| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
REVIEW |
1 Centre for Regenerative Medicine, College of Medicine & Veterinary Medicine, University of Edinburgh, Chancellor’s Building, Rm GU. 531, 49 Little France Crescent, Edinburgh, EH16 4SB, UK 2 Scottish National Blood Transfusion Service, Tissue Services, Edinburgh, UK
Correspondence should be addressed to P A De Sousa; Email: paul.desousa{at}ed.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
Realizing therapeutic aspirations for human embryo stem cells (hESCs) requires convergence of the capacity to comply with existing and emerging standards for the transplantation of cells and tissues with the evolution of scientific and technical capability to preserve the properties of these cells whilst minimizing the risk of introducing unknown pathogens. Since the unique capacity of hESC for renewal and expansion offers realistic prospects for their broad usage, standards established for the procurement and processing of these cells today within individual nations should ideally be sufficiently comprehensive to accommodate their global acceptance and utility in the future. In this paper, our focus is on the progress of the UK to achieve the safe and quality-assured evaluation of hESCs in the clinic. Within this context, we discuss underpinning fundamental principles governing donation, procurement, testing, processing, preservation, storage and distribution of embryos and cells based on experience in adult cell and tissue transplantation and European Union (EU) Directives. Given the absolute dependence of hESCs on cell culture, we also address international progress in the creation of defined and animal cell product-free culture environments to isolate hESCs for cell banking. While definition and evolution of high-throughput systems for hESC expansion and differentiation are equally important to their ultimate therapeutic use, they are beyond the scope of this paper.
|
Existing and emerging EU/UK standards governing human cells and tissues |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
While WHO recommendations to National Health Authorities are anticipated in late 2006, the intervening period has witnessed significant progress in defining and legislating improved standards by the Councils and Parliament of European Union, which in turn have been ratified or are in the process of being ratified by member states, including the UK. Specifically, in March 2004, EU directive 2004/23/EC was issued, followed in November by Royal assent of the Human Tissues Act (2004) in the UK. These have set new standards of quality and safety for the donation, procurement, testing and distribution of human tissues and cells. The first technical annex providing detailed requirements for donation, procurement and testing was published in February 2006 (directive 2006/17/EC) with the technical annex for coding, processing, preservation, storage and distribution presently in consultation in draft form (see Draft 29/03/2005). Copies of issued and draft EU directives are available online (http://europa.eu.int/). The scope of these directives encompasses blood, bone marrow, reproductive cells (i.e. eggs, sperm and embryos), cells derived from fetal and adult tissues, and embryo stem cells. The inclusion of reproductive (i.e. eggs, sperm and embryos) in this directive has generated the greatest concern amongst practitioners of assisted reproduction, who have questioned the relevance of standards normally associated with transplantation medicine. It is worth noting that autologous grafts of tissues or cells within the same surgical procedure, allogeneic transplantation of blood, blood products, and tissues or cells or animal origin are covered by other directives and recommendations (i.e. Directives 2001/83/EC & 2000/70/EC, 2002/98/EC and Recommendation 98/463/EC). Also excluded are the uses of human tissues for research, unless those tissues are used in clinical trials applied to the human body. In this context, it is possible that the non-reproductive use of embryonic cells was a contributing factor to their grouping with other cells in a single legislative document, namely EU directive 2004/23/EC.
To the credit of EU legislators and contributors involved in its drafting and consultation, Directive 2004/23/EC comprehensively identifies the multitude of factors that must be considered for quality-assured and safe therapies involving human cells. As summarized in Tables 1
and 2, it specifies provisions to be addressed by multiple parties within member states from designated authorities to tissue facilities, third parties and individuals. It not only allows for member states to impose more restrictive measures but also promotes unification of standards that ultimately benefit all participants. The implications of these provisions are significant with increased burdens of responsibility and costs to the public and private sectors and end beneficiary awaiting new treatments. While it is easy to envisage that the therapeutic use of cells in general or with hESC derivatives in specific could be achieved more expediently without these standards, the consequences associated with inconclusive or adverse results that could occur without having them cannot be overlooked. This is borne out by recent experience with gene therapy trials, or transmission of infectious pathogens via blood products or tissues (Gode & Bhide 1988, Simonds et al. 1992, MacLaren et al. 2005, Ludlam et al. 2006). While some of the complications that have been experienced could arguably be described as unforeseen on the basis of available knowledge, this in itself does not substantiate less rigorous standards. Rather, it emphasizes the need for the forethought that such standards invoke.
