Modifications to standard immunoblotting protocols - ARTICLE

References 1. Generally, conditioned media from cells generated to overexpress an antibody are run through a bead column similar to an immunoprecipitation and then the bound antibody is eluted off the column. However, how a company generates this antibody can vary (ie, using insect or mammalian cells) as well as the methods used to purify the antibody. This can make a big difference in what additional proteins may be present in the antibody as it is shipped. 2. These experiments in Figure 1 were performed with antibodies, not commercially available, that were generated by 4 different companies. All were raised to detect Receptor X and selected for in vivo use because they inhibit the binding of Receptor Xs ligand. Their use in vivo has been to test whether inhibiting Receptor X can inhibit tumor growth. In general, these antibodies are not different from those commercially available and used in Westerns yet it is possible that that these antibodies may not be as pure as those you buy commercially.

REVIEW

Stem Cells Hope or Hype for Reproductive and Regenerative Medicine?

Zubin Master1,* and Amandine Truong2
1- Department of Bioethics Dalhousie University 2- Department of Medical Biophysics University of Toronto *Correspondence: zmaster@sten.sunnybrook.utoronto.ca A stem cell (SC) is a cell from an embryo or adult that can self-renew and differentiate into specialized cells such as neurons, skin or muscle. Embryonic stem (ES) cells are considered to be blank slates as they typically exhibit little or no differentiation, and are considered to have more therapeutic potential than adult SCs. Adult SCs are found in the bone marrow, gut, and blood, whereas ES cells are obtained from the inner cell mass during early embryogenesis, resulting in the destruction of the embryo. Due to the elementary properties of SCs, research in this field shows tremendous promise for regenerating or replacing failed tissues and organs. Moreover, a recently published scientific article by Hubner and colleagues (2003) demonstrated that mouse ES cells could form oocytes in culture, expanding their potential utility from regenerative to reproductive medicine1. Assuming that this research is translatable to humans, having the ability to produce artificial, viable oocytes in culture from human ES cells would prevent the exploitation of women for egg donations, expand research on causes of infertility and improve assisted reproductive technologies. The approach taken by Hubner et al.. (2003) involved inserting a DNA construct carrying a germ cell-specific green fluorescent protein (GFP) into cultured mouse ES 1 cells . GFP was under the control of a modified Oct4 regulatory sequence deleted in conserved enhancer elements that restrict expression to germ cells. Artificially derived oocytes were characterized by the formation of follicular structures and an examination of oocyte-specific gene expression (zona pellucida (ZP) 1-3) revealed similar expression patterns between naturally and artificially derived oocytes. Although this study validated that the SCs created cells resembled oocytes, it did not demonstrate that the eggs were functional and capable of being fertilized and developing into embryos suitable for implantation. Interestingly, the authors found that only two of the three oocyte-specific ZP genes in cultured oocytes were expressed properly, which may explain why artificially derived oocytes have fragile zonae. Although the popular press has implied that similar studies can be done with human ES cells, such experiments have yet to be reported. Implications for human reproductive biology and tissue regeneration. The ability of SCs to differentiate into tissue or gametes has the potential to impact both reproductive and regenerative medicine. Assuming that human ES cells can form oocytes in culture, the Hubner et al.. (2003) study provides a model for studying egg development without using donated oocytes from women. In this sense, an unlimited source of raw material for reproductive research will be available. An unlimited source of fresh oocytes may help infertile women who have problems producing oocytes or whose oocytes are incapable of being fertilized. Using artificially derived oocytes may also obviate the need for donated eggs and the concerns that women be exploited for egg donations. Furthermore, the option of using artificially derived oocytes may be a safer alternative to other currently available options. For example, couples undergoing in vitro fertilization (IVF) require women to stimulate egg release from the ovaries using superovulation drugs, which have been reported to cause

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Stem cells and reproductive medicine - REVIEW

