growth factors, ESC form embryoid bodies, clumps of cells that resemble an embryo (Chadwick, Wang et al. 2003). These embryoid bodies will differentiate seemingly at random (unless given carefully controlled signals) into all types of tissues from the three germ layers. Cell types that have been selectively grown from embryoid bodies include cardiomyocytes, hepatocytes, and neurons (Carpenter, Rosler et al. 2003).

The transcription factors Oct-4 and Nanog are reliable markers of pluripotency. An ESC can differentiate only after germ cell nuclear factor (GCNF) is expressed; GCNF represses Oct-4, thereby releasing from its suppression FoxD3, which initiates differentiation (Sato, Meijer et al. 2004). Another way that Oct-4 can be down-regulated—and therefore differentiation triggered—is if the oxygen content of the local environment rises (Guo, Einhorn et al. 2004). Like many ESC genes, Oct-4 is a putative pseudo-oncogene; it may be stimulated by Ewing’s sarcoma protein (Lee, Rhee et al. 2005). Similarly, Nanog acts as a “gatekeeper” to differentiation, maintaining the pluripotent state of the ESC (Hyslop, Stojkovic et al. 2005).

Interestingly, some aspects of the differentiation signaling pathways are nearly opposite for mice compared to humans. Bone morphogenic protein (BMP) appears to signal increased expression of Oct-4 and Nanog in murine ESC, but BMP is antagonistic to these anti-differentiation factors in human ESC (Suzuki, Raya et al. 2006). Great care must be taken in drawing conclusions about human ESC based on research on murine ESC.

Application
Given their pluripotency, ESC could potentially be used to regenerate any tissue in the body, even to grow entire organs. An immediate challenge is to find the right combination of culture conditions and signal molecules to grow functional tissue on demand. Other challenges include finding an efficient and ethically acceptable source of ESC, preventing host rejection of an ESC transplant, and stopping the growth of teratomas in transplant hosts.

Type 1 diabetes mellitus is an autoimmune disease in which loss of pancreatic β-islet cells leads to diminished glucose sensitivity and severe resulting morbidities. Current therapies include insulin injections, which must be done multiple times a day, and transplant of β-islet cells from cadavers, which lasts at most four years and requires immunosuppression (Rother and Harlan 2004). If ESC could be induced to make allogenic β-islet cells, then a new therapy would be available. Initial reports that insulin-producing cells were made from ESC (Lumelsky, Blondel et al. 2001) generated much media excitement, but the results were not able to be reproduced. Another research group later suggested that the cells had taken up insulin from the media, where it had been used as a growth factor, rather than synthesizing it themselves (Rajagopal, Anderson et al. 2003).

A more recent example of a scientific result received enthusiastically by the public came in 2006, shortly before the US Congress voted on a bill extending federal funding for ESC research. A research group investigating spinal injury in rats transplanted ESC-derived neurons and used a cocktail of enzymes and cytokines to grow the new axons towards the denervated muscle (Deshpande, Kim et al. 2006). The transplanted neuron successfully reached the muscle, and some slight movement of the limbs was observed, leading the authors to assert that ESC transplantation “represents a potential therapeutic strategy for patients with paralysis.” The study was widely pronounced as a breakthrough in the popular press, and although the claims of the authors were not technically misrepresented in the media, the sensationalism of the story was used by ESC advocates to justify passage of the ESC bill before Congress.

One of the prominent celebrity spokespersons advocating increased funding for ESC research during the 2006 US midterm elections was the actor Michael J. Fox (michaeljfox.org). Fox suffers from Parkinson disease, a neurodegenerative disorder with debilitating motor symptoms but a very specific pathology. The Parkinson patient loses neurons in the substantia nigra that secrete dopamine, so potential ESC therapy would involve generation and transplantation of dopaminergic neurons. A recent study in a primate model of the disease showed promise; dopaminergic neurons made from monkey ESC relieved Parkinsonian symptoms when transplanted into a monkey (Takagi, Takahashi et al. 2005). The investigators sidestepped the tendency of transplanted ESC to form teratomas by differentiating the ESC first to neurospheres—multipotent neural progenitors that lack the high tumorigenic potential of their ESC precursors—before transplantation and terminal differentiation.

Other adults cells produced recently from animal ESC include osteoblasts (zur Nieden, Kempka et al. 2003), cardiomyocytes (Schwanke, Wunderlich et al. 2006), and platelets (Fujimoto, Kohata et al. 2003). Clearly ESC therapy has potential, so all the obstacles in its way are being actively investigated.

One possible technique for avoiding tumor formation from ESC transplants is to identify and suppress the genes responsible for uncontrolled growth. The pseudogene HRasp, for example, appears to be the human orthologue to the murine ERas, which encodes a Ras-like protein that is expressed in rapidly growing ESC as well as some tumors (Takahashi, Mitsui et al. 2003). Perhaps such genes can be suppressed in such a way that the modified ESC retain potential for useful growth but lose the easy ability to form cancer. Another proposed tactic for preventing cancer is to genetically engineer donor ESC to express the herpes simplex virus thymidine kinase gene (Schuldiner, Itskovitz-Eldor et al. 2003). If the transplant grew a tumor, then it could be easily destroyed with the herpes anti-viral drug ganciclovir.

