. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol. 2003 Oct;21(10):1200-7. PubMed.

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  1. Embryonic Stem Cells for Parkinson’s Disease

    This study gives further evidence that embryonic stem cells may provide a feasible source of dopamine neurons that could be used for cell replacement therapy for Parkinson’s disease (PD). Parkinson’s disease currently affects approximately one percent of people over the age of 60 worldwide. Current treatment strategies for the disease show variable success, with drugs such as L-dopa having limited efficacy later in the disease process, often with disturbing side effects to patients.

    Therefore, long-term recovery has been sought using cell transplantation therapies, using embryonic neurons to replace the dying or lost dopamine neurons in the diseased brain. While research has shown that this approach is feasible, and clinical trials have shown effectiveness of embryonic neuron grafts for more than 10 years to date, the restricted supply of fetal tissue, coupled with the ethical issues surrounding its use, means that alternative sources of cells must be found in order to make this therapy more widely available throughout the world.

    Embryonic stem cells, derived from the blastocyst stage of the early embryo, offer a potential alternative source of dopamine neurons. These cells are able to self-renew over long periods and are pluripotent, i.e., can form cell types from all three germ layers (endoderm, mesoderm, and ectoderm). ES cells have been used in previous studies to produce dopamine neurons that were shown to integrate into the brain and restore some normal function after transplantation to a mouse model of PD (Kim et al., 2002; Björklund et al., 2002). However, both earlier studies had significant drawbacks. Kim et al. required their ES cells to overexpress the gene Nurr1 to differentiate efficiently into dopamine neurons, whereas Björklund et al. saw the formation of teratomas in some of their transplants, highlighting safety issues, and the need to remove all dividing ES cells from the cultures prior to transplantation.

    The current study by Barberi and colleagues moves us a step closer to the goal of finding an alternative cell source for dopamine neurons. In this study, they use stromal feeder layers and addition of neurotrophic factors to the ES cells in culture to enhance differentiation down a particular neuronal or glial pathway. In this way, the authors are able to enrich for populations containing large numbers of dopamine neurons or other specific cell subtypes. The in-vitro studies were well-executed using a number of different measures (immunocytochemistry, electron microscopy, and electrophysiology) to define accurately the presence of functional neurons in the culture dish. In addition, transplantation studies to a mouse model of PD showed efficacy of the neurons in reduction of amphetamine-induced rotation, suggesting that the neurons had become integrated in the host brain. In addition, some of the ES cell lines were derived from cloned mouse embryos, showing that these cells are equally capable of generating dopamine neurons.

    Interestingly, when comparing these ES-derived neurons to previous studies using embryonic dopamine neuron transplants or ES cells (Björklund et al., 2002), an overcompensation of rotation was not observed despite the large numbers of dopamine neurons detected in the grafts (approximately 40,000). Therefore, it is not clear whether the neurons derived from these ES cells are fully mature or as efficiently integrated in the host brain as would be required for normal function to be restored. Equally, although no excessive growth was seen from the transplants, the limited survival period of eight weeks needs to be extended in order to confirm no long-term issues of contamination of the grafts either with dividing ES cells, or the stromal cells used in the feeder layer in culture.

    Despite these and other issues that may still need to be resolved, this study provides further evidence that ES cells may be a realistic source for dopamine neurons, and takes us a further step closer to their efficient generation to provide a reliable, reproducible, and safe cell type that could be used in future treatments for PD. Equally, the use of cloned embryos to generate ES cells which then can differentiate into dopamine neurons adds more strength to the argument that therapeutic cloning may have a real value to generate cells that could be widely used in many different disease therapies.

    References:

    Kim JH, Auerbach JM, Rodríguez-Gómez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sánchez-Pernaute R, Bankiewicz K, McKay R. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature. 2002 Jul 4;418(6893):50-6. Abstract

    Bjorklund LM, Sánchez-Pernaute R, Chung S, Andersson T, Chen IY, McNaught KS, Brownell AL, Jenkins BG, Wahlestedt C, Kim KS, Isacson O. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2344-9. Abstract

  2. This paper is an extraordinary achievement. Studer et al. have built on the work of Sasai and colleagues in Japan who showed that embryonic stem cells (ES cells) can differentiate into dopamine neurons when grown on a special cell feeder layer (Sasai, 2002). The Studer group has found what appears to be a better cell support layer than the Japanese used, the MS5 cell. They have shown that ES cells are efficiently transformed into dopamine neurons, and that those neurons can survive transplantation into a mouse model of Parkinson's disease and improve movement in the mice in a way similar to fetal dopamine cell transplants. Their manufactured cells, therefore, appear to satisfy the criteria for being authentic dopamine neurons.

