WASHINGTON (Reuters) - Injecting human embryonic stem cells into the brains of Parkinson's disease patients may cause tumors to form, U.S. researchers reported on Sunday.

Steven Goldman and colleagues at the University of Rochester Medical Center in New York said human stem cells injected into rat brains turned into cells that looked like early tumors.

Writing in the journal Nature Medicine, the researchers said the transplants clearly helped the rats, but some of the cells started growing in a way that could eventually lead to a tumor.

Various types of cell transplants are being tried to treat Parkinson's disease, caused when dopamine-releasing cells die in the brain.

This key neurotransmitter, or message-carrying chemical, is involved in movement and Parkinson's patients suffer muscle dysfunction that can often lead to paralysis. Drugs can slow the process for a while but there is no cure.

The idea behind brain cell transplants is to replace the dead cells. Stem cells are considered particularly promising as they can be directed to form the precise desired tissue and do not trigger an immune response.

Goldman's team used human embryonic stem cells. Taken from days-old embryos, these cells can form any kind of cell in the body. This batch had been cultured in substances aimed at making them become brain cells.

Previous groups have tried to coax stem cells into becoming dopamine-releasing cells.

Goldman's team apparently succeeded and transplanted them into the rats with an equivalent of Parkinson's damage. The animals did get better.

But the grafted cells started to show areas that no longer consisted of dopamine-releasing neurons, but of dividing cells that had the potential to give rise to tumors.

The researchers killed the animals before they could know for sure, and said any experiments in humans would have to be done very cautiously.

Scientists have long feared that human embryonic stem cells could turn into tumors, because of their pliability.

Opponents of embryonic stem cell research cite such threats. Many opponents, including President George W. Bush and some members of Congress, believe it is immoral to destroy human embryos to obtain their stem cells.

Scientists are aware of this pitfall and are looking for ways to control the ES cells. For example:

"There is a risk with using immortalized cells — they may be more likely to develop into tumour cells. A way around this problem is exemplified by the work with immortalized liver cells, which were generated with a 'cell-suicide' gene that could be activated by administering a drug to the rats. Strategies of this kind could allow cell numbers to be controlled and immortalized cells to become a clinically useful technology."

From:
Nature 406, 361-364 (27 July 2000) | doi:10.1038/35019186
Stem cells — hype and hope

Ron McKay

Studies of stem cells will help in understanding the development and function of organs in mammals. They may also offer a way of treating diseases ranging from liver failure to Parkinson's disease.

http://www.nature.com/nature/journal.../406361a0.html

Neural stem cells may be able to treat brain tumors:

PNAS | November 7, 2000 | vol. 97 | no. 23 | 12846-12851

Neurobiology
Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas


Karen S. Aboody*,dagger ,Dagger , Alice Browndagger , Nikolai G. Rainovdagger , Kate A. Bower*,
* Departments of Neurology, Pediatrics, and Neurosurgery, Children's Hospital; dagger Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital; and § Brain Tumor Service, Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; and Dagger Layton Bioscience, Sunnyvale, CA 94086

Communicated by Richard L. Sidman, Harvard Medical School, Southborough, MA, July 24, 2000 (received for review May 19, 2000)

One of the impediments to the treatment of brain tumors (e.g., gliomas) has been the degree to which they expand, infiltrate surrounding tissue, and migrate widely into normal brain, usually rendering them "elusive" to effective resection, irradiation, chemotherapy, or gene therapy.

We demonstrate that neural stem cells (NSCs), when implanted into experimental intracranial gliomas in vivo in adult rodents, distribute themselves quickly and extensively throughout the tumor bed and migrate uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells, while continuing to stably express a foreign gene.

The NSCs "surround" the invading tumor border while "chasing down" infiltrating tumor cells. When implanted intracranially at distant sites from the tumor (e.g., into normal tissue, into the contralateral hemisphere, or into the cerebral ventricles), the donor cells migrate through normal tissue targeting the tumor cells (including human glioblastomas).

When implanted outside the CNS intravascularly, NSCs will target an intracranial tumor. NSCs can deliver a therapeutically relevant molecule---cytosine deaminase---such that quantifiable reduction in tumor burden results.

These data suggest the adjunctive use of inherently migratory NSCs as a delivery vehicle for targeting therapeutic genes and vectors to refractory, migratory, invasive brain tumors. More broadly, they suggest that NSC migration can be extensive, even in the adult brain and along nonstereotypical routes, if pathology (as modeled here by tumor) is present.

This study is scary. No problem with the rat brain, but tumor in the mouse brain. And we are neither.

Journal of Cerebral Blood Flow & Metabolism (2003) 23, 780–785; doi:10.1097/01.WCB.0000071886.63724.FB
Host-Dependent Tumorigenesis of Embryonic Stem Cell Transplantation in Experimental Stroke

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models.

Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion.

In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells.

The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

Poor mice!

Rheumatology 2003; 42: 162-165
© 2003 British Society for Rheumatology
Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint
S. Wakitani, K. Takaoka, T. Hattori1, N. Miyazawa2, T. Iwanaga2, S. Takeda2, T. K. Watanabe2 and A. Tanigami3
Pharmaceutical Co. Ltd, Otsu, Japan.

Objective. To determine whether the joint space is a suitable environment for embryonic stem (ES) cells to grow and form cartilage.

Method. We transplanted ES cells into the knee joint and a subcutaneous space of mice with severe combined immunodeficiency.

Results. Teratomas formed in both areas. Those in the joints grew and destroyed the joints. The incidence of cartilage formation was the same in the knee joint and subcutaneous space, but the ratio of cartilage to teratoma was higher in the knee joint than in the subcutaneous space. The teratomas were proved to have been derived from the transplanted ES cells by detection of the neomycin-resistance gene that had been transfected into the ES cells.

