2007-2008 An Eventful Year In Stem Cell Science and Ethics

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By Shane K. Green, PhD

A little more than a year has passed since this magazine published a cover story on stem cell science, ethics and policy1. In that short time, international efforts in human stem cell genomics, cell biology and other research have provided a wealth of fresh insight into stem cell derivation, development and differentiation. While of tremendous significance from a scientific perspective, much of this work has escaped the notice of all but those pursuing related lines of inquiry. However, this past year has been remarkable for a number of developments in stem cell research that have garnered significant attention in the scientific and lay press alike; not only for their considerable scientific impact but also on account of their ethical implications and motivations – indeed, ethics can influence not only what research is permissible, but can also profoundly influence what scientists choose to investigate.

The quest for ‘ethical stem cells’
Driven – at least in part – by moral convictions and/or social and political influences, many stem cell researchers have endeavored to discover or develop cells that behave like embryonic stem cells (ESCs), but whose derivation would not require the destruction of human embryos. Such cells are sometimes referred to as ‘ethical stem cells’, which is an unfortunate label as it suggests in the absence of supporting consensus that conventionally-derived ESCs are, by definition, unethical.

Adult stem cells (ASCs), or the cells responsible for replacement of damaged cells in many human tissues, behave somewhat like ESCs and their derivation is uncontroversial. However, intense research efforts have yet to identify truly pluripotent ASCs – that is, ASCs with the potential to give rise to all the different cell and tissue types in the body. As a result, some researchers have taken their quest to obtain pluripotent cells without destroying embryos along paths away from ASCs; this past year, two such paths led to noteworthy discoveries.

In the early part of 2008, a study2 was published in Cell Stem Cell, in which a research team demonstrated the ability to derive human ESC (hESC) lines from single cells removed from eight-cell embryos without causing apparent harm to the embryo. The work built on an earlier proof-of-principal study by many of the same researchers3 in showing that the biopsied embryos were capable of developing into blastocysts – the embryos in the earlier study had been biopsied multiple times and destroyed in the process.

The researchers’ motivation for performing these studies was made apparent in the preamble to the more recent paper, in which the authors explicitly cite the ethical concerns, funding restrictions and legal prohibitions associated with research that causes harm to human embryos. However, as highlighted in an accompanying editorial in Cell Stem Cell4, “it is unclear whether this new technique will satisfy the ethical criterion of no harm.” The procedure they used, called blastomere biopsy, is similar to one that has been safely used for many years to enable pre-implantation genetic diagnosis (PGD) of embryos created through in vitro fertilization. Nonetheless, the risk of harm to and/or unintended destruction of an embryo from such intrusive manipulation is not negligible. With PGD, those risks may be seen as acceptable when weighed against the potential development of serious genetic disease in untested embryos; however, in this case, the counterweight is not the prevention of disease but rather the promotion of ESC research, which brings us around full circle to the issue of causing harm to human embryos for the sake of research.

Scientific importance aside, the biopsy blastomere approach is unlikely, then, to provide a reliable source of pluripotent stem cells for research and, down the road, stem cell therapies. However, another set of studies a couple of months earlier continues to be touted by many as the most promising means yet discovered of overcoming ethical concerns surrounding embryonic derivation of pluripotent stem cells.

In November 2007, research teams in Japan and the United States published landmark studies in which they showed for the first time that human dermal fibroblasts – skin cells – can be reprogrammed, or induced, to a pluripotent state through the introduction of a small number of genetic factors5,6. Interestingly, the groups each used a different cell type, viral vector and set of genes to create these induced pluripotent stem cells, or iPS cells: the US team used lentiviruses to introduce four genes (Oct4, Sox2, NANOG and LIN28) into newborn foreskin cells; the team from Japan transformed adult human skin cells into iPS cells with a different combination of four genes (Oct3/4, Sox2, Klf4 and c-Myc) using retroviruses, though subsequent work from the same group7 showed that one of the genes (c-Myc) was not, in fact, required for iPS cell generation. In both cases, the iPS cells expressed genes and cell surface markers characteristic of hESCs, possessed telomerase activity, and showed the potential to differentiate into the primary germ layers (ectoderm, mesoderm, endoderm). In other words, by all means tested, the iPS cells looked and behaved like hESCs.