    The icing on the cake of this paper is that they showed the same procedure worked with cells developed by "therapeutic cloning" techniques. They reprogrammed mouse cells via transfer of the nucleus into an unfertilized egg, created a new line of ES cells, and showed those ES cells could be made into dopamine neurons. If the same technology were applied to humans, it would be possible to create dopamine neurons that are genetically identical to the patient needing the nerve cells.

    For dopamine neurons and other brain cells, it is probably unnecessary to clone cells for a specific person, because brain cells are not ordinarily rejected. Nonetheless, if it was practical, generating cells that were immunologically identical would be desirable.

    If these same methods can be used for human ES cells, it will be possible to generate an unlimited supply of dopamine neurons for transplant into patients with Parkinson's disease and bypass the need to recover cells from aborted fetal tissue. Importantly, laboratory-produced dopamine neurons can undergo rigorous quality control during manufacturing.

    Some patients have had dyskinesias after fetal dopamine cell transplants (15 percent of patients in our study). Before they entered our study, nearly every patient had dyskinesias caused by L-dopa. Transplants reproduced the effects of L-dopa, including dyskinesias, in some of the patients. They did not cause dyskinesias de novo (see ARF related news story). It is possible that doing transplants earlier in the disease before dyskinesias develop may be a way of dealing with that complication.

  3. I regard this work as very interesting and important for showing how somatic cell nuclear transfer can be used properly for therapies, and how it can augment the potential of stem cells, particularly ES cells. The work is very well done.

  4. This is a nice paper which represents a significant step forward, in my opinion.
    Stem cells have been touted as a theoretical panacea for a variety of neurological disorders, but their practical use has been limited by issues including a ready source, methods of efficient differentiation, and the issue of rejection of non-isogenic transplants.

    The results presented in this manuscript offer a possible solution. The authors show that ES cells can be used to obtain a variety of different phenotypes by simply altering culture conditions, and that the efficiency is much higher than reported in previous publications. The authors further show that cells derived from ES cells can integrate and differentiate in a model of neurological disease and that clear functional benefits can be seen. Finally, the authors offer a possible solution of the immune rejection issue by showing that ES lines obtained after nuclear transfer can also survive, differentiate, and integrate. Thus, a possible source for cell therapy for all individuals likely exists.

    The results are intriguing and exciting. Portions of these results have earlier been reported by other groups, but this is the first report, to my knowledge, where each aspect of the process has been performed. While there is excitement in the field, and certainly this represents an important step forward, it is also important to remember that additional problems remain. The field still needs to achieve migration, integration, and removal of all unwanted phenotypes. In the case of Alzheimer’s disease in particular, the issues of whether the cells will survive in a hostile environment, will not succumb to the disease process, and will integrate with sufficient specificity to improve function remain to be determined. I note, however, that a triple-mutant model for Alzheime'rs disease has been developed where such questions could be addressed empirically (see Oddo et al., 2003).

    In the case of Parkinson's disease, I would suggest that these results bring us much closer to therapy than before. There is clear precedent for cell therapy being of benefit, although research and clinical application have been hampered by scarcity of tissue and its unpredictable availability. Having defined populations of virtually unlimited numbers of cells that have a documented therapeutic benefit makes this sort of therapy less esoteric and more of an FDA-type regulated product.

    Overall, I am impressed with the work, though it would have been nice to have obtained better quantitation of the results to better evaluate how close or far we are from a potential therapy. I await results with human ES cells to confirm that similar strategies will work there, as well.

    I note that nuclear transfer, even for therapeutic purposes, is under review. Perhaps this report, which illustrates the potential benefits of such a technology, will provide impetus to reconsider a proposed ban.

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