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Embyronic Stem Cells
A Scientific Review

Introduction
Ground-breaking scientific research is often associated with controversies and at times outright opposition. Scientists continuously work to improve the quality of life by trying to understand the mechanisms and pathology of disease and use this information to then create new drugs or treatments. However, there are those who feel that science can go too far and that certain functions must be left as nature intended. A prime example is the controversial issue of embryonic stem cell research, in which one finds a collision between supporters who see embryonics stem cells (ESC) as potential for curing disease and opponents who are concerned with the ethical issues surrounding the acquisition of these cells from an embryo. This review will not address ethical issues surrounding ESC research, but it is a brief review of the basic science of these cells and their potential use in cell therapy.

I. Background
Definition of a stem cell

A stem cell is defined as a cell capable of dividing to produce a one daughter cell that remains equivalent to the parent cell and a second daughter cell that differentiates into specific cell types (1). The former results in a population of cells termed self-renewing and the latter type results in cells that differentiate into a terminal differentiated specific cell. The differentiated cells formed via the development of an organism results in certain cellular populations that no longer replicate and the cells do replicate, the rate is slow. Examples of these cells are: neural cells, in which if damaged or if they undergo cell death, there is minimal self-repair mechanism. In some parts of the body, stem cells exist that serve as sources of cell replacement throughout life. For example, red blood cells, which have a half life in the body of 120 days (2), are replenished from the bone marrow-resident hematopoietic stem cells (1). Today, the majority of research studies involve studies on embryonic and adult stem cells.

Sources of ESC

Embryonic stem cells (ESC) are pluripotent cells found in the inner cell mass of the blastocyst derived from an embryo. Post-fertilization of an egg with a mature sperm, a structure called the blastocyst forms (Fig 1). A blastocyst consists of three structures: the inner cell mass, which contains the ESC that will form the embryo; the blastocoel; which is the hollow cavity inside the blastocyst; and the trophoblast which is the outer layer of cells that surround the blastocyst and later in development will form the placenta (3). The human ESCs used with federal funds in the USA have been left-over from in vitro fertilization (3).

Figure 1. Depicts the development of ESCs from the blastocyst and the fate of ESCs

Human ESCs were first isolated by Dr. James Thompson from the University of Wisconsin in 1998. They were removed from the inner cell mass of the blastocyst, an early stage of the embryo, four days post-conception (4). The inner cell mass contains the cells that have the potential to become cells of all three germ layers: ectoderm, mesoderm, and endoderm. The ectoderm is the outer most layer, the mesoderm is the middle layer, and the endoderm is the inner most layer of the gastrula. In turn, the three germ layers are the precursors to all the cells that eventually for a human. For instance, the mesoderm gives rise to the precursor cells, which eventually give rise to cells of the skin. The mere fact that the cells of the inner cell mass give rise to such a wide array of cell types via the aforementioned differentiation pathway makes them pluripotent (5).

History
On August 9, 2001, President George W. Bush passed a bill in which he approved federal funding for research if any of 60 ESC lines were used (6). He proposed that the government would fund research on these ESC lines if they met certain criteria. These criteria state that a) the stem cells must have been derived from an embryo created for reproductive purposes, b) the embryo was no longer needed for these purposes, c) informed consent must have been obtained for the donation of the embryo, d) no financial inducements were provided for donation of the embryo (6). According to President Bush, the cells lines that can be used legally cannot be from cloning. Therefore, the ESC lines that were derived from in vitro fertilization clinics had to be identical without manipulation.

The human ESC (hESC) lines that were approved for federal-funded research are mostly contaminated with animal products. This problem has arisen because the cells were propagated and passaged with feeder cells from mice. This caused murine chromosomes and proteins to integrate within the hESCs during expansion. Scientists are concerned that the approved hESCs have undergone mutations and chromosomal changes due to the murine feeder cells (7). The International Society for Stem Cell Research has reported that the research findings stating that sialic acid, Neu5AC, which is released by murine feeder cells, can be taken up by the human cells and expressed on the cell surface glycoproteins. This could lead to cell lysis since humans have natural antibodies that react with Neu5AC. The contamination of the hESC with the murine feeder cells raises concerns about the possible rejection of the cells after implantation. Despite these problems, there are currently 21 lines of ESC that are available for research studies (8).

