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Survival, Neuronal Differentiation, and Fiber Outgrowth of Propagated Human Neural Precursor Grafts in an Animal Model of Huntington's Disease
Abstract
Expanded neural precursor cells provide an attractive alternative to primary fetal tissue for cell replacement therapies in neurodegenerative diseases. In this study we transplanted epigenetically propagated human neural precursor cells into a rat model of Huntington's disease. Neural precursors survived transplantation and large numbers differentiated to express neuronal antigens, including some that expressed DARPP-32, indicating a mature striatal phenotype had been adopted. Neuronal fibers from the grafts projected diffusely throughout the host brain, although there was no evidence that outgrowth was specifically target directed. This study supports the contention that propagated human neural precursors may ultimately be of use in therapeutic neural transplantation paradigms for diseases such as Huntington's disease.
... This observation finds further support by a transplantation study in which another chemical HD animal model was investigated. Here, injection of quinolinic acid into the striatum leads to regional excitotoxicity and subsequent degeneration of DARPP-32-positive, medium spiny projection neurons [71][72][73]. Even though the authors referred to the implanted cells as neural precursors, corresponding in vitro analyses revealed that the isolated cells were proliferating and could give rise to both, neurons and astroglia, which suggests that they were still NSCs. ...
... Even though the authors referred to the implanted cells as neural precursors, corresponding in vitro analyses revealed that the isolated cells were proliferating and could give rise to both, neurons and astroglia, which suggests that they were still NSCs. Finally, transplantation of human fetal, striatal eminence-derived NSCs six hours after striatal injury induction led to broad migration rostral and caudal to the injection site and to robust tissue integration [73]. Analysis after 12 weeks post-transplantation revealed that the majority of NSCs indeed differentiated into neurons of which some also displayed DARPP-32 expression. ...
The adult mammalian central nervous system (CNS) is generally considered as repair restricted organ with limited capacities to regenerate lost cells and to successfully integrate them into damaged nerve tracts. Despite the presence of endogenous immature cell types that can be activated upon injury or in disease cell replacement generally remains insufficient, undirected, or lost cell types are not properly generated. This limitation also accounts for the myelin repair capacity that still constitutes the default regenerative activity at least in inflammatory demyelinating conditions. Ever since the discovery of endogenous neural stem cells (NSCs) residing within specific niches of the adult brain, as well as the description of procedures to either isolate and propagate or artificially induce NSCs from various origins ex vivo, the field has been rejuvenated. Various sources of NSCs have been investigated and applied in current neuropathological paradigms aiming at the replacement of lost cells and the restoration of functionality based on successful integration. Whereas directing and supporting stem cells residing in brain niches constitutes one possible approach many investigations addressed their potential upon transplantation. Given the heterogeneity of these studies related to the nature of grafted cells, the local CNS environment, and applied implantation procedures we here set out to review and compare their applied protocols in order to evaluate rate-limiting parameters. Based on our compilation, we conclude that in healthy CNS tissue region specific cues dominate cell fate decisions. However, although increasing evidence points to the capacity of transplanted NSCs to reflect the regenerative need of an injury environment, a still heterogenic picture emerges when analyzing transplantation outcomes in injury or disease models. These are likely due to methodological differences despite preserved injury environments. Based on this meta-analysis, we suggest future NSC transplantation experiments to be conducted in a more comparable way to previous studies and that subsequent analyses must emphasize regional heterogeneity such as accounting for differences in gray versus white matter.
... L'étape suivante, consistant à évaluer leur capacité à remplacer des cellules endommagées, se place dans une perspective de médecine régénérative. Ainsi, des transplantations de cellules souches nerveuses foetales humaines dans le SNC lésé de rongeur ou de primate non humain ont montré des améliorations significatives dans des modèles de maladies telles que l'attaque vasculaire cérébrale (Kelly et al., 2004;Roitberg et al., 2006), la maladie de Parkinson (Svendsen et al., 1996), la maladie de Huntington (Armstrong et al., 2000;Lee et al., 2005), les lésions de la moelle épinière (Tarasenko et al., 2007) ou l'épilepsie (Chu et al., 2004). ...