But while iPS cells appear indistinguishable from ESCs, we don’t yet know enough about ESCs to determine if they are indistinguishable. Further research will be required to determine if there are significant differences between iPS cells and ESCs.

In addition, the viruses used to create these iPS cells insert their genetic payloads into random sites in the skin cell’s genome; such random insertion could cause disruption of other genes or regulatory elements, and dysregulation of important genetic pathways. In a research context, this could compromise the study model; in a clinical setting, it could cause cancers to develop. Scientists will need to find safer, non-disruptive modes of generating iPS cells before they would be reliable substitutes for ESCs in research and safe for clinical use.

Moving towards – and away from –
cloned human stem cells
The use of somatic cell nuclear transfer (SCNT) to create cloned embryos from which to derive hESCs is a controversial practice, permitted in a handful of jurisdictions but prohibited in others, including Canada. Nonetheless, many researchers see hESCs cloned from persons with genetic disorders as uniquely valuable tools for research into the development of genetic diseases. Others embrace cloned hESCs for their theoretical potential to provide patient-matched stem cell therapies capable of avoiding immune rejection.

If iPS cells prove sufficiently comparable to ESCs, and if their genetic transformation did not compromise their usefulness for the study of genetic diseases, they could render cloned hESCs largely unnecessary. But until those questions are answered, researchers will continue to pursue the creation of cloned hESCs.

No one has yet succeeded in creating cloned hESCs; in fact, until recently, no one had developed cloned embryos to the blastocyst stage at which ESCs are most reliably derived. That changed in January 2008, when a US research team announced the creation of the first human blastocysts using SCNT8. Specifically, they generated five putative blastocysts via SCNT, using 29 human oocytes obtained from consenting young women undergoing oocyte retrieval for reproductive purposes.

Although they did not derive ESCs from the blastocysts, they did move the technology forward to a point where such derivation is now only a single step away. Given the focus of this article, it is also worth noting that the Methods section of their paper begins not with a description of an SCNT protocol, but with a section entitled ‘human ethics’.

But as interesting as this was, the attention it received paled in comparison to that garnered by the UK Human Fertilisation and Embryology Authority’s approval, in September 2007, of applications from two research teams to use SCNT to develop interspecies cytoplasmic hybrids, or cybrids, to study human genetic diseases. Cybrids are created by inserting genetic material from human somatic cells (e.g., skin cells) – or the cells in their entirety – into non-human oocytes (e.g., from cows) from which the nuclear genetic material had been removed. This approach, which is motivated by both practical and ethical considerations, seeks to enable the study of disease-specific hESCs while reducing researchers’ reliance on scarce human oocytes.

On a practical level, while large numbers of human oocytes are hard to come by, bovine oocytes are readily available as byproducts of the meat production industry – itself an ethically charged reality of our society – and thus could provide an abundant source of raw materials for research. Removing the bulk of the genetic material from cow egg leaves a largely, but not entirely, empty vessel. What remains unclear is the extent to which the residual bovine mitochondrial DNA and intracellular proteins may influence human gene expression and cybrid embryonic development.

Cybrids are often referred to as ‘mostly human,’ which begs the question: just how human would they need to be to provide a reliable model for the study of human genetic disease?

In light of this confounding factor, why use cybrids at all? Because significant medical and ethical obstacles stand in the way of obtaining the vast numbers of human oocytes required for research. With current technologies, the retrieval of human oocytes involves hormonal treatments and surgical interventions, which carry substantial medical risks to donors, risks that are not offset by potential rewards in those donors who would provide oocytes solely for research and not for personal use (e.g. for IVF). Rewards in the form of payments are illegal in some jurisdictions, including Canada, and where permitted can be ethically problematic, as money – or power imbalances – can amount to an undue and potentially coercive influence on donors.