Pluripotency: surface markers and functions of ESC
The characteristic pluripotency of ESCs was first discovered in mouse models. The pluripotency of murine ESCs (mESCs) were tested by injecting into a recipient murine blastocyst, and then determine the extent of chimaeras (9), which is indicated by the mixing of cells from different sources. Embryologists have studied the development of embryos and their structures by fusing cells of genetically distinct or otherwise different colored mice (10). As the area of embryonic development research grew, certain markers were used to illuminate points of cellular differentiation as they migrated to their respective germ layers. Markers for differentiation and pluripotency were established, thereby revealing how neural, cardiac, hematological are developed (9). Nevertheless, an ESC cannot be identified solely on surface markers; but in combination of function. These include: 1) symmetric and asymmetric divisions; 2) self-renewal and retention of stem properties; 3) generate specialized cells of the three germ layers.

This paragraph focuses on self-renewal and maintaining ‘stemness’ of ESCs. Notch is a delta signaling molecule in ESC that maintains their ‘stemness’ and self-renewal properties. In order to verify if a cell is a true ESC. When Notch is knockdown by siRNA method, the cell loses its ‘stemness’ and differentiate (11). Labeling of cells derived from ESC germ layer-specific antibodies such as PAX6, cardiomysin, and α-fetoprotein, which corresponds to ectoderm, mesoderm, and endoderm, respectively, can help to verify that originating cell as ESC.
The addition of specific growth factors to ESC culture media causes them to differentiate into a wide array of cell types. This occurs because the stem cells, after being exposed to these factors, form embryoid bodies that resemble an embryo. The embryoid bodies can generate cells belonging to ectoderm, mesoderm and endoderm types. When the embryoid bodies are transferred to further cell cultures supplemented with tissue specific growth factors, the mesoderm, endoderm, and ectoderm, can be forced to generate significant amounts of specific desired cell types. Laboratories have reported the generation of cardiomyocytes, hepatocytes, neurons, and many other cell types from this experimental approach (12).

Markers of pluripotency are Oct-4 and Nanog. The pathways known to contribute to the maintenance of ESC pluripotency include Wnt, and in the case of murine ESC, Leukemia Inhibitory Factor (LIF) and Bone Morphogenic Protein (Fig. 2). In murine ESC, LIF activates STAT3 proteins, which in turn activates other signaling molecules to activate Oct-4. BMP on the other hand, activates Smad, which activates Id, and that activates Oct-4. Conversely, Wnt blocks GSK-3 which will then block β-catenin that will induce Oct-4 (13). Oct-4 induces Nanog when it binds to it, and together, Oct-4 and Nanog maintains pluripotency in the ESC (14). These experiments can be done using SCID mice, which are immunodeficient mice.

Figure 2. Pathways that contribute to the maintenance of ESC pluripotency.

Murine vs. Human ESC

Both murine and human ESC express Stage-Specific Emrbyonic Antigen-1 (SSEA-1) and alkaline phosphatase activity (15). Just as the mouse, the human embryo also exhibits similar makers. According to Richards et al, ESC that have been maintained for 50 generations without differentiation and continued to display a normal karyotype, express the ES cell-associated transcription factor OCT4 and cell surface markers SSEA-3, SSEA-4, Tra-1-60 and GCTM-2 and tested positive for alkaline phosphatase activity (16). The transcription factor OCT4 maintains pluripotency in murine and human ESCs (17). The following table summarizes the markers expressed in murine and human ESCs.

MARKERS	Murine ESC	Human ESC
SSEA-1	+	--
SSEA-3	--	+
SSEA-4	--	+
Tra-1-60	--	+
GCTM-2	--	+
OCT 4	+	+
Alkaline Phosphatase	--	+

The use of murine ESCs not only demonstrates their ability to generate various differentiated cells, but also acts as a tool to understand developmental processes. Such developmental studies could prove vital to attaining a detailed understanding of cell growth and differentiation. It is certain that murine stem cells are valuable in studying this development as they are similar to the human system, however the pathway is not completely identical, and thus the murine derived stem cell system is limited. Furthermore, mouse stem cells cannot be transplanted into humans at the present time, since this would constitute a xenogeneic model, which, to date, has not been successful. At this time, the science dictates that only human sources ESCs could have potential for human implantation.