|
|
HESC lines derived in the UK under licence from the HFEA must be deposited in the UK Stem Cell Bank (UKSCB), hosted at the National Institute for Biological Standards and Controls (NIBSC). Established in 2002, this government-backed facility was the first of its kind to be established with the aim of providing a quality-assured repository for hESCs for research and therapeutic purposes (Healy et al. 2005). The bank was accredited by the MHRA in June 2004. At the time of writing, 24 lines had been approved for deposition in the bank, of which 5 had been deposited (Glyn Stacey, Personal Communication). Five of these had been derived in the UK. While at present all these lines are regarded as ‘research grade’ owing to the nature of facilities and procedures in place at the site and time of their derivation, multiple groups across the UK including ourselves are actively engaged in upgrading procedures and facilities designated for this purpose to fulfil existing and emerging regulatory standards for clinical use. International competition to attain these standards is intense, and recently one multinational effort led by the Singapore-based bio-technology company Embryo Stem Cell International, announced the derivation of two new hESC lines in facilities operating to present Good Manufacturing Practice (GMP) as defined in Singapore (for further information, see www.escellinternational.com/).
As embryologists and scientists unaccustomed to thinking in manufacturing terms, the learning curve has been steep. Key challenges have included: (1) the anticipation and implementation of elevated quality standards that are still being defined, (2) addressing the needs of transplantation medicine without compromising assisted reproduction services and (3) securing the necessary resources to achieve elevated standards for a therapeutic objective whose benefits remain speculative. The funding breach to support the latter remains an outstanding issue, with many efforts across the UK moving slowly forward with varied and piece-meal support from either the Medical Research Council, Charitable trusts, Universities and/or regional economic development agencies. Although private–public funding partnerships to support the general translation of stem cell research to the clinic have been discussed, this support has yet to be realized.
|
Lessons from haematopoietic stem cell transplantation |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
Donor consent, selection and screening
While the original scope of UK Human Fertilisation and Embryology Act (1990) and resulting HFEA code of practice addressed patient consent for the use of gametes and embryos in IVF treatment, storage by cryopreservation, disposal and the licensing of research in human reproduction and development, the production of a tangible asset such as stem cells has raised new issues in the consenting process, the impact of which are still being addressed. Based on experience with consenting for blood products, including HSCs, there is now a greater recognition for a need to provide information and obtain instruction regarding disposal of tissues, their commercial exploitation and feedback of information relevant to the health status of the donor. As noted in the first EU Cells and Tissues technical annex (summarized in Table 2
), more rigorous selection and microbiological screening of gamete donors is required, consistent with the present requirements of the UK Blood Services & NIBSC Guidelines for the Blood Transfusion Services (the Red Book). Donors will be required to complete a health and medical history questionnaire. Furthermore, on the basis of family history, the donor’s first degree relatives may have to be free of non-trivial autosomal dominant or X-linked disorders with age of onset extending beyond the age of the donor (such as Huntingdon’s chorea), autosomal dominant disorders with reduced penetrance (such as Marfan’s syndrome or Alport’s disease) and autosomal recessive disorders with high prevalence in the general population (such as cystic fibrosis, Factor V Leiden and haemochromatosis). A family history of familial disease with a major genetic component (such as cleft lip, spina bifida or congenital malformation), non-trivial Mendelian disorder (such as haemophilia, hereditary hypercholesterolaemia or neurofibromatosis), familial disease with a reliably indicated major genetic component (such as juvenile diabetes mellitus, severe hypertension or rheumatoid arthritis) or of chromosomal rearrangement may also preclude contribution to therapeutic grade cell lines. Consideration will also need to be given as to the extent to which the potential for later development of disease in donors of embryos might impact upon the clinical safety or utility of a cellular therapeutic product. The significance of this point is underscored by stipulations in the EU cells and tissues directive for maintenance of full traceability of tissues from donor(s) to recipient patient(s) for at least 30 years following the last clinical use of any therapeutic product derived from those cells.
