SEARCH

SEARCH BY CITATION

Keywords:

  • Blastocyst;
  • disease modelling;
  • drug discovery;
  • embryonic stem cells;
  • preimplantation genetic diagnosis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Embryos surplus to therapeutic requirements following preimplantation genetic diagnosis can be used to derive human embryonic stem cell (hESC) lines carrying mutations significant to human disease. These cells provide a powerful in vitro tool for modelling disease progression in a number of cell types as well as having the potential to revolutionise drug discovery. Robust and reproducible directed differentiation protocols are needed to maximise the potential of these cells. In this review, we explore the current use of hESC and induced pluripotent stem cells in disease-specific research and discuss the use of stem cell technology in drug discovery and toxicity testing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

There has been continued substantial interest in the therapeutic and scientific potential of human embryonic stem cells (hESC) since the first report of their isolation in 1998.1 Social scientists and ethicists, in addition to embryologists and stem cell biologists, have become involved in response to concerns from the public and religious groups and from pro-life factions and medical associations. Since 1998, the hESC field has expanded rapidly. Besides the potential for therapeutic benefit, various research groups have been working on derivation methods and their efficiency, on striving to remove animal products from culture to enable cells to be used in therapy, on differentiation and genetic modification studies, on the potential of hESC as drug discovery tools and for identifying novel pharmaceuticals with high-throughput drug screens in vitro. hESCs have also been proposed as invaluable clinically relevant alternatives to animal models for studying early development and degenerative diseases. In addition, investigation into significant clinically relevant genetic disorders can be facilitated by the use of disease-carrying hESCs obtained from affected embryos following preimplantation genetic diagnosis (PGD).

The origins of stem cell research

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

The first report describing embryonic stem cells (ESC) was in 1966, when Cole et al.2 described long-lived and stable cells obtained from either whole or dissected rabbit blastocysts. Different colonies of these cells expressed high alkaline phosphatase activity, underwent 200 generations while remaining diploid or displayed outgrowths containing blood islands, muscle, connective tissue, neurons, macrophages and other undefined tissues. All the cell lines they obtained possessed large nuclei and distinct nucleoli and retained typical morphological, karyotypic and enzymatic characteristics throughout successive generations. A number of seminal papers soon followed from Cambridge University, with Gardner describing the generation of the world’s first chimeric mouse with its distinctive coat-colour pattern in 19683 by the injection of inner cell mass (ICM) cells into the blastocoelic cavity, and the landmark paper by Evans and Kaufman4 describing the establishment in culture of pluripotent cells from mouse embryos.

hESCs are derived most commonly from the ICM of human preimplantation blastocysts at days 5 to 8 of development. The first report of ESC lines derived from human blastocysts was in 1998.1 Thomson et al. used 36 human embryos to derive five hESC lines. The lines were fully characterised, including expression of cell-surface markers that identify undifferentiated cells, an ability to differentiate into tissues from all three germ layers, and teratoma formation on transplantation into severe combined immunodeficient mice. From one line in 1998, there is now anecdotal evidence of over 300 lines worldwide,5 derived using a variety of methods to isolate the ICM such as immunosurgery, acid, laser or mechanical means, or by plating the whole embryo in culture and isolating cells with stem-like morphology at a later stage.

Use of embryos from routine in vitro fertilisation

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Embryos used in stem cell research most commonly are donated by patients undergoing in vitro fertilisation (IVF) procedures to alleviate infertility. In general, these embryos are reported in derivation publications as being ‘surplus’ to the IVF cycle of the donor couple being deemed unsuitable for use or cryopreservation on day 3 of development. However, morphology on day 3 is not a particularly good indicator of developmental competence of the embryo6,7 nor is it a good predictor of developmental progression to the blastocyst stage8 (Figure 1). Given that the requirement for efficient derivation of hESC is a progressive blastocyst with a visible ICM, embryos considered suitable for derivation attempts are blastocysts that would be also be suitable for transfer to patients in their pursuit of pregnancy, or for cryopreservation for their future use. Thus, in this context, it is ethically questionable whether the use of ‘surplus’ embryos for stem cell derivation is in the best interest of the IVF patients trying to maximise their chances of pregnancy. The general shift towards single blastocyst transfer, which not only carries excellent prospects for pregnancy,9 but will also address the epidemic of multiple pregnancy following IVF, adds credence to this moral stance. Another advantage to this strategy is the increase in the proportion of cycles where supernumerary embryos are available for cryopreservation,9 thereby enhancing the cumulative chance of pregnancy from a single cycle of ovarian stimulation and oocyte retrieval. As increasing numbers of patients will choose or be offered extended culture to day 5, day 3 ‘surplus’ embryos will no longer be available even for those research groups willing to use this source for stem cell derivation. Therefore, certainly in the UK, the ethical source of embryos for research will shift to those embryos that have been cryopreserved for future use by the patients who then decide they no longer wish to use them in treatment or the use of embryos unsuitable for transfer following preimplantation diagnosis of serious genetic disease. In other countries such as the USA, legislation is in place reflecting fundamental objections to any embryo research that restricts the use of human embryos regardless of the source.10

