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Medicine Meets Millennium World Congress on Medicine and Health Hannover, Germany 21 July - 31 August 2000 This text is an updated version of a chapter to be published in: Neuraltransplantation in neurodegenerative disease: Current status and new directions. Novartis Foundation Symposium 231 (J Gray and SB Dunnett, eds), John Whiley & Sons, in press Cell Replacement Strategies for Neurodegenerative Disorders Anders Björklund Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Sölvegatan 17, S-223 62 Lund, Sweden Telephone: +46-46 222 05 41 Fax: +46-46 222 05 59 Email: [log in to unmask] Abstract Cell transplantation has over the last two decades emerged as a promising approach for restoration of function in neurodegenerative diseases, inparticular Parkinson´s and Huntington´s disease. Clinical trials have so far focused on the use of implants of embryonic mesencephalic tissue containing alredy fate-committed dopaminergic neuroblasts with the capacity to develop into fully mature dopamine neurons in their new location in the host brain However, the recent demonstration that immature neural progenitor cells with multipotent properties can be isolated from both the developing and adult central nervous system (CNS) and that these cells can be maintained and propagated in culture, has provided a new interesting tool for restorative cell replacement and gene transfer therapies (see Gage 1998, Snyder 1998, Svendsen and Smith 1999). Embryonic stem cells, obtained from the early stages of embryonic development, and neural stem cells, obtained from the developing brain, may provide renewable sources of cells for therapeutic purposes, and could eventually offer a powerful alternative to primary fetal CNS tissue in clinical transplantation protocols. The purpose of this review is to discuss the prospects of the emerging progenitor cell technology for cell replacement and restorative therapies in neurodegenerative diseases, and consider some of the critical issues that must be solved in order to make progenitor cells useful in studies of brain repair. Introduction Neural transplantation as a tool for replacement of lost neurons and reconstruction of damaged circuitry in the mammalian CNS is still in its infancy. Transplantation of neuronal tissue is a classic approach in neuroembryology, and this technique has been extensively used as an experimental tool for the study of neuroregeneration and repair in submammalian vertebrates. Pioneering work in amphibians and fish, which was carried out above all by Matthey, Stone and Sperry, was the first to demonstrate that grafted neurons have a capacity to substitute both structurally and functionally for lost axonal connections, and that afferent and efferent connections can be established with a high degree of specificity between grafted neurons and denervated targets in the host brain (see, e.g., Björklund and Stenevi 1984, for review). The first attempts to apply neural grafting in animal models of neurodegenerative disease were made in the late 1970s (Björklund and Stenevi 1979, Perlow et al 1979). Subsequent studies in rodents have shown that the ability of grafted neurons to become functionally integrated into the host brain depends on not only on the developmental potential of the implanted cells, but also on the plasticity of the host environment, i.e., the capacity of the brain tissue to accept new cellular elements to become integrated into the developing or established neuronal circuitry. Both these factors are greatly dependent on the developmental stage of donor and host: The brain becomes less plastic as it matures, and neurons removed from their normal context survive and grow well after intracerebral transplantation only if they are immature, i.e. at the stage of their development when they have become terminally differentiated but before they have formed extensive axonal connections (see Dunnett and Björklund 1994, for review). In the adult brain the capacity of transplanted fetal neurons to grow, integrate and establish functional efferent and afferent connections is in many cases substantially increased when the host circuitry is damaged, suggesting that some of the plastic properties that are present during development, such as mechanisms that regulate and guide axonal growth and synaptogenesis, can be reactivated by lesions or neurodegenerative changes. These lesion-induced cellular and molecular changes, which have yet to be clarified, form the basis for the remarkable ability of the lesioned adult brain to incorporate new functional elements, and thus to a degree rebuild itself. Cell transplantation as an approach to replace lost or damaged brain cells, is thus in its most effective form a technique for reconstruction of damaged neuronal circuitry. Neuronal replacement in Parkinson´s disease Parkinson's disease has come to serve as the primary test bed for the neural transplantation technique, for several reasons. One important reason is that Parkinson's disease affects primarily a circumscribed set of neurons in the brain (the mesencephalic dopamine neurons) whose main target, striatum, is anatomically well-defined and relatively accessible surgically. Moreover, and most importantly, there are well characterized animal models, both in rodents and primates, that mimic the cardinal features of the disease. Results obtained in these animal models