In recognition of the need to improve and consolidate provision of information to assisted conception patients and consenting for embryo donation for stem cell research, the MRC has funded the co-ordination of this effort across multiple centres. This has resulted in the formation of the human Embryonic Stem Cell Co-ordinators group (hESCCO; http://ukstemcell.net). Till date, this group has worked to standardize donor information and consent forms and the consenting process in accordance with HFEA guidelines, and establish a secure database to provide solid evidence of public confidence in embryo donation for stem cell research, the latter in concert with social scientists.
With publication of EU directive 2006/17/EC, there is now more clarity on microbiological testing that is required of donors of cells or tissues (summarized in Table 2). With the exception of additional testing dependent on the donor’s travel and exposure history, it is assuring that the present screening of third party donors of gametes for assisted reproduction of an infertile woman is consistent with these requirements. However, one nuance is an EU requirement for testing at the time of donation. In the context of embryo donation for stem cell research, this could be interpreted to imply repeat blood sampling of donors of the gametes resulting in that embryo closer to the time the embryos are created.
Facilities specification and operation
Considerable attention and concern has focused on the necessity to handle gametes and embryos in an in vitro fertilization clinical setting in the same manner as cells and tissues for transplantation. Most notable have been fears of incorporating provisions for the specification and operation of clean rooms described by EC Guide to GMP as contained in Rules and Guidance for Pharmaceutical Manufacturers and Distributors 2002 (Orange guide) and updated regularly in Eudralex (http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/index.htm). Although it is clear that facilities for deriving hESCs need to operate to these standards, it is less clear that this will be the case for IVF labs. Indeed, a recent consultation document issued by the HFEA on standards for assisted conception units (May 2006) has implied lesser requirements, such as operation in unclassified or lower classification of background air. The present draft of the EU Technical Annex (Draft 29/03/2005) makes provisions for less stringent environments ‘where it is demonstrated that the mode and route of application of the tissue or cell to the recipient implies a significantly lower risk of bacterial or fungal infection than cell transplantation (e.g. insemination)’. Until clarity is achieved on this point, IVF centres across the UK supporting hESC derivation have taken a variety of approaches from focusing on the introduction of quality systems of operation to the clean room installation. The former is an absolute requirement of the EU directive, on which practically all other considerations are dependent (see Table 1). Recognition of the importance of quality systems in assisted reproduction treatment in Europe is not new, but until recently has been implemented entirely voluntarily (Wikland & Sjoblom 2000).
Irrespective of lab specification, it is clear that settings supporting IVF and hESC derivation will require quality systems comparable with those required by the MHRA for tissue establishments. This will entail a fully audited quality management system, such as the ISO9000 system (http://www.iso.org/iso/en/iso9000-14000/index.html) or other systems meeting the requirements of the HTA. These would feature:
Based on the experience provided by the quality-assured production of blood cells and products, a number of other considerations are worth noting. These include:
Evaluation of hESCs in clinical trials
Although the clinical evaluation of hESCs may be a few years away, future-proofing present standards is necessary. Many of the same factors will need to be defined and optimized as presently met by transplantation of fetal and adult cells and tissues. This includes definition of the optimum cell lineage for transplantation, the route and timing of administration, ‘dosage’ of cells and whether single or multiple administrations are required, definition of clinical subsets of disease, interplay of other medications, etc. Appropriate investigatory and clinical end points need to be defined, as well as the necessity and ethical appropriateness of randomizing or blinding the study (Cesaro 2004, Dunnett & Rosner 2004). The extensive dependence of hESCs on culture systems to expand and differentiate them, presently achieved by reliance on research grade or animal sourced reagents, arguably makes this a greater issue for the therapeutic use of these cells than for cells sourced from other tissues. Reagents need to be quality assured and traceable, and exposure to animal cell products could theoretically brand a stem cell product as a xenobiotic, impacting on the patient follow-up in a clinical trials.