Conclusions. It is currently not possible to use ES cells to repair joint tissues. Further optimization of donor ES cells to differentiate as well as inhibit tumour growth may help to meet these challenges.

I'll post this because I have access:

PNAS | February 19, 2002 | vol. 99 | no. 4 | 1755-1757

Commentary
Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson's disease?
Curt R. Freed*

Division of Clinical Pharmacology and Toxicology, University of Colorado School of Medicine, C237, 4200 East Ninth Avenue, Denver, CO 80220

Human embryonic stem (ES) cells entered the popular lexicon during the political debate of summer 2001 and the decision by President George Bush to allow federal funding for research on the 64 human ES cell lines that had been derived by the day of his speech. Before the policy decision, the National Institutes of Health did a comprehensive review of the field (http://www.nih.gov/news/stemcell/scireport.htm). Human ES cells come from preimplantation embryos, most of which are generated at in vitro fertilization clinics (1). Within days after fertilization, the embryo consists of a hollow sphere, the blastocyst, which contains a cluster of a few hundred identical cells called the inner cell mass that can eventually develop into a fetus. When removed from the blastocyst, these cells can be propagated indefinitely in specialized media (2). When the media are changed to allow differentiation, cells continue to divide and aggregate, forming embroid bodies. Although these cell clusters lack the organization of an embryo, they contain all tissue types including skin, muscle, bone, and neurons. Because ES cells can become any cell in the body, there is hope that they will lead to treatment for diseases like diabetes, Parkinson's, Alzheimer's, and heart failure. The problem is controlling cell growth and differentiation. If large numbers of ES cells are transplanted into an organ like the brain, they grow into every cell type and form tumor-like masses called teratomas, eventually killing their host. How can ES cells be restricted to produce useful cells without overgrowing? In their article in this issue of PNAS, Björklund et al. (3) have transplanted small numbers of partially differentiated mouse ES cells from embroid bodies into a rat model of Parkinson's disease and have shown that at least some of the cells become the dopamine neurons that are needed to reverse the Parkinson condition. The authors suggest that the brain environment may encourage survival of cells appropriate for treating the disease while controlling the tendency to form a tumor mass. The results were far from perfect. Of 25 rats receiving transplants of 1,000-2,000 cells, 56% of animals showed surviving grafts containing dopamine neurons whereas 20% had lethal teratomas and 24% had no cells survive. Although these proportions are not promising as a treatment for humans with Parkinson's disease, the results illustrate the principle that relatively undifferentiated cells can develop into neurons appropriate for a specific brain region without invariably leading to uncontrolled cell growth.


How can embryonic stem cells be restricted to produce useful cells without overgrowing?

The hope is that research on human ES cells may reveal methods for producing an infinite supply of dopamine neurons for transplant into patients. Parkinson's disease is caused by the death of a small number of dopamine neurons located in a nucleus in the midbrain, the substantia nigra (4). Axons project to the forebrain and supply dopamine to the putamen and caudate nucleus. Without dopamine, patients are frozen in place. In one of the most remarkable drug developments of the 20th century, Cotzias and colleagues (5) demonstrated in 1967 that the amino acid L-dopa can be taken by mouth, enter the brain, be converted to dopamine, and improve Parkinson's symptoms. By 1979, a new treatment strategy, replacement of the damaged dopamine neurons by cell transplantation, proved successful in a rat model of Parkinson's (6-8). Dopamine cells survived, grew axons and dendrites, and improved behavior of the rats. Experiments showed that only fetal dopamine cells from the midbrain at a specific developmental stage could survive transplantation---13-15 days after fertilization in the rat and 6-8 weeks after fertilization in the human. At this stage, both rat and human fetus are only 2-3 cm in overall length.

By the late 1980s, fetal dopamine cell transplantation was being performed in humans with Parkinson's (9-17). All of the principles developed in the rat have been validated in human subjects, and we have found that neurons survive for at least 8 years after transplant (Fig. 1). Neurotransplantation with fetal dopamine neurons is now a proven strategy for treatment of patients with advanced Parkinson's disease. In our double-blind, placebo-controlled surgical trial of fetal tissue implants, we found that transplants survived in 85% of patients regardless of age and without immunosuppression and improved signs of Parkinson's disease in patients under age 60 and in older patients who still had a good response to the drug L-dopa (17). Sham surgery patients had no change in their symptoms. About 15% of patients who reduced or discontinued all L-dopa had the same kind of excess movements (dyskinesias) that had been caused by their L-dopa treatment. This study has proven that neurotransplants have significant benefits and also have some of the same side effects as the drug L-dopa. Transplant methods will continue to evolve, regardless of the source of dopamine neurons used for transplant.



Fig. 1. Human fetal dopamine neuron in putamen of Parkinson's patient dying 8 years after cell transplantation. The cell is immunostained for tyrosine hydroxylase and has multiple processes growing out from the cell body to specifically reinnervate the surrounding brain region. Other staining methods used on other transplanted cells in the same patient revealed the neuromelanin pigment particles typical of mature substantia nigra dopamine neurons. Immunosuppression was used for the first year after transplant, but not for the subsequent 7 years. If human ES cells can be differentiated to a phenotype identical to fetal substantia nigra dopamine neurons, those cells should have the same stable survival seen in this patient's transplant. (Magnification: ×400.)

Two problems limit the wide applicability of fetal dopamine cell transplants. First is the difficulty in recovering fragments of fetal brain tissue from elective abortions. The second problem is poor survival of dopamine neurons. About 90-95% of transplanted neurons fail to survive with most dying by apoptotic programmed cell death (15, 17, 18). An unlimited source of dopamine cells produced in tissue culture would solve both of these problems. Because fetal neurons usually are not rejected when transplanted into an adult host brain from the same species, it is likely that cells from a small number of individuals representing different HLA types would provide tissue immunologically acceptable for most everyone. Apoptotic losses could be managed by simply increasing the number of cells transplanted. If survival of laboratory-generated cells was predictably 20%, then transplants of a 5-fold excess would deal with the cell death problem.