image

Figure 1. (A) An embryo on day 3 of development deemed of too poor quality to cryopreserve. (B) The same embryo on day 6 having successfully developed into a blastocyst.

Download figure to PowerPoint

Use of embryos from PGD

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

PGD was developed as an alternative to prenatal diagnosis for fertile couples carrying serious genetic disorders.11 A representative cell is removed from an IVF-generated embryo from the couple and tested for the specific genetic defect before implantation. Only embryos found to be unaffected by the genetic disorder, or those that are carriers in the case of recessive diseases, are replaced into the patient or cryopreserved for their later use. The first births of PGD children were reported 1990.12 In these initial cases, polymerase chain reaction (PCR) was used for gender determination for patients carrying X-linked diseases. Blastomeres are now tested using either PCR in single gene disorders or fluorescence in situ hybridisation for sex-linked diseases or translocations. The development of preimplantation haplotyping as a universal method of amplifying and testing DNA from single cell biopsies13 has significantly increased the repertoire of disorders which can now be offered for PGD.14

Embryos found unsuitable for replacement because they are at high risk of transmitting the genetic disorder would normally be discarded, despite often being of good progressive quality and capable of forming blastocysts. These blastocysts may be suitable for stem cell derivation and are free from the ethical difficulties associated with using ‘surplus’ embryos from patients seeking infertility treatment.15 For those individuals with a fundamental objection to embryo research however, these embryos are no less ethically problematic.

The potential of hESCs with mutations significant to human disease has provoked substantial interest from patient groups and scientists alike. These cells can provide a powerful in vitro tool for modelling disease progression, identifying molecular mechanisms that may be blocked to prevent this progression, studying the pathogenesis in specific cell types following differentiation as well as providing an ideal system for investigating in vitro toxicity and the efficacy of drugs. The strategy of using PGD embryos as models for studying genetic disease is predicated on the assumption that disease-carrying hESCs will differentiate faithfully and reliably into relevant differentiated cells that are functionally competent.

Generation of hESC lines from PGD embryos

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Success has already been reported in deriving these lines. Pickering et al.16 reported the generation and characterisation of a hESC line carrying the most common cystic fibrosis mutation Δ508. The line showed the same morphology and protein expression as unaffected lines and provides the opportunity to test therapies relevant to cystic fibrosis as well as investigating the progression of the disease with the differentiation of the cells to pulmonary phenotype. At around the same time, Mateizel et al.17 reported the derivation of three hESC lines carrying genetic disease—one with the mutation for myotonic dystrophy type 1, one carrying cystic fibrosis and another with Huntington’s disease (HD). Verlinsky et al. reported in 200518 the derivation of 18 hESC lines with genetic disorders, including adrenoleukodystrophy, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy, fragile-X syndrome and thalassaemia. The cells are in a repository that may be available on request. hESC lines with genetic disorders serve as an unlimited source for the study of the disease; therefore, the importance of the continued use of these embryos for stem cell derivation should not be underestimated. However, pluripotency is a double-edged sword; the same plasticity that allows hESC to differentiate into hundreds of cell types also makes them difficult to control. Differentiation protocols often result in a mixture of cell types, and expression of undifferentiated markers can persist even at the completion of a protocol. The impact of this on clinical use would be that undesired, therefore potentially deleterious, cell types could be transplanted, and that any cells retaining the ability for exponential growth could cause tumorigenesis. Therefore, it is crucial that robust and reproducible differentiation protocols continue to be developed to fully exploit the potential of hESC as disease models in a safe and efficacious manner. In addition to developing appropriate protocols to generate clinically relevant cell populations, international collaboration is needed to ensure that the route to the clinic is as rapid as possible. Examples of such alliances include the development of internationally agreed characterisation,19 universally agreed standards for dissemination of results20,21 and the formation of international working groups to address each aspect of the route from the laboratory to the patient, such as the European Human Embryonic Stem Cell Registry (www.hescreg.eu), The International Society for Stem Cell Research (www.isscr.org) and the International Stem Cell Initiative.22

