Using stem cells and iPS cells to discover new treatments for Parkinson's disease

Fetal cell transplantation can improve the symptoms of Parkinson's disease (PD) patients for more than a decade. In some patients, alpha-synuclein aggregates and Lewy bodies have been observed in the transplanted neurons without functional significance. Recently stem cells have emerged as an ethically acceptable source of cells for transplantation but, importantly, the type of stem cell matters. While the lineage restriction of adult neural stem cells limits their clinical applicability for patients with PD, human pluripotent stem cells provide an opportunity to replace specific types of degenerating neurons. Now, cellular reprogramming technology can provide patient-specific neurons for neural transplantation and problems with cell fate specification and safety are resolving. Induced pluripotent stem (iPS) cell-derived neurons are also a unique tool for interpreting the genetic basis for an individual's risk of developing PD into clinically meaningful information. For example, clinical trials for neuroprotective molecules need to be tested in presymptomatic individuals when the neurons can still be protected. Patient-specific neural cells can also be used to identify an individual's responsiveness to drugs and to understand the mechanisms of the disease. Along these avenues of investigation, stem cells are enabling research for new treatments in PD.     

Stem cell treatment for Parkinson's disease: an update for 2005

PURPOSE OF REVIEW: The hallmark pathologic feature of Parkinson's disease is loss of melanized dopaminergic neurons within the substantia nigra pars compacta coupled with depletion of striatal dopamine. This is responsible for the major motor features of the disease. Whereas dopaminergic replacement therapy is effective in the early stages of the illness, chronic treatment is associated with motor complications and development of features that do not respond to levodopa therapy. Development of cellular therapies offers the potential to provide more effective treatment for the disease without motor complications. RECENT FINDINGS: Two clinical trials of fetal nigral transplantation failed to meet their primary endpoint and were complicated by the development of dyskinesia that persisted after withdrawal of levodopa ('off-medication' dyskinesia). However, recent studies suggest that both the limited clinical response and off-medication dyskinesia may be related to partial, but incomplete, dopaminergic reinnervation of the striatum and that both might be improved by transplantation of more dopamine neurons. Stem cells offer the potential to provide a virtually unlimited supply of optimized dopaminergic neurons that can provide enhanced benefits in comparison to fetal mesencephalic transplants. Stem cells have now been shown to be capable of differentiating into dopamine neurons that provide benefits following transplantation in animal models of Parkinson's disease. However, cell survival and behavioral responses are limited. There have been numerous advances in enhancing the yield of dopamine neurons from stem cells, and promoting their survival and consequent clinical effects. SUMMARY: Stem cells offer great promise as a therapy for Parkinson's disease, but numerous hurdles remain to be overcome with stem cell therapy. The adverse event profile of transplantation must be determined, and societal and ethical issues addressed. As Parkinson's disease involves degeneration of both dopaminergic and non-dopaminergic neurons, it also remains to be determined if transplantation of even the ideal dopamine neuron will improve non-dopaminergic features of the disease or provide benefits superior to existing therapies.     

Stem cell therapy for Parkinson's disease

The aim of stem cell therapy for Parkinson's disease is to reconstruct nigro-striatal neuronal pathways using endogenous neural stem/precursor cells or grafted dopaminergic neurons. As an alternative, transplantation of stem cell-derived dopaminergic neurons into the striatum has been attempted, with the aim of stimulating local synapse formation and/or release of dopamine and cytokines from grafted cells. Candidate stem cells include neural stem/precursor cells, embryonic stem cells and other stem/precursor cells. Among these, embryonic stem cells are pluripotent cells that proliferate extensively, making them a good potential donor source for transplantation. However, tumor formation and ethical issues present major problems for embryonic stem cell therapy. This review describes the current status of stem cell therapy for Parkinson's disease, as well as future research approaches from a clinical perspective.     