Tumorigenic Potential of ESCs

The mouse has served as an efficient experimental model for stem cell research. Today with the rise of nuclear reprogramming and stem cell biology the murine serves as the system of choice of deriving and perfecting these areas of research. The technique of nuclear reprogramming, also referred as cloning, has been suggested as ideal for repairing disease and for gene therapy through an autologous system (15). Stem cells isolated from a cloned embryo would be genetically identical to the patient and pose no risk of rejection (15). Some argue that the end result may still cause an immune response, but this is more than likely not the case. For instance, in one study, the presence of allogenic mitochondrial antigens in cells derived by nuclear transfer did not appear to significantly impede the development of muscle tissue (16). The caveat with ESC research is not the possibility of an allogeneic T-cell response. In fact, it is believed that ESCs are immune privileged, although this point is controversial (18). The problem with ESCs is that they have been shown to develop into tumors. Thus, while ESC can be potential cell sources for repair medicine, they can also forming tumors.

There have been reports of ESCs developing into teratomas, in the absence of chromosomal abnormalities. Takahashi et al. reported ERAS gene as a cause of tumor formation by ESC. ERAS null cells maintained pluripotency while exhibiting reduced ESC growth and tumorigenicity (19).

Current Events

The ethical controversy surrounding hESC research is centered on the fact that their harvest requires living human embryos to be disaggregated and destroyed. In an effort to alleviate the debate and controversy over the harvesting of ESCs for research, new techniques are being proposed for the development of new lines. Studies reported on an alternative method of establishing ES cell lines by a technique of single-cell embryo biopsy from an 8-cell stage of a zygote. This technique is similar to that used in pre-implantation genetic diagnosis of genetic defects in in-vitro fertilization. The remaining 7-cell embryos progressed to generate an embryo. The studies indicated that this technique generated stable ESCs without removing the blastocysts (22). In other studies, altered nuclear transfer (ANT) used genetic modification to create genetically disabled “embryos” that cannot develop past the blastocyst stage. These cloned blastocysts were morphologically abnormal, they lacked functional trophoblast and failed to implant into the uterus (21). However, the cells were deemed to be pluripotent ESCs when the cells were explanted (21). Supporters for these techniques argue that these are moral research options because it does not entail the destruction of viable embryos. This technique has not been used in humans and its potential usefulness and applications in humans has not been reported.

Potential Clinical Application

Though ESCs have yielded a degree of positive results in regenerative medicine by replacing damaged tissues, they have also been described as a tool to study development, and thus attain a deeper understanding of the human body and physiological processes. In a study conducted by zur Nieden et. al, stem cells were differentiated into osteoblasts, which are the major bone- forming cells. By extracting stem cells from the inner cell mass of a mouse blastocyst, zur Nieden et. al, were able to stimulate the stem cells to differentiate into bone progenitors using a cocktail of ascorbic acid, β-glycerophosphate, and 1 α, 25-OH vitamin D3 in vitro. PCR was used to verify the presence of osteocalcin and bone sialoprotein genes that are associated with osteogenic cells (20). Osteocalcin is associated with bone resorption, which bone sialoprotein is associated with mineralization. Also, as part of the study, alkaline phosphatase activity was measured in the cells generated, as this membrane bound enzyme is active in functioning osteoblasts. Not only did these analyses reveal the fact that functioning osteoblasts had been generated from mouse ESCs, but also resulted in a comprehensive analysis of the expression of the aforementioned genes and enzymatic activity over a thirty-five day period, detailing the upregulation of gene expression and enzymatic activity as bone cells develop (20).

Conclusion

As the study of developmental biology in terms of in vitro fertilization (IVF) and cell biology increased in the last decade along with the advent of the human genome project, the use of ESCs as therapeutics has became a greater possibility. Experimental models have demonstrated rapid movement and colonization of ESCs to target tissue in the fetus (9). This shows the remarkable nature of the ESCs to become any cell of the germ layer. These possibilities can be shared with humans in terms of the development of idiosyncratic organs.

When considering the benefits of stem cell research, one should realize that research must progress, otherwise a potentially amazing clinical treatment for virtually all disease types may be cast aside. If benefits do in fact exist, as current research suggests, then one can talk about expanding research, and if significant benefits are not found, then one can talk about ending such research. Until sufficient information is gathered via research, one cannot accurately be a disbeliever in stem cells, and until such research is gathered, one should be hesitant to be closed-minded and set on the pro or con stances of this issue.