At present, clinical trials of experimental therapeutics need to be carried out in compliance with: (1) the EU Directive on clinical trials (2001/20/EC) and (2) the Research Governance Framework for Health and Community Care. These cover the responsibilities of the Sponsor, approval of the trial by the relevant competent authority (MHRA) and Ethics Committee(s) and the conduct of the study according to the principles of Good Clinical Practice. In addition, any clinical trial must be reviewed and monitored by an independent trial monitoring committee. Any experimental therapeutic product would be manufactured in compliance with GMP standards and systems would need to be in place for the recording and investigation of any adverse events occurring during the study and ongoing pharmacovigilance. Written informed consent would be obtained for all participants before commencement on the study and specific documented procedures would be in place for the handling of donor and patient trial samples and archive material. Patient and donor related data would be handled in accordance with the Data Protection Act 1998 and strict confidentiality would be maintained. The issue of compensation in the event of non-negligent harm would be addressed in line with Ethics Committee requirements and appropriate indemnity established.
|
Evolution of defined culture systems supporting hESC isolation |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
Eight years on from the first successful hESC derivation by Thomson et al.(1998) all successful efforts to isolate and expand these cells have continued to involve direct exposure to one or more undefined animal sourced blood or cell products. This includes the use of blood complement to ‘immunosurgically’ lyse blastocyst outer trophectodermal cells in order to recover embryo inner cell mass (ICM), mitotically inactivated mouse embryo fibroblasts (MEF) or MEF-derived extracts for embryo cell attachment and outgrowth, and supplementation of media with bovine serum or serum-derived products (Reubinoff et al. 2000, Lanzendorf et al. 2001, Hovatta et al. 2003, Mitalipova et al. 2003, Cowan et al. 2004, Heins et al. 2004, Klimanskaya et al. 2005, Ludwig et al. 2006). However, many studies have demonstrated the capacity to obviate direct exposure to one or more of these factors. Thus, at least two groups demonstrated hESC derivation from whole embryos without animal immune complement to isolate ICM (Heins et al. 2004, Simon et al. 2005). In place of MEFs, human fibroblasts derived from neonatal foreskin and adult placentas have also been exemplified to serve as feeders for the outgrowth of embryo ICM and whole blastocysts respectively (Hovatta et al. 2003, Simon et al. 2005). The prospect of feeder-free isolation has also been exemplified by growth of embryo ICM on extracellular matrix (ECM) extracted from MEFs (Klimanskaya et al. 2005) and most recently on a combined preparation of human sourced collagen IV, fibronectin, laminin and vitronectin (Ludwig et al. 2006). In our own recent efforts, we have isolated six new hESC lines from whole blastocysts, without exposure to animal immune complement, on an ECM substrate of purified human laminin and transitional reliance on mitotically inactivated human fibroblast feeder cells. Most notably, one of these lines was isolated using a serum-free medium (SFM) containing only human sourced and recombinant proteins (De Sousa et al. 2005, Fletcher et al. 2006). To our knowledge, this is the first exemplification of a new hESC line derived without direct exposure to any animal cell product, and as such represents another significant sequential step in the generation of conditions suitable for the derivation of therapeutic grade stem cells with reduced risk for transmitting zoonotic pathogens.