Approaches to disease models and stem cell therapy

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

There are several approaches to develop models for human disease. Primary cultures of relevant cell types can be isolated from patients, but they are generally difficult to obtain and is a challenge to obtain fully matched control cells. Samples obtained postmortem only represent a single point in the disease and are commonly advanced disease stages; presymptomatic or early-stage specimens are rare. There is a limited range of tissues from which primary cells can be obtained and the replication span of the cells before senescence is reached, or before unwanted transformation occurs, can be brief.

Alternatively, genetically engineered animals are used to generate research models, of which the most popular is the use of mouse models. These models have generated valuable data and enabled the rapid accumulation of knowledge for many diseases. In mouse models, specific human genes or gene fragments can be ‘knocked-in’ or endogenous genes ‘knocked-out’ to study the outcome with respect to disease pathophysiology and progression. In addition, combinations of transgenic mice can be generated through breeding patterns to establish a genotype and/or phenotype relevant for disease studies. Whole animal models have the advantage of allowing simultaneous study of the disease effect in multiple organs/cell types, and particularly when using mice, large numbers of animals can be made available relatively quickly for experimentation. However, experiments in mice are long, costly and severely limited in the extent to which pathways can be manipulated. Unfortunately, many of these models do not faithfully represent the abnormal phenotype as manifested in humans, and therapeutic achievement in animal models is not always successfully repeated in human studies. Moreover, the insertion of gene fragments to cause overexpression to recapitulate the disease in animal models (e.g. in HD)23 tends to lead to a shortened time span before disease onset, but can also lead to premature death of the animal and loss of pathologic specificity. In response to these concerns, ESC have been suggested as an alternative disease model to complement animal studies. As a model of disease progression, naturally occurring mutations in hESCs from PGD embryos represent an even more relevant model than genetically engineered ESC or mouse models for elucidating the pathophysiological mechanisms that underlie a genetic disease as the mutant protein is expressed in its normal physiological context and range of expression pattern.

hESC as disease models

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

The suitability of ESC as models has been validated for CAG repeat diseases, the most common of which is HD. Using genetically engineered murine ESC (mESC), research has shown that mESC expressing long CAG motifs are able to differentiate into neurons but are characterised by stunted neurite outgrowth, low efficiency of neuronal formation, decreased survival during differentiation and exhibit redistribution of polyQ-containing proteins consistent with those observed in CAG repeat disorders when compared with ESC lines without the expanded CAG mutation.24 Many models of CAG repeat disorders are based on the overexpression of mutant proteins, in many cases transiently, that may not accurately reflect the pathology in human neural populations. By using modified ESC, stable and relevant population of neuronal cells have been successfully generated for study.24 Similarly, the feasibility of generating spinal muscular atrophy (SMA) cell-based assay using neural lineages derived from hESC with transient transfection of proliferating neuroprogenitors with plasmid DNA or adenoviral vectors has been assessed. Results indicated that hESC-derived neuroprogenitors provided a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.25

However, it is important to address basic but crucial questions with regard to the use of hESC in disease research. Bearing in mind the late onset of diseases such as HD, it is essential to assess whether neurons and neuroprogenitors differentiated in vitro from HD-hESCs faithfully express Huntington’s phenotypes as assayed by cell-based analyses, transcriptomics and proteomics (Figure 2). Similarly, it needs to be confirmed that muscle cells, including cardiomyocytes, differentiated in vitro from, for example, DMD-hESCs and from SMA-hESCs, faithfully express their phenotypes. Furthermore, it must be established whether, for example, neuronal cells differentiated from HD-hESCs and muscle cells from DMD-hESCs and SMA-hESCs are equivalent or superior to the respective animal disease models. It then remains to be seen whether HD-SMA neurons and DMD myocytes and SMA myocytes accelerate clinically relevant drug discoveries, while reducing toxicity, because the initial screen is performed with human (rather than typically rodent) cells and tissues.