Dopamine neurons generated in tissue culture should be identical to dopamine neurons native to the substantia nigra. They must synthesize, release, take up, and catabolize dopamine. Because many in vitro manipulations can induce temporary expression of the dopamine synthetic enzyme tyrosine hydroxylase, candidate cells should be evaluated with a panel of markers such as the dopamine transporter, ptx-3, nurr-I, and aldehyde dehydrogenase. They also should have the ability to project axons to target nuclei like the putamen. While the molecular mechanisms for target recognition by dopamine neurons are unknown and because in vitro conditions do not mimic conditions in the brain, cells must be transplanted into the dopamine-depleted striatum of experimental animals to see whether phenotype is preserved and whether neurites can connect with appropriate target cells. Björklund et al. (3) met these criteria, at least in the 56% of animals that showed surviving dopamine neurons without teratomas.

Will ES cells provide an infinite and safe supply of dopamine neurons? It is too early to say. To reduce the risk of teratomas, cell differentiation should take place in vitro and not after transplant into brain. At the National Institutes of Health, Lee et al. (19) have used a progressive expansion, selection, and differentiation strategy to convert mouse ES cells to a mixed population of mature neurons in tissue culture with up to 30% having the characteristics of dopamine cells. Lee et al., like Björklund et al., started by predifferentiating ES cells to embroid bodies. To increase the fraction of cells that were neural, they continued the differentiation process in neuron-selective media. The combination of expansion and selection of ES-derived cells will likely be fundamental to the safe production of selected phenotypes.

Using a somewhat different approach, Kawasaki et al. (20) have been able to generate dopamine neurons from mouse ES cells without embroid body formation by exposing the cells to factors generated by PA6 stromal cells. ES cells grown on a layer of either live or paraformaldehyde-fixed PA6 cells led to a mixed population of differentiated cells of which 16% were tyrosine hydroxylase-positive. The cell cultures produced dopamine. Importantly, the tyrosine hydroxylase phenotype was preserved after transplant into rats. To prevent tumor formation from residual ES cells, Kawasaki et al. treated their cell preparation with mitotic inhibitors. Although the differentiation factor or factors secreted by PA6 cells are unknown, the neural fate could be blocked by treatment with BMP4, an antineuralizing agent. The reports from Lee et al. and Kawasaki et al. indicate that a number of strategies will likely be successful for converting ES cells to dopamine neurons.

What about producing dopamine neurons from stem cells found in skin, blood, bone marrow, or umbilical cord blood? Recent evidence suggests that some stem cells from each of these sources have the potential to become neurons (21-23). Producing specific cell types from a patient's own stem cells would have immunologic advantages. It remains to be seen whether specific neuronal phenotypes can be generated efficiently and economically.

Immortalization of cells during normal development offers another route for producing useful dopaminergic neurons. Using the simian virus 40 large T antigen, we have immortalized fetal rat dopamine neurons. These cells express phenotypic markers of dopamine neurons and have behavioral effects after transplant into the rat model of Parkinson's (24). While the cells divide continuously in vitro, factors in brain inhibit tumor formation after transplant (25). Cells with a dopaminergic phenotype have been produced from a v-myc immortalized mouse stem cell line in which the nurr-I orphan receptor gene has been expressed and after cells have been exposed to astrocytes derived from ventral mesencephalon (26).

How will human ES cells fit into cell replacement strategies? Diseases treatable with ES cells will likely fall into two categories. First is replacement of specific cell types, like dopamine neurons for Parkinson's disease. There is no evidence that dopamine cells can arise spontaneously from a stem cell population in the adult brain. Thus, creation of dopamine neurons from ES cells would offer a therapeutic strategy not possible even with normally functioning intrinsic stem cells. The second category of treatment is replacement of failed host stem cells. A common example is bone marrow transplantation for aplastic anemia. Novel stem cell populations are still being revealed. Cardiac myocytes, previously thought to be fixed in number in the adult, have been shown recently to be replaceable by host tissue in the setting of cardiac transplantation, indicating that myocardial stem cells exist (27). Progenitor cells capable of generating neurons have been detected in adult human hippocampus as well as in the subventricular zone (28, 29). We have shown that human neural stem cells can be integrated into the germinal zone of the developing monkey brain in utero and can join in region-appropriate neurogenesis (30). If diseases like Alzheimer's prove to be the result of neural stem cell failure in some patients, transplants of neural stem cells may be therapeutic. For human ES cells to be useful for stem cell replacement, they should be partially differentiated into lineage-restricted stem cells to eliminate the pluripotency responsible for teratomas. This goal is being achieved (31-34).

In summary, there are a number of strategies for producing dopamine neurons in tissue culture that may lead to cells appropriate for transplantation into humans with Parkinson's disease. Björklund et al., Lee et al., and Kawasaki et al. have shown that generation of dopamine neurons from mouse ES cells is possible. Hopefully, a similar outcome will be demonstrated for human ES cells. Alternatively, stem cells derived from brain, blood, bone marrow, or skin may be convertible to dopamine neurons. Immortalized dopamine neurons may be useful if cell replication after transplant can be controlled. The risk of tumor formation is present in all ES and immortalized cell sources. Eliminating the possibility of uncontrolled proliferation is fundamental to the development of this field. The winners in this lottery are unknown, but the characteristics of the successful cell preparation can be specified. The cells should be identical to authentic fetal human midbrain dopamine neurons capable of reinnervating the dopamine-denervated striatum. Must the cell preparation be uniformly dopaminergic? Probably not. Clinically effective transplants of human fetal mesencephalon contain many cell types, with only a small fraction being dopamine cells. Whether transplants require the presence of cell types other than dopamine neurons is unknown. The successful generation of an unlimited supply of dopamine neurons will make neurotransplantation widely available for patients with Parkinson's disease. Embryonic stem cells are opening an exciting era in human therapeutics.