ovarian cancer2. A continuous source of oocytes will also advance the science of understanding egg development. By knowing how oocytes develop naturally, scientists can design more effective means of contraception or advance our understanding of some forms of infertility in women. Combining artificially produced oocytes with somatic cell nuclear transfer (SCNT) to obtain cloned ES cells allows scientists to derive oocytes cloned from an individual. SCNT involves transferring the nucleus of a skin cell into an enucleated oocyte and through parthenogenic activation (a phenomenon where oocytes undergo embryonic development without fertilization) of the oocyte, embryonic development will begin and cloned ES cells can be obtained from the inner cell mass of the embryo. The ES cell population can be cultured to differentiate into oocytes from the cloned individual, which in one example could be an infertile mother who would want to have a genetically related child. However, this procedure fails to escape destruction of the embryo to derive ES cells, but only has to be done once to generate an endless supply of cloned oocytes. Unfortunately, this procedure would not help treat cases where the infertility is caused by genetic defects from the mother or in situations where the infertility is caused by epigenetic factors. This procedure also raises a number of ethical concerns surrounding the use of cloning technology irrespective of its use in either therapy or reproduction. By performing SCNT using a male nucleus, scientists can derive ES cells and differentiate them in culture to produce oocytes with a male genome. Through IVF, male sperm can inseminate the artificially derived oocytes to produce a child from two men. Interestingly, the popular press reports a Japanese laboratorty creating sperm by culturing ES cells. Atrificially derived sperm could revolutionize research on sperm development and male fertility, as well as perhaps lead to the design of a contraceptive pill for men by inhibiting sperm production/release or by lowering the ability of sperm to penetrate the ovum. In addition, by using SCNT technology, artificial sperm cells derived from a female could be produced thereby resulting in a child conceived from the genetic material of two women. Despite the forthcoming role of SC science in reproductive medicine, the promise of SCs has always been believed to help in tissue regeneration as ES cells have been shown to differentiate into almost all cell types except for germ cells. The Hubner (2003) study demonstrates the totipotency and endless differentiating capabilities of ES cells into various cell types for tissue regeneration. Although some scientists believe that ES cells have greater potential in regenerative medicine than adult SCs, some, such as hematopoietic SCs, are extremely efficient for the regeneration of peripheral blood 4 Numerous leukocytes in lethally irradiated animals . scientific reports have demonstrated the efficiency of adult SCs in pancreatic5, heart6, vascular7 and neural8 regeneration in various animal models. Recently, doctors attempted an experimental procedure in which adult bone marrow SCs were transplanted into a 16 year-old boy to repair a heart defect9. This procedure was reported to 9 have regenerated heart performance . The utility of SCs in regenerative medicine has also been combined with gene transfer to increase the survival of transplanted mesenchymal SCs. For example, in one case, overexpression of PKB/Akt increased myocardial performance post injury when compared to SCs expressing the vector control6. Thus, numerous studies clearly demonstrate the potential value of adult and ES cells in regenerative medicine. Stem cells and public policy. At present, there are no specific guidelines on SC research in Canada. However, in 1998, the Medical Research Council, Social Science and Humanities Research Council, and National Science and Engineering Research Council released the Tri-Council Policy Statement (TCPS), which broadly states what types of embryo research are ethically acceptable10. The Canadian Institute for Health Research (CIHR) adopted similar ethical guidelines as TCPS, but specific to SC research and is in the process of forming a stem cell oversight committee to complement work from research ethics boards in reviewing all SC grants prepared for any 11 of the three Councils . Failure to comply with CIHR and TCPS guidelines may result in the loss of federal funding from these granting agencies. However, none of these guidelines limits funds for private research on SC that do not abide by CIHR ethical standards. Bill C-1312 is the most current legislation that restricts human embryo and SC research consistent with TCPS and CIHR policies that apply to both federally and privately funded human research. Despite the many advantages that come with SCs, many feel that using ES cells is merely paving a path to begin experimentation on embryo cloning. In 1996, the US Congress imposed a ban on federal funds for all human embryonic research whether the embryos were created for research, donated or discarded from IVF clinics13. In 14 and in 2001, 1998, the first ES cell was isolated President Bush mandated that federal funds only be available for existing SC lines, thereby preventing the 15 creation of new ES cells from discarded human embryos . Convinced by scientists and his scientific advisors, President Bush stated that the 60 existing human ES cell lines available at the time would provide great insight in SC research15. To date, only eleven ES cell lines are routinely available for research16. Placing the ethical concerns of cloning aside, several scientists feel that the technology of cloning is unsafe since many cloned animals exhibit medical problems, however, it remains to be determined if the defects in cloned animals are due to the procedure itself17. Some believe that cloning human embryos may be impossible because the procedure may cause the formation of disarrayed mitotic spindles resulting in unequal chromosome segregation producing aneuploid, and therefore non-viable, embryos18. The technology of cloning may also affect genomic imprinting, perhaps leading to a differential expression of genes that have been shown to cause growth abnormalities in cloned animals19. Future Studies. The ethical concerns versus the medical advantages of ES cells and cloning research will be an endless debate. However, advances in science may bypass the requirement of ES cells and eliminate the moral issue of embryo destruction. For example, future studies on the biology of SCs to identify which genes maintain ES cells in an undifferentiated state and which