Neuromodulation for Parkinson's disease]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of midbrain dopaminergic (DA) neurons and a subsequent reduction in striatal dopamine. As a treatment for advanced Parkinson's disease, deep brain stimulation (DBS) of the thalamus was introduced in 1987 to treat tremor, and was applied in 1993 to the subthalamic nucleus. Now high-frequency stimulation of the subthalamic nucleus has become a surgical therapy of choice. Another surgical treatment is a cell replacement therapy. Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief, however, the technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue have limited the application of this therapy. Then, neural precursor cells and embryonic stem (ES) cells are expected to be candidates of potential donor cells for transplantation. We induced DA neurons from monkey ES cells, and analyzed the effect of transplantation of the DA neurons into MPTP-treated monkeys as a primate model of Parkinson's disease. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced neurological symptoms. DA neurons have also been generated from several human ES cell lines. Furthermore, functional recovery of rat PD models after transplantation was observed. One of the major problems in ES cell transplantation is tumor formation, which is caused by a small fraction of undifferentiated ES cells in the graft. So, it is essential for undifferentiated ES cells to be eliminated from the graft in order for transplantation to be feasible. These efforts will lead to clinical application of ES cell transplantation to the patients with PD.     

如何提高帕金森病中脑细胞移植中的细胞存活率？

虽然胚胎中脑细胞移植对帕金森病的疗效已得到广泛证实,但在这项技术广泛应用于临床之前仍有一些问题亟待解决。其中主要是移植物存活率低和宿主纹状体神经支配恢复有限。迄今为止,人们尝试了很多方法来解决这些问题,包括神经营养因子的广泛应用,以及神经和(或)非神经来源组织的联合移植。本文将对目前的胚胎中脑细胞移植术中所用的神经保护手段及其局限性进行简要的介绍。     

Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival of dopamine neurons in a variety of in vitro and in vivo model systems. Therefore, it constitutes an important therapeutic protein with the potential to ameliorate dopamine neuronal degeneration in Parkinson's disease or to support dopamine neuronal replacement strategies. However, biophysical and practical considerations present obstacles for the direct delivery of the GDNF protein to CNS neurons. Here we show that rodent neural precursor cells isolated and expanded in culture as neurospheres (NS) can be genetically modified to express green fluorescent protein (GFP) or to release GDNF using lentiviral constructs. GDNF-NS increased the fibre outgrowth of primary embryonic dopamine neurons in cocultures, showing that the protein was released in biologically significant quantities. Furthermore, after transplantation into the 6-hydroxydopamine-lesioned rat striatum, GDNF-NS significantly increased the survival of cografted primary dopamine neurons. However, this was not reflected in behavioural recovery in these animals. We found that, by 6 weeks, few cells expressed GDNF or GFP, suggesting either that transgene expression was down-regulated over time or that the cells died. This may explain the initial effects on dopamine neuronal survival within the graft but the lack of long-term effect on subsequent fibre outgrowth and behaviour. Providing sustained levels of neural precursor-mediated transgene expression can be achieved following transplantation in the future; this approach may prove beneficial as an alternative therapeutic strategy in the cell-based management of Parkinson's disease.     

如何提高帕金森病中脑细胞移植中的细胞存活率?

虽然胚胎中脑细胞移植对帕金森病的疗效已得到广泛证实,但在这项技术广泛应用于临床之前仍有一些问题亟待解决.其中主要是移植物存活率低和宿主纹状体神经支配恢复有限.迄今为止,人们尝试了很多方法来解决这些问题,包括神经营养因子的广泛应用,以及神经和(或)非神经来源组织的联合移植.本文将对目前的胚胎中脑细胞移植术中所用的神经保护手段及其局限性进行简要的介绍.     

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.     

Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5 × 10⁵) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.     

Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into parkinsonian mice

Clinical trials have provided proof of principle that new dopamine neurons isolated from the developing ventral midbrain and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue is likely to be a contributing factor in the variable outcomes. Dissections of ventral midbrain used for transplantation will variously contain progenitors for different dopamine neuron subtypes as well as different neurotransmitter phenotypes. The overall impact of the resulting graft will be determined by the functional contribution from these different cell types. The A9 substantia nigra pars compacta dopamine neurons, for example, are known to be particularly important for motor recovery in animal models. Serotonergic neurons, on the other hand, have been implicated in unwanted dyskinesias. Currently little knowledge exists on how variables such as donor age, which have not been controlled for in clinical trials, will impact on the final neuronal composition of fetal grafts. Here we performed a birth dating study to identify the time-course of neurogenesis within the various ventral midbrain dopamine subpopulations in an effort to identify A9-enriched donor tissue for transplantation. The results show that A9 neurons precede the birth of A10 ventral tegmental area dopamine neurons. Subsequent grafting of younger ventral midbrain donor tissue revealed significantly larger grafts containing more mitotic dopamine neuroblasts compared to older donor grafts. These grafts were enriched with A9 neurons and showed significantly greater innervation of the target dorso-lateral striatum and DA release. Younger donor grafts also contained significantly less serotonergic neurons. These findings demonstrate the importance of standardized methods to improve cell therapy for Parkinson's disease and have significant implications for the generation and selectivity of dopamine neurons from stem cell based sources.     