J. Clin. Invest. 115:102-109 (2005). doi:10.1172/JCI200521137.
Copyright ©2005 by the American Society for Clinical Investigation
Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell–derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell–derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

FULL ARTICLE:

http://www.jci.org/cgi/content/full/115/1/102

First published online February 2, 2006
Stem Cells Vol. 24 No. 6 June 2006, pp. 1458-1466
doi:10.1634/stemcells.2005-0413; www.StemCells.com
© 2006 AlphaMed Press

Embryonic Stem Cell-Derived Neuronally Committed Precursor Cells with Reduced Teratoma Formation After Transplantation into the Lesioned Adult Mouse Brain
Marcel Dihnéa,b, Christian Bernreuthera, Christian Hagelc, Kai O. Weschea, Melitta Schachner

The therapeutic potential of embryonic stem (ES) cells in neurodegenerative disorders has been widely recognized, and methods are being developed to optimize culture conditions for enriching the cells of interest and to improve graft stability and safety after transplantation.

Whereas teratoma formation rarely occurs in xenogeneic transplantation paradigms of ES cell-derived neural progeny, more than 70% of mice that received murine ES cell-derived neural precursor cells develop teratomas, thus posing a major safety problem for allogeneic and syngeneic transplantation paradigms.

Here we introduce a new differentiation protocol based on the generation of substrate-adherent ES cell-derived neural aggregates (SENAs) that consist predominantly of neuronally committed precursor cells.

Purified SENAs that were differentiated into immature but postmitotic neurons did not form tumors up to four months after syngeneic transplantation into the acutely degenerated striatum and showed robust survival.

...Cell therapy is a promising approach to treat neurodegenerative diseases [1–3]. Whereas primary tissue transplants are limited in number and thus not applicable to the numerous individuals suffering from neural disorders, in vitro expandable populations of neural precursors represent an attractive alternative source for neural transplantation [4]. Embryonic stem (ES) cells can proliferate extensively in an undifferentiated state, thus providing an almost unlimited source of neural precursors after predifferentiation [5–7]. However, ES cell-derived neural precursor cell populations have been observed to be tumorigenic, particularly after allogeneic or syngeneic transplantations [8]. Thus, there is a need to predifferentiate ES cells such that their tumorigenic potential is minimized.

We have developed an optimized protocol for predifferentiation of ES cells and isolation of ES cell-derived neural aggregates, resulting in a high yield of neuronally committed cells in vitro and after transplantation in vivo without tumor formation in a syngeneic transplantation paradigm in which predifferentiated ES cell-derived neural aggregates from C57BL/6J mice were injected into the quinolinic acid-lesioned striatum of adult C57BL/6J recipient mice.

DISCUSSION:

In this study, we developed a protocol for generating and enriching SENAs. In contrast to ES cell-derived neural precursors differentiated with the conventional 5-stage protocol [12, 13], cells within SENAs differentiated into an almost pure (> 90%) population of neurons in vitro.

For transplantation experiments, we harvested SENAs at a stage of differentiation at which SENAs consist predominantly of postmitotic ß-tubulin+ neurons (> 75%) that do not yet express mature neuronal markers. When undissociated SENAs of this stage were syngeneically grafted into the quinolinic acid-lesioned striatum, no teratomas were found for up to 4 months after transplantation—the latest time point tested—whereas neural precursors generated with the conventional embryoid body-based, 5-stage differentiation protocol led to the formation of teratomas in 70% or 17% of syngeneic transplantation experiments, depending on their state of maturation.

When SENAs start to form in stage 4 at 6+, they consist mainly of proliferating nestin+, Pax-6+, and NCAM+precursors (from 6+ until 12+), which subsequently (from 12+ until 18+) differentiate into immature, postmitotic, ß-tubulin+/NF–/GABA– neurons (about 76% at 18+). After continued differentiation at 18+/6–, the fraction of ß-tubulin+ neurons within SENAs, generated from the EGFP+C57BL/6J or R1 ES cell lines, had increased further, to more than 90% with nearly half the cells expressing mature neuronal markers, such as NF-200, GAD-6, SV-2, or ChAT, indicating the predominantly neuronal commitment of SENAs (only ~8% are glial cells). In comparison, when applying the 5-stage differentiation protocol, the EGFP+ C57BL/6J or R1 ES cell lines, in agreement with previous studies [12, 13], yielded only about 13% or 55% neurons, respectively, whereas 33%–43% glial cells were detected pointing to an approximately equally sized potential of stage 4 cell populations to generate neurons or glial cells. To additionally compare the differentiation potential of SENAs with another type of neural precursor cells in their aggregated state, we generated neurospheres from the central nervous system of 14-day-old embryos and found, also in agreement with previous studies [17], a predominantly astrocytic commitment of neurospheres (> 70%). Thus, a clear increase in neuronal yield is achieved when applying the SENA differentiation protocol, indicating that the SENA-based differentiation strategy that can be applied to different ES cell lines is able to generate highly enriched, neuronally committed cell populations. In addition, it could be shown that ES cell-derived SENAs are fundamentally different from CNS-derived neurospheres, as SE-NAs cannot be generated in a free-floating fashion and as SENA cells are predominantly neuronally committed.