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genetic and environmental factors make them differentiate into specific cell types will be most valuable for reproductive and regenerative medicine. In this sense, expression of key genes may make adult SCs behave more like ES cells. An example of this was seen as a recently discovered homeoprotein, Nanog, maintains the pluripotency of mouse ES cells maintaining them in an 20 undifferentiated state . While the Hubner et al.. (2003) study provided insight into the true potential of ES cells; their use in medicine requires further scientific investigation. Advances in SC biology may help fine-tune the ethics surrounding the use of ES cells and cloning in medicine. This information will help public policy-makers generate proper regulatory oversight and maintain public trust in SC research while simultaneously making progress in both medical and scientific discoveries.
References 1. Hubner K et al. (2003) Science. 300:1251-6. 2. Venn A et al. (1999) Lancet 354:1586-1590; Wakeley KE et al. (2000) Current Opinion in Obstetrics and Gynecology 12:4347. 3. (no author) New Scientist. May 10, 2003; Westphal SP (2003) Comment, Editorial pg 3, New Scientist. May 10, 2003. Davies JA Davies. News, This Week pg 4, July 26 2003. 4. Wagers AJ et al. (2002) Science 297:2256-9. 5. Hess D et al. (2003) Nature Biotechnology. 21:763-70. 6. Mangi AA et al. (2003). Nature Medicine. 9:1195-201. 7. Bailey AS et al. (2003). Blood. Ahead of Print. t 8. Bjorklund LM et al. (2002) Proc Nat Acad Sciences USA. 99:2344-9. 9. Wade N (2003). New York Times Section A, Column 20 p3. 10. The Tri-Council Policy Statement. Ethical Conduct for Research Involving Humans. 1998. MRC Canada, NSERC Canada, SSHC Canada. Public Works and Government Services Canada. Ottawa. 11. CIHR. Human Pluripotent Stem Cell Research: Guidelines for http://www.cihrCIHR-Funded Research. 2003.
irsc.gc.ca/publications/ethics/stem_cell/stem_cell_guidelines_e.shtml

12. Bill C-13. An Act respecting assisted human reproductive technologies and related research. 2002. Second Session, Thirty-seventh Parliament, House of Commons of Canada. 13. NARAL, Human Embryo Research, History of Federal Embryo Research Funding Bans. January 21, 2003. 14. Thomson JA et al. (1998). Science 282:1145-7. 15.http://www.whitehouse.gove/news/releases/2001/08/20010809-2.htmlhttp://www.cnn.com/2001/ALLPOLITICS/08/09/stem.cell.bush

16.Kennedy D (2003). Science 300:865. 17.Kuehn BM (2003). J Am Vet Med Assoc 222:1060-1; Tamashiro KL et al. (2002). Nature Medicine 8:262-7. 18. Simerly C et al. (2003). Science 300:297. 19. Humpherys D et al. (2001). Science 293:95-7; Young LE et al. (2001). Nature Genetics 27:153-4 ; Humpherys D et al. (2002). Proc Nat Acad Science USA 99:12889-94. 20. Chambers I et al. (2003). Cell 113:643-655; Mitsui K et al. (2003). Cell 113:631-642.

Notes added in proof

Since the acceptance of this manuscript, the Canadian govenrment has enacted legislation to allow stem cell research on human embryos

REVIEW

The homologous origin of analogous structures: A tale of an eye for an eye in evolutionary biology
Cecilia DAlterio1 and Mariano Loza-Coll2,*
1. Department of Zoology University of Toronto 2. Department of Medical Biophysics - University of Toronto *Correspondence: lozacoll@sten.sunnybrook.utoronto.ca That same old analogy-homology dichotomy. Evolutionary biologists, like good historians, do not stop at merely describing events that happened hundreds of millions of years ago. Instead, they try and learn from the past, in a very human attempt to come to grasps with our present and have a shot at predicting certain aspects of the future. Because we cannot go back in time and tweak our biological variables of interest, evolutionary biologists are limited to speculate why things happened in one way over others based on fossil records and present day phylogenetic evidence. This article deals with just one aspect of the ever-present struggle to discriminate analogies from homologies among organisms. Analogous structures are those that serve similar functions among organisms, even though they do not share an immediate common ancestor. In simpler terms, analogous structures do the same thing but do not originate from the same embryonic precursor. The classic textbook example of analogous structures is that of wings in the animal kingdom. The wings of your favorite butterfly, a bat and a blue jay originate from very different embryonic structures, via very different developmental mechanisms. However, one can easily agree on that they are somehow structurally akin and that they are quite similar from a mechanistic viewpoint, conferring these three individuals with the ability to fly. Homologous structures, on the other hand, are almost the opposite; they are those that share the same embryonic origin, regardless of what their function in the fully developed adult is. If learned embryologists can bear

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