... la maladie de Huntington(Armstrong et al., 2000;Lee et al., 2005;McBride et al., 2004), les lésions de la moelle épinière(Iwanami et al., 2005; Tarasenko et al., 2007) ou l'épilepsie(Chu et al., 2004). Globalement, les cellules survivent de façon prolongée, migrent spécifiquement en direction des régions cibles, se différencient et établissent des connexions permettant une récupération fonctionnelle partielle.Ces résultats encourageants doivent cependant être nuancés en vue des applications futures car un grand nombre de paramètres semblent influencer la survie, la migration et/ou la différenciation des cellules lors des transplantations. ...
Les folates (vitamine B9) et la vitamine B12 interviennent comme cofacteurs dans le métabolisme des monocarbones qui régule les réactions de transméthylation impliquées dans les mécanismes épigénétiques. Un déficit en folates et/ou B12 réduit la production de méthionine à partir de l'homocystéine, un acide aminé toxique dont l'accumulation a été associée à la survenue de pathologies du système nerveux central aux différents stades de la vie (spina bifida, maladie d'Alzheimer...). Afin d'explorer les mécanismes cellulaires et moléculaires impliqués dans la réponse à la carence en ces micronutriments, nous avons développé deux nouveaux modèles cellulaires. Ainsi, nous avons étudié les effets d'une déficience en folate sur la prolifération, la différenciation et la plasticité neuronale de progéniteurs neuronaux issus de l'hippocampe d'embryons de rat, la lignée H19-7. Le second modèle correspond à un projet innovant visant à obtenir une déplétion cellulaire en B12 par séquestration membranaire. Il a été obtenu par transfection stable de la lignée de neuroblastome murin NIE-115 dans le but d'induire l'expression d'une protéine chimère contenant le transporteur plasmatique de la vitamine B12, la transcobalamine II, et une protéine d'ancrage membranaire. L'ensemble de ces travaux montre que les altérations du métabolisme des monocarbones associées aux carences répriment la neurogenèse et induisent des troubles de la différentiation neuronale. Ceci suggère l'existence de mécanismes précis par lesquels le déficit en folates, en vitamine B12 et/ou l'homocystéine peuvent affecter le fonctionnement du cerveau et sa plasticité.
... Previous studies have shown that NSCs isolated from fetal or adult mammalian brain can propagate in vitro [110] and be subsequently implanted in the brain of animals presenting human-like neurological disorders, including HD [111,112]. In HD animal models, some cells differentiated in vivo into DARPP32+ neurons, replacing neurons primar- ily targeted in this disorder [111]. ...
... Previous studies have shown that NSCs isolated from fetal or adult mammalian brain can propagate in vitro [110] and be subsequently implanted in the brain of animals presenting human-like neurological disorders, including HD [111,112]. In HD animal models, some cells differentiated in vivo into DARPP32+ neurons, replacing neurons primar- ily targeted in this disorder [111]. ...
Background & objective:
Huntington's disease (HD) is an autosomal-dominant, progressive neurodegenerative disease characterized by selective loss of neurons in the striatum and cortex, which leads to progressive motor dysfunction, cognitive decline and behavioral symptoms. HD is caused by a trinucleotide (CAG) repeat expansion in the gene encoding the protein huntingtin. Despite the fact that the HD gene was identified over 20 years ago, there is no effective disease-modifying therapy for HD and only symptomatic therapies are available to date. Recently, new agents and procedures have been investigated for HD and many of them have focused on immunomodulatory and/or anti- inflammatory strategies.
Conclusion:
The objective of the current review is to summarize data on the therapeutic strategies to treat HD that are based on immunomodulatory effects.
... Stem cells, which have amazing potential to develop into many different cell types in the body during early life, may offer new therapeutic approaches for treating HD disease [7][8][9]. Fetal neural grafts, neural progenitor cells (NPCs) and mesenchymal stem cells (MSCs) have already been used in several preclinical and even in preliminary clinical trials [10][11][12][13][14]. ...
... NPCs derived from rodent ESCs have similar beneficial effects as human NPCs when transplanted into the chemical rodent model. They are able to differentiate into neurons and the animals that receive rodent NPC transplantation show rotation behavior improvement as compared with untreated animals [13,77,92]. ...
... An alternative source of cell transplantation in HD would be neural stem cells (NSCs) that participate in normal central nervous system (CNS) development and differentiate into regionally appropriate cell types in response to environmental factors [36]. In this way, previous studies have shown that NSCs isolated from foetal or adult mammalian CNS can be propagated in vitro [37] and subsequently intracerebrally implanted in animal models of human neurological disorders including HD (Table 1) [38,39]. In the latter case, some cells differentiated in vivo into DARPP32þ neurons replacing neurons primarily targeted in this disorder [38]. ...