In the absence of a detailed understanding of cell-culture requirements, serum, normally of bovine origin, is a common media supplement. It provides a range of growth factors capable of stimulating both growth and differentiation and is a valuable nutritional source of protein such as albumin, which has the added side benefit of facilitating cell attachment to substrates. Accordingly, almost all efforts to derive hESC till date have involved supplementation of minimal essential medium with bovine-sourced serum (Thomson et al. 1998, Reubinoff et al. 2000, Lanzendorf et al. 2001, Hovatta et al. 2003, Mitalipova et al. 2003, Pickering 2003, Stojkovic et al. 2004) or bovine serum replacement (bSR; marketed as KNOCKOUT SR, Invitrogen) (Cowan et al. 2004, De Sousa et al. 2005, Klimanskaya et al. 2005, Simon et al. 2005), of which albumin is a major component (Price et al. 1998). The SFM used in our own recent work was based on X vivo 10 (Cambrex, Wokingham Berkshire, UK), a product marketed for human therapeutic applications involving haematopoietic cells, to which basic Fibroblast Growth Factor (bFGF) alone was added (De Sousa et al. 2005, Fletcher et al. 2006). This was based on its capacity to support hESC self-renewal in SFM as well as bSR supplemented media under feeder-free culture conditions (Xu et al. 2001, Li et al. 2005). Although the base composition of this media is proprietary, human insulin, transferrin and albumin are specified in available product information. Recently, Ludwig et al.(2006) published details of their own animal protein-free media supporting hESC derivation. In addition to containing a broad range of inorganic salts, trace minerals, energy substrates, lipids, amino acids and vitamins, this media contains a number of human growth factors and proteins, including bFGF, TGF-ß1, GABA, pipecolic acid, glutathione, insulin, transferrin and albumin. Previous studies developing feeder-free culture environments to support established hESC lines have suggested that TGFß1 and leukaemia inhibitory factor (LIF), and the bone morphogenetic protein (BMP) antagonist Noggin, synergize with bFGF to suppress differentiation and sustain self-renewal in non-conditioned medium supplemented with bSR (Amit et al. 2004, Xu et al. 2005). How universal variations in culture environment and handling are for deriving and sustaining hESC lines remains to be formally evaluated and could be dependent on genetic or epigenetic heterogeneity in the cell lines themselves. Another issue affecting the derivation and expansion of undifferentiated hESCs appears to be the requirement to maintain cell association during passaging. This is reflected initially by a requirement for manual dissection and freezing of cells at high density in order to ensure recovery of cells post-thawing. This is further reflected by poor cloning efficiencies of less than 1% of established lines on or off of feeders (Amit et al. 2000, 2004).
While our and others recent success (Fletcher et al. 2006, Ludwig et al. 2006) in isolating hESCs in media containing only human-sourced recombinant and purified proteins is significant, indirect exposure to animal-sourced tissues used in reagent preparation remains an outstanding safety issue for therapeutic objectives. An example of this is the fact that culture of human fibroblast feeders still normally involves exposure to Low Serum Growth Supplement (LSGS; Cambrex, Berkshire, UK) containing 2% (v/v) fetal bovine serum. Fortunately, this serum is sourced from transmissible spongioform encephalopathy (TSE)-free herds in New Zealand that have been certified by the MHRA and comparable health agencies through the world. However, there is of course no guarantee that this source does not carry other unknown pathogens. Similarly, procedures used by manufacturers in the purification of human proteins such as albumin or ECM proteins from tissues undoubtedly still utilize animal-sourced reagents (i.e. trypsin or collagenase), since these are the most commonly available to manufacturers of research grade material. The future production of therapeutic grade stem cells will thus necessitate reliance on therapeutic grade reagents, whose production may in turn necessitate implementing novel strategies for organic synthesis of molecules or the use of recombinant gene technology in evolutionarily distant species (i.e. plant and insect).
A perception of a prevalence of TSE in the UK in the form of CJD has been an issue which has resulted in US bans on the therapeutic use of blood cell products derived from prospective donors that have resided or been transfused in the UK (i.e. http://www.fda.gov.cber/gdlns/cjdvcjd/pdf). This has the potential to impact on the worldwide acceptance of UK-derived hESCs irrespective of their compliance with emerging UK and EU standards. However, according to recent WHO guidelines on Tissue Infectivity Distribution in transmissible spongioform encephalopathies (September 2005), no detectable TSE infectivity has been reported in reproductive tissues such as testis, prostate, semen, ovary, uterus, placental fluids, fetuses and embryos (http//www.who.int/bloodproducts/tse/WHO%20TSE%20Guidelines%20FINAL-22%20JuneupdatedNL.pdf). Thus, while this remains the case, there is no rationale for discriminating against UK-derived hESCs or products providing that any human or animal serum or serum products used in their culture are obtained from TSE-low risk or free sources.