have repeatedly proved to have good predictive value with respect to the human disease. Neuronal replacement may be most likely to work for those types of systems which have non-specific modulatory functions in the brain. The nigrostriatal dopamine neurons are good examples of this type. These neurons, which normally are located in the midbrain substantia nigra, provide a dense, diffuse innervation of one of the principal motor control centers of the forebrain, the striatum. Dopamine released from the nigrostriatal terminals acts in a tonic, level-setting manner to regulate motor behavior. Lesions of the nigrostriatal dopamine neurons induce a profound akinetic state which is at least in part due to an increased threshold for activation of the striatal output system and initiation of movement. Neural transplantation in Parkinson's disease is based on the idea that dopamine producing cells implanted into the denervated striatum might be able to substitute for those mesencephalic dopamine neurons that have been lost as a consequence of the disease process. The grafted neurons are proposed to function either by a "pharmacological" type of action, whereby the released dopamine is able to diffuse over sufficient distances to activate the denervated striatal receptors, and/or through functional reinnervation of the denervated target neurons by the outgrowing axons of the implanted neuroblasts, which allows released dopamine to exert its action at defined synaptic sites (see Björklund 1992, Herman and Abrous 1994, for review). Intrastriatal transplants of fetal nigral neurons can reverse or ameliorate impairments in both drug-induced and spontaneous motor behaviors induced by damage to the nigrostriatal system. In rodent and primate models of Parkinson’s disease grafted nigral neurons can re-establish a functional dopamine innervation of the previously denervated striatal target neurons and restore dopaminergic neurotransmission in the area reached by the outgrowing axons. In vivo data show that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites, although largely independent of any regulatory afferent inputs. Reconstruction of basal ganglia circuitry by striatal transplants The striatal GABAergic neurons provide an inhibitory control of two major striatal output structures, globus pallidus and pars reticulata of the substantia nigra. Loss of this striatal inhibitory output system, as occurs in animals with striatal lesions or in patients with Huntington’s disease, induces a hyperkinectic syndrome in combination with cognitive deficits. Since the striatal output neurons are functionally integrated in an intricately organized cortico-striato-thalamic circuitry, however, proper functioning of the striatal output neurons will depend not only on the establishment of appropriate efferent connections but also on their access to regulatory afferent inputs. On the neurochemical level, striatal neuron transplants can restore GABA synthesis and release both within the lesioned striatum and within the adjacent, denervated globus pallidus. More importantly, however, the implanted striatal primordium develop a striatum-like structure at the site of implantation, and part of the cells develop into fully differentiated striatal projection neurons. These cells, which are of the medium-size densely spring type, grow to establish normal synaptic contracts with the output neurons in the globus pallidus, as well as the appropriate afferent synaptic inputs from both thalamus, cortex and substantia nigra. Indeed, in vivo electrophysiological and microdialysis studies in rats show that the activity of the grafted striatal neurons is under the control of both cortical glutamatergic and nigral dopaminergic afferents (see Björklund et al 1994, Dunnett1995, for review). There is thus compelling evidence that fetal striatal neurons, implanted into the lesioned striatum, can reconstruct at least some critical elements of the damaged striatal circuitry in animal models of Huntington´s disease. Proof of concept in clinical trials Since 1987 about 250 patients with advanced PD have received transplants of mesencephalic dopamine neurons, obtained from 6–9 week old cadaver embryos at several centra in Europe and America (Olanow et al. 1996, Lindvall 1997). There is now convincing data to show that embryonic human nigral neurons, taken at a stage of development when they have started to express their dopaminergic phenotype, can survive, integrate and function over a long time in the human brain (i.e. in a tissue environment with an ongoing disease process). Positron emission tomography (PET) scans have shown significant increases in 18F-fluorodopa uptake (i.e. dopamine synthesis and retention) in the areas reinnervated by the grafted cells. This increase has been maintained for at least 6-10 years in several of the longest-studied patients. Consistent with the imaging data, good survival of grafted dopamine neurons and extensive reinnervation of the surrounding host striatum have been demonstrated by immunohistochemistry in two patients that have come to autopsy at 18 months after surgery (Kordower et al. 1999). Long-lasting symptomatic improvement has been observed in about 2/3 of the grafted patients, and in the most successful cases L-dopa treatment has been possible to withdraw (see Lindvall 1997). In Huntington´s disease, clinical trials using transplants of striatal primordial, obtained