That is not to say, however, that cybrids are uncontroversial. The creation of interspecies embryos is deeply troubling to some. Just as we may ask how human the cybrid ESCs need to be to provide reliable research tools, some may ask how human a cybrid embryo needs to be before we must contend with the ethics of manipulating or destroying it.

The biggest caveat of them all… scientifically speaking
Each of the aforementioned developments, while exciting, has been accompanied by scientific and/or ethical caveats. Arguably the biggest of them all is that hESCs have yet to be definitively characterized. That is, we do not yet understand – at the molecular level – pluripotency and the path to cell specialization, or ‘stemness’, and it is upon this understanding that the ultimate success of stem cell science will depend.

A wealth of ongoing research is aimed at providing such understanding, and holistic, high-throughput approaches (e.g., genomics9, proteomics, cellular microarrays, large-scale screens10) are helping to drive this important research forward. Research supported by the Ontario Genomics Institute into the genome-wide characterization of mammalian gene regulation and cancer stem cells (CSC), for example, is helping to provide new insight into what gives stem cells their unique abilities. Using mouse and human ESCs, the International Regulome Consortium (IRC)11 will map the genetic circuit board that regulates the formation and function of cells (i.e. the ‘regulome’), which will provide a much needed genetic description of how and why stem cells develop into particular cell types during embryonic development. And although CSCs – also known as cancer initiating cells – are fundamentally distinct from ASCs or ESCs, their comprehensive characterization will nonetheless provide fresh insight into the genetic and other factors that contribute to ‘stemness’.

Not surprisingly, both the IRC and CSC research were front and centre in another of the year’s big stem cell stories here in Ontario when, in May 2007, California Governor Arnold Schwarzenegger came to Toronto’s MaRS Discovery District to announce an agreement between the IRC and the University of California at Berkeley to coordinate their stem cell research activities. The occasion was also marked by an announcement from Ontario’s Premier, Dalton McGuinty, of the creation of the Cancer Stem Cell Consortium, a Canada-California collaborative effort seeded with a $30M contribution from the Ontario Institute of Cancer Research. With this kind of political and funding support for stem cell researchers here in Canada and abroad, we are surely in for many more remarkable years ahead.

References
1     Green, SK. Biotechnology Focus. 2007; 10(1): 10-12.
2     Chung, Y et al. Cell Stem Cell. 2008; 2: 113-7.
3     Klimanskaya, I et al. Nature. 2006 Nov 23; 444(7118): 481-5. Epub 2006 Aug 23.
4     Johnson, MH. Cell Stem Cell. 2008; 2: 103-4.
5     Takahashi, K, et al. Cell. 2007; 131: 861-72.
6     Yu, J, et al. Science. 2007 Dec 21; 318(5858): 1917-20. Epub 2007 Nov 20.
7     Nakagawa, M, et al. Nat Biotechnol. 2008 Jan; 26(1): 101-6. Epub 2007 Nov 30.
8     French, AJ, et al. Stem Cells. 2008 Feb; 26(2): 485-93. Epub 2008 Jan 17.
9     Burks, C. Biotechnology Focus. 2007; 10(5): 41-43.
10     Underhill, GH & Bhatia, SN. Curr Opin Chem Biol. 2007 Aug; 11(4): 357-66. Epub 2007 Jul 25
11     http://www.internationalregulomeconsortium.ca/

Shane K. Green has a doctorate in medical biophysics from the University of Toronto (U of T) and has studied and taught bioethics and research ethics through the U of T Joint Centre for Bioethics and the American Medical Association in Chicago, IL. He has served on the Research Ethics Boards of two major health research centres in Toronto. Shane is currently the Director of Outreach and lead GE3LS (genomics-related ethical, economic, environmental, legal and social issues) advisor at the Ontario Genomics Institute (OGI) in Toronto (www.OntarioGenomics.ca). OGI is supported by operating funds from Genome Canada and Ontario’s Ministry of Research and Innovation.