|
Conclusion |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
|
Acknowledgements |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionExisting and emerging EU/UK...Lessons from haematopoietic stem...Evolution of defined culture...ConclusionAcknowledgementsReferences |
|---|
Amit M, Carpenter MK, Inokuma MS, Chiu C-P, Harris CP, Waknitz MA, Itskovitz-Eldor J & Thomson JA 2000 Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental Biology 227 271–278.[CrossRef][ISI][Medline]
Amit M, Shariki C, Margulets V & Itskovitz-Eldor J 2004 Feeder and serum free culture of human embryonic stem cells. Biology of Reproduction 70 837–845.
Barratt CL, John JC St & Afnan M 2004 Clinical challenges in providing embryos for stem cell initiatives. Lancet 364 115–118.[CrossRef][ISI][Medline]
Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A & Reubinoff BE 2004 Transplantation of human embryonic stem cell derived neural progenitors improves behavioural deficit in Parkinsonian rats. Stem Cells 22 1246–1255.
Bjorklund LM, Sanchez-Pernaute R, Chung S, Andersson T, Ching Chen IY, McNaught KSP, Brownell A-L, Jenkins BG, Wahlestedt C, Kim K-S & Isacson O 2002 Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. PNAS 99 2344–2349.
Cesaro P 2004 The design of clinical trials for cell transplantation into the central nervous system. NeuroRx 1 492–499.[CrossRef][Medline]
Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D & Melton D 2004 Derivation of embryonic stem-cell lines from human blastocysts. New England Journal of Medicine 350 1353–1356.
De Sousa PA, Fletcher JM, Ferrier PM, Gardner JO, Harkness L, Dhanjal S, Harper J, Delhanty J, Serhal P, Lebkowski J, Mandalam R & Wilmut I 2005 Isolation of new human embryonic stem cell lines without direct exposure to animal cell products. 3rd Annual Meeting of International Society for Stem Cell Research. San Fransisco, USA, June 23–25. Abstract no. 227.
Dunnett SB & Rosner AE 2004 Cell therapy in Huntington’s disease. NeuroRx 1 394–405.[CrossRef][Medline]
Fletcher JM, Ferrier PM, Gardner JO, Harkness L, Dhanjal S, Serhal P, Harper J, Delhanty J, Brownstein DG, Prasad Y et al. 2006 Variations in humanised and defined culture conditions supporting derivation of new human embryo stem cell lines. Cloning and Stem Cells [in press].
Gode GR & Bhide NK 1988 Two rabies deaths after corneal grafts from one donor. Lancet 2 791.[ISI][Medline]
Healy L, Hunt C, Young L & Stacey G 2005 The UK Stem Cell Bank: its role as a public research resource centre providing access to well-characterised seed stocks of human stem cell lines. Advanced Drug Delivery Reviews 57 1981–1988.[CrossRef][ISI][Medline]
Heins N, Englund MCO, Sjoblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C & Semb H 2004 Derivation, characterisation and differentiation of human embryonic stem cells. Stem Cells 22 367–376.
Hovatta O, Mikkola M, Gertow K, Stromberg A-M, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M & Ahrlund-Richter L 2003 A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Human Reproduction 18 1404–1409.
Kim J-N, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee S-H, Nguyen J, Sanchez-Pernaute R, Bankiewicz K & McKay R 2002 Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 418 50–56.[CrossRef][Medline]
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD & Lanza R 2005 Human embryonic stem cells derived without feeder cells. Lancet 365 1636–1641.[CrossRef][ISI][Medline]
Kofidis T, Lebl DR, Swignenburg R-J, Greeve JM, Klima U, Gold J, Xu J & Robbins RC 2006 Allopurinol/uricase and ibuprofen enhance engraftment of cardiomyocyte-enriched human embryonic stem cells and improve cardiac function following myocardial injury. European Journal of Cardio-thoracic Surgery 29 50–55.[CrossRef][Medline]
Lanzendorf SE, Boyd CA, Wright DL, Muasher S, Oehninger S & Hodgen GD 2001 Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines. Fertility and sterility 76 132–137.[CrossRef][ISI][Medline]
Li Y, Powell S, Brunette E, Lebkowski J & Mandalam R 2005 Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnology and Bioengineering 91 688–698.[CrossRef][ISI][Medline]
Lie S, Qu Y, Stewart TJ, Howard MJ, Chakrabortty S, Holekamp TF & McDonald JW 2000 Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. PNAS 97 6126–6131.