... In this way, previous studies have shown that NSCs isolated from foetal or adult mammalian CNS can be propagated in vitro [37] and subsequently intracerebrally implanted in animal models of human neurological disorders including HD (Table 1) [38,39]. In the latter case, some cells differentiated in vivo into DARPP32þ neurons replacing neurons primarily targeted in this disorder [38]. Also, genetically modified NSCs producing neurotrophic factors have been used to protect striatal neurons against excitotoxic insults [40]. ...
... Human neural precursor cells (HNPCs) have in several studies proved an excellent capacity to generate neurons after in vivo intracerebral transplantation [15][16][17]. Such cells have been studied in animal models of neural degenerative diseases such as Huntington's and Parkinson's diseases with a remarkable ability to form site-specific neurons and to some extent have a functional effect [17][18][19][20]. ...
... Human neural precursor cells (HNPCs) have in several studies proved an excellent capacity to generate neurons after in vivo intracerebral transplantation [15][16][17]. Such cells have been studied in animal models of neural degenerative diseases such as Huntington's and Parkinson's diseases with a remarkable ability to form site-specific neurons and to some extent have a functional effect [17][18][19][20]. In addition, the possibility to culture human spiral ganglion tissue to obtain neurons indicates a presence of HNPCs also in the adult human auditory nervous system [21]. ...
Objectives:
To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation.
Methods:
We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of β-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact.
Results:
Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus.
Conclusion:
Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN).
... Replacement of lost cells is, however, a greater challenge. Although human neural stem cells can differentiate into neurons after im- plantation [10,11], improvements of functional deficits by fetal striatal transplants into a lesioned rat striatum is associated with DARPP-32 neurons within the trans- plants121314. Despite good survival and differentiation of human neurons in the rats, differentiation of cells into DARPP-32 neurons remains a challenge [10,15] . ...
... Although human neural stem cells can differentiate into neurons after im- plantation [10,11], improvements of functional deficits by fetal striatal transplants into a lesioned rat striatum is associated with DARPP-32 neurons within the trans- plants121314. Despite good survival and differentiation of human neurons in the rats, differentiation of cells into DARPP-32 neurons remains a challenge [10,15] . Predifferentiation of cells prior to implantation into a DARPP-32 phenotype therefore could potentially result in an improved outcome [16]. ...
Background
Cell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology.
Results
In this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3.
Conclusions
Although the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line’s therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.
... Under these conditions, differentiated cells die, and more primitive cells proliferate as free-floating spheres of cells, the so-called -neurospheres‖ (154). Continuously dividing immortalized human NSCs with switch-on oncogenes have advantageous features including homogeneous nature and the same clone (6,154). It requires significant advances in our understanding of the developmental signals for proliferation, differentiation, and integration of NSCs and to determine favorable conditions of the host brain environment for implanted NSCs to survive, prosper, and restore the damaged brain by stem cell transplant therapy. ...
... This was initially shown in rodents (Reynolds et al., 1992) and since then numerous papers have demonstrated similar findings in both rodent and human (Murphy et al., 1990(Murphy et al., , 1994Bartlett et al., 1995;Fricker et al., 1999). There are clear species differences in the responses of FNPs to specific proliferation media, with murine cells tending to proliferate for much longer periods of time than rat and porcine cells Ciccolini et al., 1998;Armstrong et al., 2000Armstrong et al., , 2002. In addition, if hFNPs could survive cryopreservation this would ease current practical constraints associated with scheduling the neurosurgery and would also permit at least some standardisation of the cells, which cannot currently be achieved for primary hWGE. ...