image

Figure 2. (A) Passage 2 of a hESC line derived from a PGD embryo affected with HD. (B) CAG repeat analysis showing 47 repeats in the HD-hESC line. (C) Neural precursors differentiated from HD-hESC stained for the neural marker nestin (red) and nuclear marker DAPI (blue).

Download figure to PowerPoint

Pharmaceutical research and stem cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Because of their fundamental attributes of unlimited expansion and pluripotency, hESC, particularly those carrying clinically relevant mutations, have gained considerable interest from the biopharmaceutical sector. Investigators have begun to consider stem cells as a new source of predictive, cell-based assays in drug discovery and significant advances in the application of these cells have been reported. These advances are aligned with three important stages of pharmaceutical research: target discovery and validation, identification of efficacious chemical leads and drug safety pharmacology.26 The current pharmaceutical discovery process is universally accepted as being time consuming and inefficient with an accompanying high financial burden. Improvements to the discovery phase of new compounds will come through the application of more appropriate, disease-orientated cellular screens, for both therapeutic target validation and optimisation of compounds following their discovery through high-throughput screens.27 High-throughput screening (HTS) is an approach to drug discovery that uses the process of assaying a large number of potential effectors of biological activity against targets. The goal of HTS is to accelerate drug discovery by screening large libraries often composed of hundreds of thousands of drug candidates. Using robotics, data processing and control software, liquid handling devices and sensitive detectors, HTS allows the rapid running of millions of biochemical, genetic or pharmacological tests. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology or disease progression. High-throughput clonal microarrays compatible with screens for proliferation, subcellular protein localisation, cell morphology, cell signalling and differentiation have already been developed for investigating functional genomics of hESC.28 In HD studies, HTS has been designed to isolate compounds that inhibit stages of protein misfolding and aggregation, with one identifying the benzothioazoles as a potentially interesting class of compounds in an anti-aggregation screen.29

The completed sequence of the human genome has resulted in target opportunities to deliver the next generation of medicines. The challenge is to identify the right target mechanism and the right molecule to modulate that target in a clinically safe and effective manner.30 Within the toxicological field, ESC are already being used.31 The EST test for developmental toxicology is an in vitro embryotoxicity test in which test reagents are assessed according to three end-points: inhibition of the differentiation of ESC into contracting myocardium, cytotoxicity in ESC and cytotoxicity of mouse 3T3 fibroblasts that serve as a comparison for differentiated cells. hESC carrying known mutations will be important for exploring pathophysiological effects produced by gene-dosage anomalies and how these might in the future be addressed to ameliorate the phenotype in conditions such as trisomy 21.32 In addition, both mouse and hESC-like derivatives of particular lineages can be used for early-stage assessment of drug adsorption, metabolism and toxicity. Stem cells for Safer Medicine is a public–private collaboration with the aim ‘…to enable the creation a bank of stem cells, open protocols and standardised systems in stem cell technology that will enable consistent differentiation of stem cells into stable homogenous populations of particular cell types, with physiologically relevant phenotypes suitable for toxicology testing in high throughput platforms’ (www.sc4sm.org).

Induced pluripotent stem cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Although hESC lines derived from affected PGD embryos are not subject to the same moral controversies as those derived during fertility treatment, the use of human embryos in research generally causes ethical objections that could hinder the universal application of hESC disease models or therapy. In addition, while the number of diseases able to be screened with PGD is ever growing, stem cell models of some late-onset degenerative diseases such as Parkinson’s, motor neuron disease or Alzheimer’s disease, for which no single predictive gene has been identified, will not be available through PGD. Successful reprogramming of human somatic cells into a pluripotent state would allow the creation of disease-specific stem cells for a huge range of human diseases. Furthermore, such technology would enable the production of patient-specific cells, which are required to fully exploit the application of stem cell therapy. Somatic cell nuclear transfer is the alternative approach to generating patient-specific cells for therapy. The creation of blastocysts has been achieved with this method but derivation attempts to produce ESC have not yet proven successful.33,34