Purified SENAs at 18+/3– were chosen for transplantation into the acutely lesioned striatum of adult mice. Thus, SENA grafts consisted of numerous committed, immature neurons, but only a few nestin+ proliferating precursors. In comparison, stage 4 grafts, and, to a lesser degree, stage 5 grafts, consisted predominantly of proliferating nestin+ , Pax-6+, and NCAM+ precursors (Table 1Go). Although neurons are generally regarded to be more vulnerable toward toxic influences at the lesion site in comparison to immature nestin+ cells [18], most likely due to expression of NMDA receptors, which make them sensitive to excitotoxic insults [19], survival of cells in SENA grafts was robust, as indicated by the low percentage of caspase+ cells, which was comparable to that of stage 5 grafts. Indeed, determination of the number of NF+ (~57,000) and ß-tubulin+ (~60,000) cells in SENA grafts revealed neuronal survival of approximately 80% of all grafted neurons. In comparison, neuronal survival of 3%–5% or 22% has been reported in previous experiments [20, 21]. Robust neuronal survival in SENA grafts might be related at least in part to the grafting of nondissociated cellular aggregates. As ~50,000 transplanted SENA-derived cells had adopted a GABAergic phenotype, as assessed by GAD-65/67 immunohistochemistry (~83% of all SENA-derived neurons), SENAs provide a particular neuronal subtype after transplantation that can be used for neurodegenerative diseases with a loss of this cell type.

An additional benefit of SENAs was the absence of teratoma formation in the syngeneic transplantation paradigm used in this study. Erdö and colleagues [8] reported that murine ES cells and ES cell-derived stage 4 neural precursors formed tumors in 86% of recipient mice but not in rats, questioning the applicability of allogeneic or syngeneic transplantation strategies in humans. The reasons for tumor formation after syngeneic transplantation but not after xenogeneic transplantation are not known, but it is likely that immune rejection of the tumor occurs after xenogeneic transplantation. Assessment of possible teratoma formation after xenogeneic transplantation into immunodeficient recipients, for instance into nude rats [22], should clarify the role of a potentially teratoma-preventing immune response. In agreement with the results of Erdö and colleagues, we found that stage 4 grafts formed teratomas in 70% of recipient mice but not in recipient rats (unpublished observations), suggesting that the therapeutically most relevant allogeneic or syngeneic transplantation paradigms [23] can reveal the tumorigenic potential of ES cell-derived neural progeny that would not be recognized in xenogenic transplantations.

In our study, teratoma formation after syngeneic transplantation was decreased to 17% when predifferentiated stage 5 cells were transplanted, suggesting a relation between graft maturity and tumor formation. This notion was further supported by the observation that syngeneic SENA grafts did not generate teratomas during a post-transplantation period of up to 4 months. A plausible reason for the reduced teratoma formation of SENAs might be that some immature teratoma-forming ES cells may have escaped pre-differentiation toward stages 4 or 5 but not toward the SENA stage. However, assessment of possible Oct-4+ cells within stages 4 or 5 and SENA cell populations revealed that immature ES cells can be found only rarely in all of these cell populations, with less than 5 Oct-4+ cells/100,000 stage 4, stage 5, or SENA cells. Thus, it is unlikely that the very few immature ES cells in stages 4 and 5 and in SENA populations would give rise to teratomas only after grafting stage 4 or 5 cells.

Alternatively, one may argue that proliferating, immature, nestin+ /Pax-6+ /NCAM+ neural precursors in stage 4 and 5 grafts, induced by cues from the host tissue, regress more easily into teratoma-forming cells than ß-tubulin+ neurons within SENA grafts. Our assessment of the proliferation and maturity of stage 4 and 5 cells and cells in SENAs before transplantation revealed a high correlation between proliferation and maturity in vitro and tumor formation in vivo, supporting the possibility that at least some ES cell-derived nestin+ neural precursor cells can be re-programmed in vivo toward an immature, teratoma-forming state. Thus, this study suggests that even purification of ES cell-derived nestin+ /Pax-6+ /NCAM+ neural precursors does not prevent teratoma formation and that ES cells should rather be differentiated toward a more mature state. However, as also SENAs contain some proliferating nestin+ cells, it has to be speculated if they also have a minimal tumorigenic risk.

Our observations are thus noteworthy with regard to long-term safety and efficacy of ES cell therapy in humans. Although most transplantation studies have used immature, nestin+ neural precursors or slightly predifferentiated neural precursors we have grafted ES cell-derived aggregates consisting predominantly of ß-tubulin+ neurons. Our study demonstrates that the state of maturity of ES cell-derived transplants critically determines tumorigenicity and provides a platform for further experiments aimed at using ES cell-derived transplants to treat neurological disorders.

Another study showed a method to prevent tumor formation in monkeys:

http://stemcells.alphamedpress.org/c...005-0391v1.pdf

another article:

Journal of Neuroscience Research

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Volume 83, Issue 6, Pages 1015-1027

Published Online: 21 Feb 2006


Research Article
Generation of graftable dopaminergic neuron progenitors from mouse ES cells by a combination of coculture and neurosphere methods
Asuka Morizane 1, Jun Takahashi 1 *, Mizuya Shinoyama 1, Makoto Ideguchi 1, Yasushi Takagi 1, Hitoshi Fukuda 1, Masaomi Koyanagi 1, Yoshiki Sasai 2, Nobuo Hashimoto 1


Parkinson's disease is characterized by a loss of midbrain dopamine (DA) neurons and is generally viewed as a potential target for stem cell therapy. Although several studies have reported the generation of postmitotic DA neurons from embryonic stem (ES) cells, it is unknown whether the proliferative progenitors of DA neurons can be isolated in vitro. To investigate this possibility, we have developed a combined approach in which ES cells are cocultured with PA6 stromal cells to expose them to stromal cell-derived inducing activity (SDIA) and are then cultured as neurospheres.