Huntington's disease (HD) is a neurodegenerative disease that offers an excellent paradigm for cell replacement therapy because of the associated relatively focal cell loss in the striatum. The predominant cells lost in this condition are striatal medium spiny neurons (MSNs). Transplantation of developing MSNs taken from the fetal brain has provided proof of concept that donor MSNs can survive, integrate and bring about a degree of functional recovery in both pre-clinical studies and in a limited number of clinical trials. The scarcity of human fetal tissue, and the logistics of coordinating collection and dissection of tissue with neurosurgical procedures makes the use of fetal tissue for this purpose both complex and limiting. Alternative donor cell sources which are expandable in culture prior to transplantation are currently being sought. Two potential donor cell sources which have received most attention recently are embryonic stem (ES) cells and adult induced pluripotent stem (iPS) cells, both of which can be directed to MSN-like fates, although achieving a genuine MSN fate has proven to be difficult. All potential donor sources have challenges in terms of their clinical application for regenerative medicine, and thus it is important to continue exploring a wide variety of expandable cells. In this review we discuss two less well-reported potential donor cell sources; embryonic germ (EG) cells and fetal neural precursors (FNPs), both are which are fetal-derived and have some properties that could make them useful for regenerative medicine applications.
... However, advanced studies with NSCs in injured animal models have shown the regenerative potential of these cells. 15,45,46 NSCs are self-renewing cells able to generate neurons, glial cells and astrocytes. NSCs can be derived from embryonic, fetal or adult tissues. ...
Huntington's disease (HD) is a fatal genetic disorder, which causes the progressive breakdown of neurons in the human brain. HD deteriorates human physical and mental abilities over time and has no cure. Stem cell-based technologies are promising novel treatments, and in HD, they aim to replace lost neurons and/or to prevent neural cell death. Herein we discuss the use of human fetal tissue (hFT), neural stem cells (NSCs) of hFT origin or embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs), in clinical and pre-clinical studies. The in vivo use of mesenchymal stem cells (MSCs), which are derived from non-neural tissues, will also be discussed. All these studies prove the potential of stem cells for transplantation therapy in HD, demonstrating cell grafting and the ability to differentiate into mature neurons, resulting in behavioral improvements. We claim that there are still many problems to overcome before these technologies become available for HD patient treatment, such as:
a) safety regarding the use of NSCs and pluripotent stem cells, which are potentially teratogenic;
b) safety regarding the transplantation procedure itself, which represents a risk and needs to be better studied; and finally
c) technical and ethical issues regarding cells of fetal and embryonic origin.
... No proven medical treatments are currently available to counteract the devastating course of the disease. Over the past decades, studies in both HD animal models (Peschanski et al., 1995;Armstrong et al., 2000) and pilot clinical trials (Bachoud-Lévi et al., 2006;Reuter et al., 2008;Gallina et al., 2010;Zuccato et al., 2010;Onorati et al., 2014;Paganini et al., 2014) have demonstrated that replacement of degenerated striatum and repair of circuitries by grafting fetal striatal primordium is feasible, safe and may counteract disease progression, thus prospecting an effective strategy to treat HD patients. The achievement of this practice depends on the ability of the grafted cells to proliferate, differentiate, and re-establish impaired circuitries (Tuszynski, 2007). ...
Over the past decades, studies in both Huntington's disease animal models and pilot clinical trials have demonstrated that replacement of degenerated striatum and repair of circuitries by grafting fetal striatal primordium is feasible, safe and may counteract disease progression. However, a better comprehension of striatal ontogenesis is required to assess the fetal graft regenerative potential. During neuronal development, neurotrophins exert pleiotropic actions in regulating cell fate and synaptic plasticity. In this regard, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are crucially implicated in the control of fate choice of striatal progenitor cells. In this study, we intended to refine the functional features of human striatal precursor (HSP) cells isolated from ganglionic eminence of 9–12 week old human fetuses, by studying with electrophysiological methods the effect of BDNF and FGF2 on the membrane biophysical properties and the voltage-dependent Ca2 + currents. These features are particularly relevant to evaluate neuronal cell functioning and can be considered reliable markers of the developmental phenotype of human striatal primordium. Our results have demonstrated that BDNF and FGF2 induced membrane hyperpolarization, increased the membrane capacitance and reduced the resting total and specific conductance values, suggesting a more efficient control of resting ionic fluxes. Moreover, the treatment with both neurotrophins enhanced N-type Ca2 + current amplitude and reduced L- and T-type ones. Overall, our data indicate that BDNF and FGF2 may help HSP cells to attain a more functionally mature phenotype.
... for differentiation are best candidates for such replacement cell therapy [1,4,11,12] Numerous diseases of the central nervous system of a Stem and multipotent cells exist in germinative (venmetabolic, genetic or inflammatory nature are accompanied tricular and subventricular) brain structures not only in by dysfunction or degeneration of a significant proportion embryonal and early postnatal period of ontogenesis of neuronal population. A promising approach for curing [8,10,12], but during the whole life span of human and such diseases is transplantation of neural cells. ...