Differentiation of induced pluripotent stem cells into functional oligodendrocytes

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.     

Re-examining the ontogeny of substantia nigra dopamine neurons

Recently, the need to detail the precise ontogeny of nigrostriatal dopamine neurons has grown significantly. It is now thought that the gestational day on which the majority of these neurons are born is important not only for maximizing the yield of primary cells for transplantation but also for extracting suitable dopamine neural precursors (as stem cells) for expansion in vitro. Historically, peak ontogeny of substantia nigra pars compacta (SNc) dopamine neurons in the rat has been considered to occur around embryonic day (E)14. However, such a concept is at odds with recent studies that reveal not only that substantial numbers of tyrosine hydroxylase-immunopositive cells reside in the ventral mesencephalic region of rats at E14 but that many of these cells have matured extensive axonal projections to the ventral forebrain. Here, then, the ontogeny of SNc neurons in rats commonly used as a source of donor tissue for experimental cell transplantation in animal models of Parkinson's disease has been re-examined. Using a combination of bromodeoxyuridine (BrdU) administration at E11, E12, E13 or E14 with immunocytochemical stainings for both BrdU and tyrosine hydroxylase after 4 weeks of postnatal development, this characterization reveals that the vast majority (perhaps 80%) of SNc dopamine neurons are probably born on E12 in Sprague-Dawley rats. Such findings are important in refining the use of embryonic tissues for primary cell transplantation and may provide more precise timing for identifying the cellular and molecular events that drive neural stem cells toward a dopaminergic phenotype during development.     

Effects of sertoli cell-conditioned medium on ventral midbrain neural stem cells: A preliminary report

The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.     

Induced pluripotent stem cells and promises of neuroregenerative medicine

First created in 2006 from adult somatic cells by a simple molecular genetic trick, induced pluripotent stem cells (iPS) system is the latest platform in stem cell research. Induced pluripotent stem cells are produced by nuclear reprogramming technology and they resemble embryonic stem cells (ES) in key elements; they possess the potentiality to differentiate into any type of cell in the body. More importantly, the iPS platform has distinct advantage over ES system in the sense that iPS-derived cells are autologous and therefore the iPS-derived transplantation does not require immunosuppressive therapy. In addition, iPS research obviates the political and ethical quandary associated with embryo destruction and ES research. This remarkable discovery of cellular plasticity has important medical implications. This brief review summarizes currently available stem cell platforms, with emphasis on cellular reprogramming and iPS technology and its application in disease modeling and cell replacement therapy in neurodegenerative diseases.     

Models of repair mechanisms for future treatment modalities of Parkinson's disease

Parkinson's disease is one of the most likely neurological disorders to be fully treatable by drugs and new therapeutic modalities. The age-dependent and multifactorial nature of its pathogenesis allows for many strategies of intervention and repair. Most data indicate that the selectively vulnerable dopaminergic neurons in the substantia nigra of patients that have developed Parkinson's disease can be modified by protective and reparative therapies. First, the oxidative stress, protein abnormalities, and cellular inclusions typically seen could be dealt with by anti-oxidants, trophic factors, and proteolytic enhancements. Secondly, if the delay of degeneration is not sufficient, then immature dopamine neurons can be placed in the parkinsonian brain by transplantation. Such neurons can be derived from stem cell sources or even stimulated to repair from endogenous stem cells. Novel molecular and cellular treatments provide new tools to prevent and alleviate Parkinson's disease.     