An additional challenge for ESC therapy is managing the host response to any foreign transplant; immune cells recognize MHC I molecules on donor cells as foreign and reject the graft. Some research suggests, however, that rejection may not be an issue for ESC. The cells and their differentiated progeny appear to have low immunostimulatory capacity despite expression of foreign MHC I (Li, Baroja et al. 2004). If this characteristic proves reliable upon continued study, then a major benefit of using ESC instead of adult organ transplants will be the lack of need of immunosuppressant therapy.

If allogeneic ESC turn out to be immunogenic after all, then autologous ESC could perhaps be generated for each patient using therapeutic cloning (Munsie, Michalska et al. 2000), also known by the less provocative name of somatic cell nuclear transfer. In therapeutic cloning, the nucleus of an adult cell (say, a skin cell) is inserted in an enucleated oocyte, which is then given appropriate signals to divide as if it had been fertilized. The resulting blastocyst contains ESC with DNA identical to the donor of the adult nucleus, so the ESC can be harvested and transplanted into the patient, hopefully, without fear of rejection. This technique has very low efficiency, however, and recent hope of better yields was dashed when a breakthrough paper out of South Korea was retracted (Chong and Normile 2006). More important than current technical limitations is the failure of cloning to address widespread ethical concerns surrounding ESC research and therapy.

The harvesting of ESC is perhaps the biggest challenge to the development of ESC therapy, because this issue deals with ethics and politics beyond the science. So far, the only techniques that have successfully harvested human ESC have involved destroying a blastocyst that otherwise might have matured into a fetus and eventually a human baby. One proposed solution is to make blastocysts incapable of forming a placenta and therefore completely unable to form a viable fetus (Meissner and Jaenisch 2006). The technique of altered nuclear transfer is similar to somatic cell nuclear transfer with the addition of siRNA to knock down the product of gene cdx2, the expression of which is required for differentiation into the placenta-forming trophoblasts. If the embryo created by cloning has no possible future as a viable fetus, supporters of altered nuclear transfer argue that a potential human life has not been destroyed.

Another new technique, which received popular attention, is based on diagnosis procedures used during in vitro fertilization. When a couple is at high risk for genetic disease, each embryo is genotyped before the expensive task of implantation is attempted. A single cell is removed from the 8-cell zygote for analysis, and this tiny loss does not seem to affect the health of the embryo. If this one cell could be expanded in vitro, then this routine biopsy could be used both to diagnose the zygote and to generate a new ESC line (Chung, Klimanskaya et al. 2006). Although the authors failed to preserve any of the embryos used in their study, they showed that the technique has promise.

A different solution to the harvesting problem is identification of another versatile stem cell type that is easier to access. The placenta is an embryonic organ discarded at birth, so the discovery of placenta-derived multipotent cells is encouraging (Yen, Huang et al. 2005). These cells displayed many of the same surface markers as ESC, and could be induced to differentiate into adipose, bone, and neural tissues. Another possibility is stem cells isolated from amniotic epithelial cells (Miki, Lehmann et al. 2005), which express many ESC surface markers and transcriptional factors, though not telomerase. If any such alternatives are found to have as much therapeutic potential as ESC, then the thorny ethical dilemmas can be sidestepped easily.

Availability
On 9 August 2001, President George W. Bush approved US federal funding for research on certain previously-derived ESC lines (escr.nih.gov). Not all ESC lines were approved, but only those derived from a donated embryo that was originally created for reproductive purposes, i.e., embryos created but unused for in vitro fertilization. In order to discourage the creation and destruction of more embryos, President Bush restricted funding only to those lines already in use as of his announcement, which at the time was estimated to be at least sixty viable lines. This decision was hailed as a great compromise between the practical need to fund promising science and the ethical imperative to prevent further destruction of embryos.

Unfortunately, most of the 2001 lines are now unusable for research or therapy; at most 21 viable lines remain (isscr.org). Since the lines approved for funding in 2001 were created early in the history of research on human ESC, they were not cultured under conditions now known to be ideal. The most prevalent problem is a result of the common practice of growing ESC in vitro on murine feeder cells. Many cell lines grown this way express the murine sialic acid Neu5AC on their surface, for which humans express antibodies (Martin, Muotri et al. 2005). Therefore, these cells will never be appropriate for therapy, because they will be quickly rejected by any host.

Newer human ESC lines are available that were not grown on animal feeder cells, but research on those lines cannot be connected in any way to US federal funds. State governments, other countries, and private organizations offer funding, but none can match the size of NIH grants, nor do non-NIH sources all require reporting of research results to the public.

Conclusion
Given the passion and tension permeating debates over ESC use, scientists have an especial responsibility to be informed well enough to teach the public about the exciting potential and realistic limitations of ESC research. The magnitude of the possible benefits from ESC therapy precludes a simple ban on all research, so scientists must work with the public to find ethically acceptable ways to move forward with ESC. At the same time, work on adult stem cells and possible ESC alternatives should be pursued with equal vigor, because the hurdles of ESC harvesting and use may prove too great to overcome without disrespecting the human life we all are trying to enrich.

Acknowledgements

This summary, written in 11/2006 by Tim Kreider, was based on a paper written by Olufunke Amele, Shreya Chakravarti, Colin Craig, Michael Ricardo, and Anthony Shoo in 11/2005. Figure 1 was created by the 2005 authors (TA: Kelly Corcoran). The summary was edited by Mariann Galdass and Nirmala Hariharan, and it was reviewed by Steve Greco and Dr. Pranela Rameshwar.

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