from the ganglionic eminences from the developing forebrain from either human or porcine embryonic donors, have been initiated in four different centers. Although initial reports have described signs of improvements in parameters of motor function and cognition, it is still too early to tell if any of these changes indeed are due to the function of the transplanted cells. The best indications that embryonic striatal transplants indeed may work also in the larger and more complex primate brain come from two recent studies in monkeys, which show that striatal transplants can induce significant recovery in both motoric and cognitive function in animals with toxin-induced destruction of the striatum, i.e. in the best models of HD that we have available today (Palfi et al. 1998, Kendall et al. 1998). Neuronal progenitors A major limitation of the fetal cell transplantation procedure is the low survival rate of the grafted dopamine neurons (in the range of 5–20%) which makes it difficult to obtain sufficient amounts of cells grafting in patients. Currently, mesencephalic fragments from at least 6-8 embryos are needed for transplantation in one PD patient. Moreover, the ethical, practical and safety issues associated with the use of tissue from aborted human fetuses are problematic, and severely restricts the possibility to apply the procedure outside highly specialized centers. The emerging possibilities to use stem cells or neuronal progenitors as a renewable source of cells provides an attractive approach to solve these problems. Differentiation of immature neural progenitors into fully mature neurons depends on an interaction between extrinsic signals, present in the cells local environment, and intrinsic signals operating in a cell autonomous manner within the cells themselves. With time during development neural progenitors will gradually acquire independence from extrinsic signals and as the cells become more restricted in their developmental potential cell-autonomous programs of differentiation will take over (see Edlund and Jessell 1999, for review). This is observed also in transplantation experiments: Cells taken at a stage of development when they have become committed to a specific neuronal fate (which appears to be around the time of the last cell division in case of nigral dopamine neurons) will carry on their normal development also in the fully mature brain, or when transplanted to ectopic sites (such as the anterior eye chamber or the kindey capsule). Uncommitted neural progenitors are able to integrate, differentiate and develop appropriately after transplantation to the brain in fetuses (Brustle et al. 1995, Fishell 1995, Campbell et al. 1995), but the capacity of the host brain environment to direct the development of uncommitted progenitors in a region-specific manner is gradually lost during the late fetal and early postnatal development in rodents. This indicates that critical extrinsic signals, necessary for terminal differentiation of neuronal precursors, are lost or downregulated as the brain matures. There are two notable exceptions to this rule: The olfactory bulb and the dentate gyrus of the hippocampal formation where active neurogenesis continues throughout life (Gage 1998, McKay 1998). In both these regions there is a continuous production of new neurons (of the small, short-axoned interneuron type) from a pool of endogenous undifferentiated progenitors located in the subventricular zone, which supplies cells to the olfactory bulb, and in the subgranular zone of the dentate gyrus. Indeed, undifferentiated progenitors transplanted into either of these sites will migrate, integrate and differentiate, along with the endogenous cells (Lois and Alvarez-Buylla 1994, Gage et al. 1995, Suhonen et al. 1996, Flax et al. 1998, Fricker et al. 1999). In these two brain regions, at least, neurogenic signals of the type present during development continues to be expressed also in the adult. Expansion of dopamine neuron precursors in vitro Along the path to fully differentiated neurons the precursor cells pass a critical event, i.e., they stop to divide and cannot re-enter the cell cycle. Determination of cell fate may take place several divisions before cell cycle exit, or – as is probably the case for the mesencephalic dopamine neurons – at the time of or shortly after the last cell division. In the rat ventral mesencephalon dopamine neurons are generated over a 5-day period, from about E11 to about E15, and phenotypic markers (e.g. tyrosine hydroxylase, TH) are expressed at the time of, or shortly after the last cell division (see Bouvier and Mytilineou 1995). In transplants of fetal mesencephalic tissue, grafted to the adult striatum, Sinclair et al (1999) have shown that in grafts taken at E14, i.e. during active neurogenesis, virtually all surviving dopamine neurons are derived from precursors that have undergone their last cell division prior to transplantation, and hence already been committed to a dopaminergic neuronal fate. These results are consistent with the view that those extrinsic signals which are necessary for the induction of mature neuronal phenotypes from uncommitted precursors may not be present normally in mature non-neurogenic brain regions, such as the adult striatum. Bouvier and Mytilineou (1995) and Studer et al (1998) have shown that mesencephalic dopamine neuron precursors can be expanded in a