Stem cell therapy for Parkinson's disease

Transplantation of human fetal dopamine (DA) neurons to patients with Parkinson's disease (PD) has given proof of the principle that new neurons can survive for at least a decade, and then functionally integrate and provide significant symptomatic relief. Unfortunately, the ethical, technical, and practical limitations of using fetal DA neurons as the source for cell transplantation in PD, in combination with the development of unwanted grafting-related side effects, have put a halt to the spread of this treatment into clinical practice. Hopefully, recent advances in the fields of stem cell biology and adult neurogenesis research will lead totamen in new exciting ways to better understand and control the biological parameters necessary for achieving safe and successful neuronal replacement in PD patients.     

The role of the locus coeruleus in the development of Parkinson's disease

In Parkinson's disease, together with the classic loss of dopamine neurons of the substantia nigra pars compacta, neuropathological studies and biochemical findings documented the occurrence of a concomitant significant cell death in the locus coeruleus. This review analyzes the latest data obtained from experimental parkinsonism indicating that, the loss of norepinephrine in Parkinson's disease might worsen the dopamine nigrostriatal damage. Within this latter context, basic research provided a new provocative hypothesis on the significance of locus coeruleus in conditioning the natural history of Parkinson's disease. In particular, the loss of a trophic influence of these neurons might be crucial in increasing the sensitivity of nigrostriatal dopamine axons to various neurotoxic insults. In line with this, recently, it has been shown that locus coeruleus activity plays a pivotal role in the expression of various immediate early genes and in inducing the phosphorilation of cyclic adenosine monophosphate response element-binding proteins, suggesting a role of the nucleus in sustaining a protective effect.     

Generation of dopaminergic neurons from embryonic stem cells

Neuronal transplantation is considered to be a promising therapeutic approach to neurodegenerative diseases. In addition to fetal tissues and neural stem cells, embryonic stem cells are good candidates for the creation of neurons. We have recently identified a stromal cell-derived inducing activity that promotes neural differentiation of mouse embryoric stem cells. This activity accumulated on the surface of PA6 stromal cells and induced efficient neuronal differentiation of co-cultured embryonic stem cells under serum-free conditions without the use of either retinoic acid or embryoid bodies. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained. Induction of neurons with stromal cell-derived inducing activity may be a useful new method for basic neuroscience research and therapeutic applications, including cell transplantation therapy for Parkinson's disease.     

Stem cell therapy for Parkinson's disease: safety and modeling

For decades,clinicians have developed medications and therapies to alleviate the symptoms of Parkinson’s disease,but no treatment currently can slow or even stop the progression of this localized neurodegeneration.Fortunately,sparked by the genetic revolution,stem cell reprogramming research and the advancing capabilities of personalization in medicine enable forward-thinking to unprecedented patient-specific modeling and cell therapies for Parkinson’s disease using induced pluripotent stem cells(iPSCs).In addition to modeling Parkinson’s disease more accurately than chemically-induced animal models,patient-specific stem cell lines can be created,elucidating the effects of genetic susceptibility and sub-populations’differing responses to in vitro treatments.Sourcing cell therapy with iPSC lines provides ethical advantages because these stem cell lines do not require the sacrifice of human zygotes and genetically-specific drug trails can be tested in vitro without lasting damage to patients.In hopes of finally slowing the progression of Parkinson’s disease or re-establishing function,iPSC lines can ultimately be corrected with gene therapy and used as cell sources for neural transplantation for Parkinson’s disease.With relatively localized neural degeneration,similar to spinal column injury,Parkinson’s disease presents a better candidacy for cell therapy when compared to other diffuse degeneration found in Alzheimer’s or Huntington’s Disease.Neurosurgical implantation of pluripotent cells poses the risk of an innate immune response and tumorigenesis.Precautions,therefore,must be taken to ensure cell line quality before transplantation.While cell quality can be quantified using a number of assays,a yielding a high percentage of therapeutically relevant dopaminergic neurons,minimal de novo genetic mutations,and standard chromosomal structure is of the utmost importance.Current techniques focus on iPSCs because they can be matched with donors using human leukocyte antigens,thereby reducing the severity and risk of immune rejection.In August of 2018,researchers in Kyoto,Japan embarked on the first human clinical trial using iPSC cell therapy transplantation for patients with moderate Parkinson’s disease.Transplantation of many cell sources has already proven to reduce Parkinson’s disease symptoms in mouse and primate models.Here we discuss the history and implications for cell therapy for Parkinson’s disease,as well as the necessary safety standards needed for using iPSC transplantation to slow or halt the progression of Parkinson’s disease.