Current status of research using human embryonic stem cells.

Stem cells are undifferentiated and pluripotent--they are able to transform into a number of mature cell types, given the correct environment. A stem cell culture is capable of self-replication and, hence, has a theoretically infinite capacity for self-renewal. In essence, stem cells can be obtained from three sources: adult, fetal and embryonic. While there are many sources of adult stem cells, including bone marrow, brain, intestine, liver and smooth muscle, current research suggests that these can only differentiate within their own lineage. Most information is available from studies on hemopoietic stem cells, which can produce different types of cells within the hemopoietic system but cannot transdifferentiate into other lineages. (1) In contrast, as one moves toward the earlier stages of fetal and embryonic development, the capacity of stem cells to achieve totipotentency--that is, to differentiate into multiple mature lineages of endodermal, mesodermal and ectodermal origin--becomes evident. Development of antibodies to stage specific cell surface markers (2) and specific markers for ectodermal, endodermal and mesodermal developmental pathways has allowed early characterization of cell lines derived from the blastocyst inner cell mass.

A major plank in the platform of research initiatives was the establishment of the National Stem Cell Bank at the National Biologic Standards Board in Hertfordshire, announced by the UK Medical Research Council after the passage of the Human Fertilization and Embryology (Research Purposes) Regulations Bill through the House of Lords in 2001. The United Kingdom Stem Cell Bank acts as a repository for human stem cell lines of all types and is designed to supply well-characterized cell lines under appropriate and accredited quality systems, both for basic research and for clinical applications. There are now 24 human embryonic stem cell lines maintained within the Bank, from sources within the United Kingdom, Australia, Spain and Sweden (C. Hunt, personal communication, January 12, 2006). These cell lines are available to researchers around the world. The Bank will become a major resource for research as the number and variety of lines increases. The long term aim is to develop novel therapies for severe and currently incurable human diseases, a prospect that has stimulated research groups over the last decade. Differentiation of stem cells into specified tissue types offers the possibility of tissue regeneration and replacement, with many possible therapeutic benefits.

Stem cells derived from nonembryonic sources, including umbilical cord blood, bone marrow and elsewhere, are already used clinically to treat a wide spectrum of diseases. (3) Advantages of adult stem cells include the opportunity for their being used in autologous transplantation, avoiding risk of rejection, and need for immune suppression. However, attempts to induce transdifferentiation in adult stem cells have been largely disappointing, despite early enthusiasm, (4-6) whereas many groups have now reported derivation of mature cell lines of all three lineages from embryonic stem (ES) cells. (7-15) Embryonic stem cell derivation and differentiation have become the focus of many research groups and a huge investment. However, accepting that we can now manipulate ES cell lines to differentiate into practically any tissue of choice, there remain substantial obstacles before the therapeutic potential of ES cells can be exploited. These fall into three categories; namely, problems of derivation of cell lines to Good Manufacturing Practice in cell-free culture conditions, problems of immunogenicity and rejection, and problems of possible teratogenicity and karyotypic abnormality.

Good Manufacturing Practice

Good Manufacturing Practice (GMP) refers to the development of pharmaceutical grade agents for use in human clinical trials to defined standards of reproducibility and safety. The particular problem presented in attempts to derive therapies from cultured cells is in the development of cell-free culture media. Most ES cell lines have been derived using feeder cell layers and additives from animal sources, such as fetal calf serum. Risks of introduction of xenobiotic viral or prion proteins into the human proteome require that cell lines for use in clinical trials be free of such cells and supplements. Recent publications suggest that some of the problems posed by this stringency are being overcome, (16) and others have successfully cultured hES cells using feeder cells of human origin, from foreskin fibroblasts or endometrial cells. (17,18) However, further work is needed to define the optimal culture conditions for hES cells in completely defined media.

Developing stable human embryonic stem cell lines to Good Manufacturing Practice requires more than a xenobiotic-free environment. Cell lines must be derived and maintained in a defined environment, with the same level of air quality, laboratory systems, and process management and quality control as used in manufacturing of pharmaceuticals and tissue banking. Applying these rules is not easy within the more relaxed environment of a research laboratory. The forthcoming European Union Technical Directive, (19) covering processing, preservation, storage and distribution of human embryos for in vitro fertilization (IVF), will set the lowest acceptable standards for quality management in the IVF laboratory and will cover all aspects of practice in assisted conception, from donor selection and testing, laboratory handling of gametes and embryos, tracking of cells from donated embryos with linkage to specific recipients, and a system for reporting of adverse events. This will be the starting point for derivation of ES cells to GMP, with maintenance of the derived lines within the required environment at all stages up to and including the National Stem Cell Bank. To achieve this standard will require substantial investment, alterations in laboratory practice and acceptance of process mapping and compliance with standard operating procedures at all stages (see http://www.dh.gov.uk/PolicyAndGuidance/HealthAndSocialCareTopics/Tissue/TissueGeneralInformation/TissueGeneralArticle/fs/en? CONTENT_ID = 4077127&chk = 8MjvZR for further information). Once embryos have been donated to the stem cell derivation laboratory, further regulation will come into play, with governance by the Medicines and Healthcare products Regulatory agency (MHRA), to ensure compliance with the requirements of GMP. This system of tight regulation should ensure that clinical trials, once begun, have the lowest possible chance of resulting in adverse outcomes for patients. This cautious approach contrasts starkly with the rapid growth of unregulated "stem cell treatment centers" in other parts of the world. (20,21)