pre-differentiated state in culture provided that they are plated at a time close to their last cell division, but before they express a definitive dopaminergic phenotype (i.e., the TH enzyme). These lineage-restricted precursors do not continue to divide spontaneously in culture but can be maintained in an undifferentiated dividing state for about a week under stimulation with a growth factor, FGF-2. At the end of this expansion period, Studer et al (1998) estimated that the total number of cells in the FGF-2 treated cultures had increased 10-fold, and upon removal of the mitogen the yield of differentiated TH-positive neurons was increased about 30-fold over the non-expanded controls. Studer and collaborators went on to show that these in vitro expanded precursors can survive transplantation to the dopamine-denervated striatum. However, the overall yield of surviving dopamine neurons in the grafts was very low, above all due to the excessive loss of cells in the differentiation and grafting steps of the procedure (over 95%), where the expanded cells were first removed from the culture dishes and then allowed to differentiate into dopamine neurons in free-floating aggregate cultures prior to transplantation. This cell survival problem should be possible to solve, however, by improvements in the handling of the cells in the differentiation step. The cells expanded in the Studer et al (1998) study differentiated spontaneously to TH-positive neurons upon removal of the mitogen, which suggests that they were committed dopamine neuron precursors. Stimulation by FGF-2 was able to induce cell division in this precursor cell population and delay terminal differentiation, but only for about 7-8 days (Bouvier and Mytilineou 1995). Mesencephalic progenitors can be expanded for longer periods of time in neurosphere cultures. The neurosphere cells express features of multipotent progenitors, and can differentiate into both neurons and glia, but they do not spontaneously differentiate into dopamine neurons when placed in monolayer cultures in the absence of the mitogen (Svendsen and Rossor 1995, Ling et al 1998). Carvey and collaborators (Ling et al 1998, Potter et al 1999) have shown that these uncommitted progenitors can be induced to differentiate into a dopaminergic neuronal fate in the presence of a combination of cytokines, mesencephalic membrane fragments and striatal culture-condition medium. About 50% of all neurons, and 20–25% of all cells, in the differentiated cultures were seen to express the TH marker. Interestingly, the cytokine effect was seen with mesencephalic but not striatal progenitors, suggesting that this combination of factors acted to induce differentiation in a lineage-restricted mesencephalic precursor cell population (Potter et al 1999). These cells expressed not only the TH enzyme but also DOPA decarbocylase, the dopamine transporter and dopamine itself, suggesting that they represent a mature dopaminergic neuronal phenotype. To what extent these cells can survive and function after transplantation is not yet clear. Engineering cells for transplantation In vitro expanded neural progenitors may provide a highly useful source of cells for intracerebral transplantation in Parkinson’s disease provided that we can reliably control or direct their differentiation towards a specific dopaminergic neuronal fate. Ideally, these cells should not only be dopamine-producing but they should also possess the specific features of mature nigral dopamine neurons. This implies factors that convey both region-specific and transmitter-specific identity on the developing precursors. Local extrinsic signals, such as sonic hedgehog and FGF-8 (Ye et al 1998) and cell specific transcription factors, such as Nurr1 (Zetterström et al 1997), are likely to be critically involved in this process. Wagner et al (1999) have recently reported that overexpression of Nurr1 in combination with (as yet unidentified) factors from local type 1 astrocytes is sufficient to induce a dopaminergic neuronal phenotype in undifferentiated cells from the C17-2 immortalized neural stem cell line. With the protocol used, over 80% of the induced cells expressed the TH enzyme, as well as two other phenotypic markers, ADH-2 and c-ret, characteristic for ventral mesencephalic dopamine neurons. These results point to an important role of glia-derived factors in the process leading to the maturation of the dopaminergic neuronal phenotype. Indeed, in primary cultures the differentiation of mesencephalic precursors into dopamine neurons coincides with the appearance of astrocytes in the FGF-2 treated cultures (Bouvier and Mytilineou 1995). Moreover, it has been shown that cortical astrocytes, stimulated with FGF-2, secrete factors that stimulate differentiation of mesencephalic dopamine neurons (Gaul and Lubbert 1992), and that glia-conditioned media (in combination with FGF-2) can induce TH expression in in vitro expanded neural progenitors (Daadi and Weis 1999). These data suggests that neurons and glia may cooperate in both regional specification and the induction of specific neuronal identities of developing precursors. If so, the ideal cell preparation for transplantation in the Parkinson model will consist of a mixture of committed neuronal precursors and regionally specified glial cells in a stage of development where they can help to convey regional indentity to the developing neuroblasts.