References

1. Cooper GM and Hausman RE: The Cell: A Molecular Approach. 3rd ed. Washington: ASM Press. 621-62.

2. “Red Blood Cell. 2005. Med Help International. 18 Nov. 2005 http://www.medhelp.org/glossary2/new/GLS_3972.HTM

3. “Stem Cell Basics.” Stem Cell Information. August 12, 2005. National Institute of Health. 15 Nov. 2005 http://stemcells.nih.gov/info/basics/basics3.asp

4. “A Stem Cell Time Line.” Embryonic Stem Cells. 2003. Research at the University of Wisconsin-Madison. 9 Nov. 2005 <http://www.news.wisc.edu/packages/ stemcells/images/timeline.txt>

5. Dazert S, Aletsee C, Brors D, Sudhoff H, Ryan AF, Muller AM: Regeneration of Inner Ear Cells from Stem Cell Precursors – A Future Concept of Hearing Rehabilitation? DNA and Cell Biology 2003, 22,9: 565-570.

6. “NIH Human Embryonic Stem Cell Registry” Stem Cell Information August 12, 2005. National Institute of Health. 12, Nov. 2005 http://escr.nih.gov

7. Curtis MG: Cloning and Stem Cells: Processes, Politics, and Policy. Current Women’s Health Report 2003, 3: 492-500.

8. International Society for Stem Cell Research October 24, 2003. Accessed on 12 Nov. 2005 http://www.isscr.org

9. Edwards RG: Stem Cells Today: An Origin and Potential of Embryo Stem Cells. Reprod. Biomed Online Mar 2004, 8(3): 275-306.

10. “Research Chimerism” Chimera December 21, 2005. Wikipedia: The Free Encyclopedia, 23 Nov. 2005 http://en.wikipedia.org/wiki/Chimera_(animal)

11. Zwaka TP, Thompson JA: Differentiation of human embryonic stem cells occurs through symmetric cell division. Stem Cells 2005; 23: 146-9.

12. Carpenter MK, Rosler E, Rao MS: Characterization and Differentiation of Human Embryonic Stem Cells. Cloning and Stem Cells 2003; 5(1): 79-88.

13. Nakashima K, Colamarino S, Gage FH: Embryonic stem cells: staying plastic on plastic. Nature Medicine 2004: 10(1): 55-63.

14. Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T, Lako M: Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells 2005; 23(8):1035-43.

15. Munsie MJ, Michalska AE, O'Brien CM, Trounson AO, Pera MF, Mountford PS: Isolation of Pluripotent Embryonic Stem Cells from Reprogrammed Adult Mouse Somatic Cell Nuclei. Current Biology 2000; 10(16): 989-92.

16. Hawley RG, Sobieski DA: Stem cell bouillabaisse-potpourri. Stem Cells 2002; 20(5):360-363.

17. Hay DC, Sutherland L, Clark J, Burdon T: Oct-4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast differentiation Markers in Human and Mouse Embryonic Stem Cells. Stem Cells 2004; 22(2):225-235.

18. Li L, Baroja ML, Majumdar A, Chadwick K, Rouleau A, Gallacher L, Ferber I, Lebkowski,
J, Martin T, Madrenas J, Bhatia M: Human embryonic stem cells possess immune-privileged properties. Stem Cells 2004; 22(4): 448-56.

19. Takahashi K, Mitsui K, Yamanaka S: Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 2003; 423(6939): 541-5.

20. Zur Nieden, NI, Grazyna K, Hans JA: In Vitro Differentiation of Embryonic Stem Cells into Mineralized Osteoblasts. Differentiation 2003; 71: 18-27.

21. Meissner A, Jaenisch R: Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature 2005: Epub ahead of print.

22. Chung Y, Klimanskaya I, Becker S, Marh J, Lu SJ, Johnson J, Meisner L, Lanza R:Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 2005; Epub ahead of print

Acknowledgements

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:
Olufunke Amele, Shreya Chakravarti, Colin Craig, Michael Ricardo, Anthony Shoo (In alphabetical order)

Teaching Assistant: Kelly Corcoran

The review was edited by two stem cell biologists.