Mouse ES cell colonies were detached from PA6 feeder cells after 8 days of SDIA treatment and then expanded as spheres for another 4 days in serum-free medium supplemented with fibroblast growth factor-2. The spheres exhibited neural stem cell characteristics and contained few DA neurons at this stage of culture. After being induced to differentiate on polyornithine/laminin-coated dishes for 7 days, these spheres generated DA neurons in vitro at a relatively low frequency.

Intriguingly, addition of PA6 cell conditioned medium to the sphere culture medium significantly increased the percentage of DA neurons to 25-30% of the total number of neurons. Transplantation of conditioned medium-treated day 4 spheres, which contained DA neuron progenitors, into the mouse striatum resulted in the generation of a significant number of graft-derived DA neurons. These findings suggest that progenitors of DA neurons are generated and can proliferate in ES cell-derived neurospheres induced by serial SDIA and PA6 conditioned medium treatment.

DISCUSSION


Here, we demonstrate that differentiating ES cells can be enriched for neural progenitor cells by culturing them on PA6 feeder cells, which provide a source of SDIA, and by using a sphere culture method. The addition of PA6 cell CM into the sphere culture medium enriches for proliferating DA neuron progenitors in spheres and results in the efficient differentiation of ES cells into DA neurons both in vitro and in vivo (Fig. 7). Thus, this method produces an efficient graftable cell source for cell transplantation. We also clearly demonstrate that the proliferating cells that gave rise to tumors were derived from undifferentiated ES cells or early ectodermal cells.

Figure 7. In vitro neural induction and dopaminergic differentiation by a combination of SDIA treatment and sphere culture. Neural induction was achieved by coculturing ES cells on PA6 feeder cells. By sphere culture in serum-free medium supplemented with B27 and FGF2, the cells proliferate while retaining characteristics of neural precursors. The CM of PA6 cells supports the survival or proliferation of DA neuron progenitors in spheres, resulting in efficient dopaminergic differentiation in vivo after transplantation.
[Normal View 37K | Magnified View 112K]

ES cell colonies growing on PA6 feeders are heterogeneous and consist of undifferentiated ES cells, neural stem/progenitor cells, differentiated neural cells, and nonneural cells, such as epidermal cells. When colonies are maintained on noncoated dishes in a serum-free medium supplemented with B27 and FGF2, neural stem/progenitor cells form spheres and proliferate in a manner comparable to that of mouse brain-derived neural stem cells (Gritti et al., [1996]), whereas other cell types tend to adhere to the bottom of the culture dish. Insofar as serum-free medium is not permissive for nonneural cell growth and ES cells are diverted into a neural lineage in the absence of LIF, it seemed likely that neural cell types would be enriched by the sphere culture method. Indeed, most of the cells in the spheres were immunoreactive for nestin or other neural cell markers and gave rise to neurons, astrocytes, and oligodendrocytes after differentiation. Previous work indicated that spheres exhibiting neural stem cell characteristics could be obtained from single mouse ES cells in the presence of leukemia inhibitory factor (LIF; Tropepe et al., [2001]) and from human embryoid bodies in the presence of FGF2 (Reubinoff et al., [2001]; Zhang et al., [2001]). In addition, dissociated mouse or human ES cells cultured on the bone marrow-derived stromal feeder cell line MS5 can form similar spheres in the presence of FGF2 (Barberi et al., [2003]; Perrier et al., [2004]).

When spheres were cultured at low density, the percentage of sphere-derived TH-positive cells decreased significantly. This finding suggests that ES cell-derived neuropheres may secrete soluble factors that promote dopaminergic differentiation, such as Wnts (Castelo-Branco et al., [2003]). Interestingly, among the soluble factors we tested, the CM of PA6 cells most efficiently induced DA neurons from spheres, yielding a percentage of TH-positive cells similar to that with continuous coculture on PA6 feeder cells. The CM increased the expression of En1 and En2, which are expressed in proliferating DA neuron progenitors (Arenas, [2002]; Burbach et al., [2003]), and mildly decreased expression of an earlier midbrain marker, Pax2, in CM spheres compared with control spheres. Thus, the CM of PA6 cells seems to promote dopaminergic differentiation or to support proliferation and/or survival of DA neuron progenitors. Although the molecular nature of SDIA is unknown, two possibilities have been proposed (Kawasaki et al., [2000]). One is that SDIA consists of two different neural-inducing factors, one that is anchored to the cell surface and one that is secreted. Another scenario is that secreted factors that are secondarily tethered to the cell surface are responsible for the activity. The facts that PA6 cells retained neural-inducing activity even after being fixed with paraformaldehyde and their CM could not elicit significant neural induction (Kawasaki et al., [2000]) suggest that there are two steps for the generation of DA neurons from ES cells: neural induction and maturation of DA neurons. The present results suggest that, although membrane-bound factor(s) might be necessary for neural induction, soluble factor(s) secreted from the ES cell-derived neurospheres and/or PA6 cells are sufficient for proliferation and/or survival of DA neuron progenitors.

FGF8 and Shh have been shown to be important for the production of DA neurons at the mid-/hindbrain boundary during development (Ye et al., [1998]). These factors also induce differentiation of DA neurons from mouse ES cells when added during the expansion phase of nestin-positive cells (Lee et al., [2000]) or when ES cells are grown on MS5 feeder cells (Barberi et al., [2003]). ES cell-derived neural progenitors express the FGF8 receptor FGFR3 as well as the Shh receptors Ptc and Smo (Lee et al., [2000]). The mild increase in the TH/TuJ1-positive cell ratio that was observed when the spheres were treated with FGF8 and Shh is compatible with these earlier findings. In addition, 17-estradiol has been reported to play a versatile role in the differentiation/maturation and survival of DA neurons (Behl, [2002]; Sawada et al., [2002]). Although the effect of estradiol on ES cells remains unknown, rat neural stem cells express estrogen receptors and (Brännvall et al., [2002]). IL-1 and GDNF were also reported to promote dopaminergic differentiation from rat mesencephalic progenitors (Ling et al., [1998]), and GDNF has been shown to be a survival factor for DA neurons (Lin et al., [1993]; Beck et al., [1995]; Tomac et al., [1995]). The spheres that we derived from SDIA-treated ES cells have characteristics of neural stem cells or DA neuron progenitors, so it is possible that 17-estradiol, IL-1, and GDNF play a role in the induction and maintenance of DA neurons.