Immunogenicity

The most clinically appealing use for tissues derived from hES cells is in transplantation. ES cells are particularly suited to this role, given their pluripotency which should allow derivation of almost any cell and tissue type under the correct conditions. However, while undifferentiated ES cells are not immunogenic, with low levels of Class 1 Major Histocompatibility Complex (MHC) expression, MHC molecules appear to be expressed on the cell surface as they begin to differentiate. (22-24) Hence, human leukocyte antigen (HLA) matching between donor and recipient may be necessary, as in conventional transplant therapy. In addition, ABO blood group matching may be required. Immunosuppressive therapy may have to be used, although alternative approaches include induction of "tolerance" to the stem cell allograft, transplantation into immunologically privileged sites to avoid risk of rejection, or genetic alteration of stem cells to reduce MHC expression or enhance molecular protection of the graft. (25) More recent studies have suggested that, although hES cells express MHC antigens, the immunostimulatory potential of hES cells and differentiated hES cell derivatives is low, at least in an in vitro system, suggesting that rejection in vivo might be prevented with low-dose, low-toxicity immunosuppressive regimes. (26) Such work requires confirmation in whole animal studies before extrapolation to human transplantation.

If HLA matching does become necessary for regenerative therapies, then this will involve the establishment of large hES cell banks which could "dispense" matched stem cell derivatives according to the patient's HLA type. Again, this would be costly and resource intensive. An alternative approach has been to attempt to derive HLA-identical stem cells using somatic cell nuclear transfer--so-called therapeutic cloning. This involves transfer of a donor nucleus into an enucleated oocyte followed by activation of the nucleus to induce cell growth and division. The process has been used successfully to create stem cell lines from ungulates and rodents. (29-30) Early attempts at human somatic cell nuclear transfer led to nuclear activation but early arrest of embryonic development. (31) Later work purporting to describe derivation of stable human ES cell lines after somatic cell nuclear transfer has been retracted. (32,33) However, blastocyst derivation after heterologous nuclear transfer into enucleated donated oocytes has been described by a group working in the UK (34) and work to derive stable stem cell lines from such blastocysts is in progress. Should this work prove fruitful, then one practical problem to be overcome is the shortage of ethically derived human oocytes. While the Korean group were able to pay oocyte donors to undergo superovulation and oocyte collection with transvaginal ultrasound guidance, this would not be permitted by the UK Human Fertilization and Embryology Authority. Alternative sources of human oocytes would have to be found if this proceeds to be a viable route to derivation of autologous stem cell products for transplantation.

Karyotypic Stability

Therapeutic use of human ES cell derivatives could only be undertaken if the transplanted cells maintained their differentiated state after transplantation. The intracorporeal regulation of cellular differentiation and rate of growth has been extensively studied, since breakdown of this complex and tightly controlled system in vivo will lead to uncontrolled cell growth and development of a cancer. Little is known of the ability of hESC-derived tissues to maintain normal growth in vivo. However, several groups have now reported observations of alterations in the karyotype of hES cells after repeated passages in culture. Induced karyotypic abnormalities include trisomy 12, trisomy 17 (35) and failure of X chromosome inactivation. (36) It seems likely that these karyotypic aberrations may result from selection pressures imposed by the abnormal environment to which the cells are exposed in laboratory culture, rather than being due to an inherent epigenetic instability.

Other Uses for Human Embryonic Stem Cells

The concept of regenerative medicine using stem cell transplantation has captured the public imagination. However, for the reasons given above, such "miracle cures" may be some distance away from realization. In the meantime, less glamorous research targets are coming within reach. Pharmaceutical companies have been using murine stem cell models for screening of possible new drugs for some years. Introduction of human embryonic stem cells as an adjunct to the murine system offers new possibilities for high-throughput screening to test efficacy and safety of possible therapeutic agents. (37) Scientifically, the massive injection of research resources into the study of early embryonic development will provide new insights into mechanisms of activation of the embryonic genome, regulation of embryonic cell division and differentiation, and optimum conditions for embryo culture. There may well be considerable spin-off from this research into other clinical areas of interest, including improving IVF success rates, understanding of causes of early pregnancy loss and implantation failure, and disorders of pregnancy, including preeclampsia and fetal growth retardation/placental failure.

Conclusion

Research in human embryonic stem cell science will undoubtedly bring benefits. Such work should continue in tandem with studies on stem cells from other sources. The time scale from derivation of stem cell lines to clinical application is unavoidably long, and there are many methodological pitfalls along the way. Translation to clinical application must be undertaken carefully, with preliminary studies in animals as well as in vitro, to avoid adverse outcomes. Meanwhile, there are already tangible contributions to our understanding of human embryology and to drug development.

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Clocks slay time ... time is dead as long as it is being clicked off by
little wheels; only when the clock stops does time come to life.
--William Faulkner

William L. Ledger, MA, DPhil

From the Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, United Kingdom.

Reprint requests to William L. Ledger, Professor of Obstetrics and Gynecology, Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Room 5, Level 4 Jessop Wing, Tree Root Walk, Sheffield, S10 2SF, United Kingdom. Email: [e-mail]