Human neural stem cells (HNSCs) are used in studies of neural development and differentiation, and are regarded as an alternative source of tissue for neural transplantation in degenerative diseases. Selection and standardization of HNSC samples is an important task in research and clinical approaches. We evaluated embryonal brain matter obtained from human 8–12-week-old fetuses by means of flow cytometry on a panel including: nestin; vimentin; NeuN; GFAP; b-tubulin III; CD56; N-Cad; OB-Cad; HLA-ABC; HLA-DR; CD34, and annexin. Samples from embryos of even the same gestation differ dramatically regarding neural cell development, their phenotype and viability. The samples containing the highest proportion of stem cells and multipotent progenitors of neural types, and the least of definitive cells and antigens of histocompatibility, were selected for further expansion in serum-free medium. Secondary phenotyping 14 days later revealed again a marked heterogeneity of the cultures. For the final culturing for 24 h in a serum-containing medium we selected only samples having following phenotype: nestin1, and vimentin1 no less than 25%; HLA-DR1 and CD341 no more than 5%; GFAP1 no more than 10%; b-tubulin1 no more than 20%; CD561, N-Cad1, OB-Cad1, HLA-A,B,C1, and annexin1 no more than 15%; cell viability no less than 60%. Immunocytochemical study of selected samples proved that numerous neural stem cells, and neuro-and glioblasts necessary for transplantation were present. Our results demonstrate that the flow cytometry phenotyping allows the screening and standardization of HNSC samples for further expansion and transplantation.
... Injection of neural stem cells into the damaged rat striatum could cure HD if the injected cells survive the dissociation/transplantation and differentiate in the brain (Svendsen, 1996;Armstrong et al., 2000;Bjorklund and Lindvall, 2000). In this context, neural stem cells were isolate from rat embryos, expand in culture, pre-labeled fluorescently with Orange Tracker, and transplanted into the striatum of the HD rat. ...
Stem cells provide an important means for regenerative medicine due to the capacity to generate multiple types of differentiated cells and at the same time to maintain self-renewal. To identify the therapeutic effect of the transplantation of neural stem cells, differentiation and migration capacity of the neural stem cells that were isolated from E14 rat embryo and maintained in culture were examined after transplantation to the striatum of the quinolinic acid (QA)-induced Huntington's disease rat model. in vitro co-culture of the neural stem cells with the mixture of primary neurons and astrocytes promoted the maturation and the synapse formation of neuronal progenies of neural stem cells. Following the implantation, the neural stem cells survived, differentiated, and migrated in the damaged striatum region, exhibiting immunoreactivities against nestin, Tuj-1, GFAP, GAD67 and synapsin 1 to a varying degree. These data provide clear evidence supporting that the neural stem cells isolated from the rat embryo and maintained in the primary culture have a multiple capacity to differentiate into neurons or glial cells both in vitro and in vivo.
... This meant that a striatal phenotype had been achieved. The neurons were found to have diffused throughout the brain of the rats [46]. ...
... Our studies have indicated that acute intrastriatal injections of the endogenous N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QA), can mimic some of the neuroanatomical and behavioral deficits of HD (Maksimović et al., 2002). Intrastriatal injections of QA have been shown to produce an HD-like pattern of neurodegeneration, including loss of striatal projection neurons (Armstrong et al., 2000). The disease causes widespread loss of cells from the corpus striatum, which causes the motor disturbance (Watts and Dunnett, 1998). ...
The aim of this study was to examine the effects of intrastriatally injected nitric oxide synthase (NOS) inhibitors, N omega-nitro-l-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), on quinolinic acid (QA)-induced toxicity in selective vulnerable brain regions of adult Wistar rats. QA was administered into the striatum unilaterally, in a single dose of 150 nM/L with a stereotaxic instrument. The other two experimental groups were pretreated with L-NAME and 7-NI, respectively. The control group of animals was treated with 0.154 mM/L saline solution. The animals were decapitated seven days after the treatment. Samples of both striatum and forebrain cortex were prepared for measurement of acetylcholinesterase (AChE) activity. QA injection revealed a significant increase in AChE activity in both the ipsi- and contralateral striatum and forebrain cortex compared to the control animals. Treatment with NOS inhibitors, followed by QA, very clearly demonstrated lower levels of AChE bilaterally in these brain structures, compared to the QA-treated group.