As mentioned above, CM of PA6 cells was able to enrich for DA neuron progenitors in spheres, resulting in good survival of DA neurons in vivo. If the donor cells are too mature, as were the colonies used in the present study, the survival rate of the DA neurons decreases, likely because mature neurons are more vulnerable to mechanical damage, inflammatory cytokines, and neurotrophic factor insufficiency. Thus, the developmental stage of the donor cells is one of the keys to successful transplantation. Among the 20,000 cells injected into the mouse striatum, we observed 1,506 surviving TH-positive cells in the graft, or 75.3 TH-positive cells per 1,000 cells grafted. This figure is almost same as that from the previous report of mouse ES cell allografts (75.0 by Barberi et al., [2003]). These numbers are higher than those from allografts of rat embryonic ventral mesencephalon, for which efforts were made to improve cell survival using 21-aminosteroids (34.6 by Nakao et al., [1994]), GDNF infusion (29.9 by Rosenblad et al., [1996]), caspase inhibitor (24.0 by Schierle et al., [1999]), or simple allografts of rat neural stem cells (3.5 by Studer et al., [1998]; 0.8 by Carvey et al., [2001]). Furthermore, all the experiments except for ours used 6-OHDA-lesioned animals as hosts, in which depletion of DA neurons within the substantia nigra might promote DA neuronal differentiation and survival (Nishino et al., [2000]). As shown here, ES cells can be manipulated to contain a substantial number of proliferating progenitors, resulting in the generation of abundant TH-positive cells in vivo.

Our in vivo studies revealed that a decrease in the number of undifferentiated ES cells in the spheres was correlated with an absence of tumor formation. Previous efforts to transplant naïve mouse ES cells have resulted in the formation of teratomas or teratocarcinomas, even in the case of xenografts (Björklund et al., [2002]; Erdö et al., [2003]). In contrast, when mouse ES cells were differentiated before transplantation by stepwise treatment with cytokines (Kim et al., [2002]) or by culturing them on MS5 feeder cells in the presence of cytokines (Barberi et al., [2003]), the donor cells did not form any tumors. In the allograft of naïve mouse ES cells, transplants of 4,000 cells led to abundant tumor formation, whereas transplants of 400 cells showed a propensity to form teratomas only in the somatosensory cortex (Harkany et al., [2004]). These findings suggest that the cause of tumor formation is undifferentiated ES cells. We first demonstrated this clearly by showing the colocalization of SSEA-1 and E-cadherin in Ki67-positive proliferating cells. All the previous tumors derived from ES cells have been described as teratomas or teratocarcinomas (Björklund et al., [2002]; Erdö et al., [2003]; Harkany et al., [2004]; Fukuda et al., [2005]). The tumors in the present study, however, were undifferentiated or intermediately differentiated neuroectodermal tumors, probably because we grafted spheres of predifferentiated cells instead of using low- or single-cell-density grafts. We suggest that undifferentiated ES cells might be influenced by the surrounding neural cells in the sphere, giving rise to neural stem/progenitor cells in vivo. In this context, it is noteworthy that Musashi1-positive cells were observed around the Ki67/E-cadherin/SSEA-1-positive cells.

In summary, the combination of SDIA treatment and sphere culture with the CM of PA6 cells significantly increased the number of induced DA neurons in vitro and in vivo by enriching for their proliferating progenitors in the spheres. However, it also partially restored the propensity of these cells to form tumors. Single ES cells are able to proliferate on PA6 feeders, so it is possible that the PA6 cell CM supported the survival and/or proliferation of not only DA neuron progenitors but also undifferentiated ES cells. As a result of these findings, the determination of the molecular mechanism of SDIA and the complete elimination of undifferentiated ES cells at an early stage of neural induction (Fukuda et al., [2005]) will be indispensable for the successful transplantation of ES cell-derived neural stem/progenitor cells.

full article:

http://www3.interscience.wiley.com/c...8892/HTMLSTART

This is my first posting on the Brain Talk forum, although I’ve been reading it for a long time. I wanted to comment on the report of the University of Rochester study being described as a “setback” (or worse) for embryonic stem cell research. Looking at a number of the media reports, each one reported the results of the study and their implications for future stem cell research a little differently.

What was left out of many of the news articles was that this study utilized a new and different technique to produce stem cells - in an attempt to maximize the number of stem cells created - and that might have been the cause of the tumors. Also it is possible that using "old" stem cells - from one of the federally approved lines -- may have caused the problem. In any case this study calls for further research, not that ESCR (especially for Parkinson’s) should be abandoned, as some will be claiming, and citing this study to back up their claims. I think we all realize that a stem cell cure is “not ready for prime time” and that is in good part because all the roadblocks the Bush administration has set up over the last 6 years have slowed its progress.

I thought this was a good, balanced account, from the
Toronto Globe and Mail
Oct. 23, 2006
A sobering setback in stem-cell research
by CAROLYN ABRAHAM

“The progress of science is paved with stories of high hopes and heartbreaks. But in a busy lab at the University of Rochester the two extremes have met in one dazzling yet devastating experiment.
Researchers there have for the first time essentially cured rats of a Parkinson's-like disease using human embryonic stem cells. But 10 weeks into the trial, they discovered brain tumours had begun to grow in every animal treated.