Loeffler M & Potten CS 1997 Stem cells and cellular pedigrees – a conceptual introduction. In Stem Cells, pp 1–27. Ed. CS Potten. London: Academic Press.
Ludlam CA, Powderly WG, Bozzette S, Diamond M, Koerper MA, Kulkarni R, Ritchie B, Siegal J, Simmonds P, Stanley S et al. 2006 Clinical perspectives of emerging pathogens in bleeding disorders. Lancet 367 252–261.[CrossRef][ISI][Medline]
Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS et al. 2006 Derivation of human embryonic stem cells in defined conditions. Nature Biotechnology 24 185–187 (1 Jan Epub ahead of print).[CrossRef][ISI][Medline]
MacLaren RE, Ali RR & Thrasher AJ 2005 Risks of gene therapy should be weighed against lack of alternatives for many diseases. BMJ 330 791.
McDonald JW, Liu X-Z, Qu Y, Lie S, Mickey SK, Turetsky D, Gotlieb DI & Choi DW 1999 Transplanted embryonic stem cells survive, differentiate, and promote recovery in injured rat spinal cord. Nature Medicine 5 1410–1412.[CrossRef][ISI][Medline]
Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A & Stice S 2003 Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21 521–526.
Parker NR, Barralet JH & Bell AM 2006 Q fever. Lancet 367 679–688.[CrossRef][ISI][Medline]
Pickering S 2003 Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reproductive Biomedicine Online 7 353–364.[Medline]
Price PJ, Goldsborough MD & Tilkins ML 1998 Embryonic stem cell serum replacement. In International Patent Application WO 98/30679.
Reubinoff BE, Pera MF, Fong C-Y, Trounson A & Bongso A 2000 Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nature Biotechnology 18 399–404.[CrossRef][ISI][Medline]
Simon C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, Asensi A, Sanchez E, Explugues J, Fisher S & Pellicer A 2005 First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertility and Sterility 83 246–249.[CrossRef][ISI][Medline]
Simonds RJ, Holmberg SD, Hurwitz RL, Coleman TR, Bottenfield S, Conley LJ, Kohlenberg SH, Castro KG, Dahan BA, Schable CA et al. 1992 Transmission of human immunodeficiency virus type 1 from a seronegative organ and tissue donor. New England Journal of Medicine 326 726–732.[Abstract]
Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA & Martin F 2000 Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49 157–162.[Abstract]
Stojkovic M, Lako M, Stojkovic P, Steward R, Przyborski S, Armstrong L, Evans J, Herbert M, Hyslop L, Ahmad S et al. 2004 Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22 790–797.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM 1998 Embryonic stem cell lines derived from human blastocysts. Science 282 1145–1147.
Weiss RA 2003 Cross-species in fections. Current Topics in Microbiology and Immunology 278 47–71.[ISI][Medline]
Wikland M & Sjoblom C 2000 The application of quality systems in ART programs. Molecular and Cellular Endocrinology 166 3–7.[CrossRef][ISI][Medline]
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD & Carpenter MK 2001 Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology 19 971–974.[CrossRef][ISI][Medline]
Xu RH, Peck RM, Li DS, Feng X, Ludwig T & Thomson JA 2005 Basic FGF and suppression of BMP signalling sustain undifferentiatied proliferation of human ES cells. Nature Methods 2 185–190.[CrossRef][ISI][Medline]
This article has been cited by other articles:
 |
|
 |
|
  L. E Young Focus on stem cells in reproduction. Reproduction, November 1, 2006; 132(5): 671 - 672. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