In a groundbreaking publication Yamanaka and colleagues described the induction of pluripotent stem cells from adult human fibroblasts by defined factors.35 By retroviral insertion of Oct4, Sox2, Klf4 and c-Myc into human dermal fibroblasts, the resulting reprogrammed induced pluripotent stem (iPS) cells were similar to hESC in morphology, proliferation, surface antigens, gene expression and differentiation ability. However, iPS cells were not identical; DNA microarray analyses detected differences between the two pluripotent stem cell lines. The claim that the immense complexity of nuclear reprogramming could be reduced to the action of four factors was understandably met with scepticism. However, in less than 1 year, Yamanaka’s methods have been validated in multiple laboratories and the efficiency improved dramatically.36 Indeed, the same group have reported the generation of iPS cells from adult mouse hepatocytes and gastric epithelial cells,37 and demonstrated that there were no common retroviral integration sites between the iPS clones. This suggests that iPS cells are generated by direct reprogramming and that retroviral integration into specific sites is not required.

iPS cells as disease models

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

The first report of the use of iPS cells as a model for the study and treatment of genetic disease has been published by Hanna et al.38 By using a humanised sickle cell anaemia mouse model, they showed that mice can be rescued after transplantation with haematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle haemoglobin allele by gene-specific targeting. This provides proof of principle for using transcription-factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The proof of principle that iPS cells can be generated directly from elderly patients with chronic disease using material that has been exposed to disease-causing agents for a lifetime has been reported.39 iPS cells were produced using skin fibroblasts obtained from an 82-year-old patient diagnosed with a familial form of amyotrophic lateral sclerosis (ALS) and used to generate patient-specific motor neurons and glia, the cell types implicated in ALS pathology.

Indeed, all the advantages of using PGD hESC as disease models also apply to the use of iPS cells with the added attraction of a greater number of diseases, the easier availability of starting material, the use of samples from the diseased tissue and from donors in the age range when the disease occurs. Being able to determine minimum factors needed for successful dedifferentiation will inevitably shed light on the maintenance of pluripotency in hESC culture. These cells will enable investigation into the rigidity of lineage relationships. For example, it may not be necessary to dedifferentiate the cells all the way back to an embryonic phenotype; it may perhaps be possible to switch lineages before this point. However, there are serious drawbacks with the current methods of iPS production even when normal cells are used. The genes are inserted into the adult cells by use of a retrovirus, clearly precluding the use of these cells clinically until an alternative vector is validated. C-Myc and klf4 are proto-oncogenes that govern the balance between cellular proliferation and differentiation. Hence, neoplasia is of major concern with a real danger of carcinogenesis if proliferation and differentiation is not (or cannot be) controlled in this cell population. Indeed, 20% of mice created by germ line transmission of iPS cells developed tumours attributable to reactivation of c-myc.40 iPS cells have subsequently been generated without the use of c-Myc, albeit at a lower efficiency.41 Although these are likely to be temporary issues, further studies are essential to determine whether human iPS cells can replace hESC in medical applications.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References

Many investigators no longer see cell therapy as the first goal of hESC research.42 Instead, it is considered that mutant hESC or iPS cells may serve as an excellent research tool to study the mechanism of disease. As both hESC and iPS cells can be differentiated into a wide variety of cell types with increasing efficiency and specificity, the expression pattern and pathophysiology of a disease, and drug and toxicity testing can be studied in a variety of cells types from the same line carrying the relevant disease. The creation of new hESC and iPS cell lines, with or without known genetic mutations, is therefore of utmost importance for further research into the fundamental biological processes in these diseases.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The origins of stem cell research
  5. Use of embryos from routine in vitro fertilisation
  6. Use of embryos from PGD
  7. Generation of hESC lines from PGD embryos
  8. Approaches to disease models and stem cell therapy
  9. hESC as disease models
  10. Pharmaceutical research and stem cells
  11. Induced pluripotent stem cells
  12. iPS cells as disease models
  13. Conclusion
  14. Disclosure of interests
  15. Contribution to authorship
  16. Details of ethics approval
  17. Funding
  18. References