"Here we have this method that works so well to reverse the symptoms of Parkinson's," said lead investigator Steven Goldman, "But no matter how you look at it, it's an expanding mass and that's bad news."

None of the cells growing out of control were cancerous tumours. But as Dr. Goldman pointed out, "In the brain, nothing's benign."

The work, published today in the journal of Nature Medicine, is a sobering setback for plans to use stem cells from human embryos to grow tissues for human transplant.

"My hopes are still high, but I think this injects real caution," said Dr. Goldman, who spent four years on the experiment and a 23-year career building up to it. "Some folks are portraying this as
imminently useful and it's not. There's still a lot that has to be sorted out."

By definition, human embryonic stem cells have the almost mythical, immortal power to grow and divide indefinitely as they become the various tissues that make up the body. As a result, scientists have always known that any stem cell therapy could result in an uncontrolled growth of cells that could give rise to cancer.

But that risk has remained largely theoretical since there have been few attempts to transplant tissues grown from stem cells into live animals. The work is difficult, expensive and tricky to pull off when several countries -- Canada included -- have enacted tough laws that
limit research on stem cells from human embryos.

"A lot of the representations of stem-cell research have resulted from the initial excitement and speculation of what can be achieved. But we're still in that early stage, we haven't seen real clinical breakthroughs," said Tim Caulfield, director of the Health Law Institute at the University of Alberta. "This [experiment] shows the incredible potential of the field, but it also sheds a more realistic light on the near-future potential."

For Mick Bhatia, scientific director of the Cancer and Stem Cell Research Institute at McMaster University, it's a bit of déjŕ vu. In 2004, he succeeded in growing human blood cells from embryonic stem cells but found transplanting them into mice wasn't simple.

"I pushed the program back," Dr. Bhatia said. "We need to do more on the basic biology."

Still, few scientific fields are hotter than stem cells as researchers everywhere investigate the possibility of using them to grow replacement parts -- cardiac cells for heart patients or islet
cells for diabetics.

Parkinson's has been considered one of the prime candidates for a stem-cell therapy because just a single cell type is needed -- one that produces dopamine.

Neurons that make dopamine are crucial for movement and degenerate in people with Parkinson's, often leaving them stiff, unable to control their physical gestures and suffering from tremors.

Twenty years worth of studies have tried treating Parkinson's with dopamine-cell transplants, in rodents, primates and people. In the 1990s, after a Swedish study found some benefit to patients who received dopamine-cell transplants from aborted fetal tissue, large human trials began in both Canada and the United States.

"These failed," Dr. Goldman said. "It made things worse; patients suffered movement abnormalities." In part, he explained, this was because the transplants contained all sorts of cells. Less than 10 per cent of the cells, and in some cases, less than 1 per cent, produced dopamine.

For this reason, Dr. Goldman, a neurologist and chief of cell and gene therapy at the University of Rochester, and many others, considered embryonic stem cells a possible source of growing only the cells that would be needed and they were right. But no one had been able to grow enough for a transplant.

In this experiment, however, Dr. Goldman and his team overcame the volume problem by tricking the embryonic stem cells into behaving like they were growing in the developing brain.

To do this, they harvested glial, or brain-support cells, from the precise brain region of an aborted fetus that would, at 11 to 22 weeks gestation, trigger the development of the dopamine neurons needed.

The researchers then used a retrovirus as a courier to deliver into the DNA of these cells a gene, known as telomerase, which would immortalize them. This way, Dr. Goldman explained, the support cells would continue to grow endlessly and continuously give off the chemical cues to keep stem cells maturing into dopamine cells.

The dopamine cells had first been grown from the limited number of human embryonic stem-cell lines that U.S. President George W. Bush made available to government-funded researchers in 2000.

Culturing the immortalized glial cells alongside the stem-cell derived dopamine neurons -- although not touching each other -- resulted in a growth of dopamine cells three to 10 times what is normally seen.

But Dr. Bhatia, who read the report, said this step might have contributed to the uncontrolled growth of the cells. Even though the immortalized glial cells and dopamine cells were not touching each other, there could have been chemical cues exchanged that affected the implanted cells, he said.
"The number of cells they're getting is incredible," said Dr. Bhatia after reading the report. "But at what cost did they gain that efficiency?" In a series of trials, the researchers next implanted
tissues of 500,000 cells each into the midbrain region of dozens of rats, which, as a result of a chemical injection, suffered a condition similar to Parkinson's. Seventy per cent of the cells
transplanted made dopamine.

Before the treatment, rats with the Parkinson's-like disease suffered from constant tremors and a lack of co-ordination. But four weeks after the transplants, Dr. Goldman said, they showed a marked improvement from the control group of untreated rats.

"It was really a complete recovery on the part of the animal. By six to eight weeks, they're normal," he said. "That's a more powerful effect than ever seen before."

But at 10 weeks, when the animals were autopsied, the researchers discovered that the implanted tissue had given rise to more than just dopamine cells.

Several cells had begun to divide at a fairly steady pace, the hallmark of cells growing into a tumour.

Where the dopamine cells once made up 70 per cent of the tissue implanted, at the autopsy they made up only about 25 per cent.

"They were undifferentiated neural cells that were expanding and dividing, and those are cells you don't want there. You don't need to be a neuro-oncologist to say, that's the start of a tumour," Dr. Goldman said.

Dr. Bhatia also raised the possibility that the years-old and scant stem lines available to government researchers in the United States may also have had tumourigenic properties from the start that skewed the experiment.
Dr. Goldman and his team are now redoing the experiment on the basis that neural cells other than dopamine-cells in the transplanted tissue led to the tumour growth.

"We are going to have to absolutely purify the cell type of interest," Dr. Goldman said. "This really pushes this [kind of transplant work] back in terms of clinical use.

"It's not ready for prime time, that's for sure."