... Various studies deriving these cells from embryonic or adult tissues have shown that these cells can be directed to give rise to cells having neuronal phenotype with the help of exogenous addition of different neurotrophic factors like brain derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) etc. [31]. This fundamental development and maintenance of these cells have been tried and tested in number of neurodegenerative disease animal models and have demonstrated a great deal of success in forming the CNS cellular phenotypes and expressing related signaling characteristics [32]. The HNPCs that are used in the present study have been well described previously. ...
... coordination, as measured on the accelerod, and that late-stage transplantation of iPSCs can promote behavioral recovery. Transplantation of pluripotent cells, isolated from fetal tissue, has demonstrated similar preservation of motor function in animal models of HD [23][24][25][26][27][28], but our findings provide the first demonstration of iPSC-induced functional recovery in a HD rodent model do date. ...
Induced pluripotent stem cells (iPSCs) show considerable promise for cell replacement therapies for Huntington's disease (HD). Our laboratory has demonstrated that tail-tip fibroblasts, reprogrammed into iPSCs via two adenoviruses, can survive and differentiate into neuronal lineages following transplantation into healthy adult rats. However, the ability of these cells to survive, differentiate, and restore function in a damaged brain is unknown. To this end, adult rats received a regimen of 3-nitropropionic acid (3-NP) to induce behavioral and neuropathological deficits that resemble HD. At 7, 21, and 42 days after the initiation of 3-NP or vehicle, the rats received intrastriatal bilateral transplantation of iPSCs. All rats that received 3-NP and vehicle treatment displayed significant motor impairment, whereas those that received iPSC transplantation after 3-NP treatment had preserved motor function. Histological analysis of the brains of these rats revealed significant decreases in optical densitometric measures in the striatum, lateral ventricle enlargement, as well as an increase in striosome size in all rats receiving 3-NP when compared with sham rats. The 3-NP-treated rats given transplants of iPSCs in the 7- or 21-day groups did not exhibit these deficits. Transplantation of iPSCs at the late-stage (42-day) time point did not protect against the 3-NP-induced neuropathology, despite preserving motor function. Transplanted iPSCs were found to survive and differentiate into region-specific neurons in the striatum of 3-NP rats, at all transplantation time points. Taken together, these results suggest that transplantation of adenovirus-generated iPSCs may provide a potential avenue for therapeutic treatment of HD.
... With the recent explosive progress of stem cell biology, application of stem cell-based therapy in HD appears a promising prospect. Three kinds of stem cells: embryonic stem cells, bone marrow stem cells and neural stem cells have previously been utilized in cell therapy in animal models of neurological disorders [18][19][20]. However, neural stem cells were preferably used by investigators in experimental HD studies, since they seemed to have the capacity to become neurons or glial cells after intracerebral or intravenous transplantation [21,22]. ...
... Mesenchymal stem cells (MSCs) have been utilized for the treatment of diverse
diseases, including neuropathies such as Parkinson's disease,1 Huntington's disease,2 multiple sclerosis,3, 4 amyotrophic lateral
sclerosis,5 ischemic
stroke,6, 7 and non-neurological diseases such as myocardial
infarction,8, 9 and graft-versus-host diseases.10 The therapeutic effects of MSCs are
ascribed to their paracrine functions that include the secretion of beneficial
molecules,11, 12 anti-inflammatory factors,13, 14 or extracellular
matrix.15 ...
Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.
... 67 Excitotoxic animal models induced by QA, which stimulates glutamate receptors, and resembles the histopathologic characteristics of HD patients, were utilized for cell therapy with mouse embryonic stem cells, mouse neural stem cells, mouse bone marrow mesenchymal stem cells and primary human neural precursor cells, and resulted in varying degrees of clinical improvement. [68][69][70][71][72][73] We have recently injected human NSCs intravenously in QA-HD model rats and demonstrated functional recovery in HD animals. 72,73 The systemic transplantation of NSCs via an intravascular route is probably the least invasive method of cell administration. ...
Human neurodegenrative diseases such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are caused by a loss of neurons and glia in the brain or spinal cord. Neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and stem cell-based cell therapies for neurodegenerative diseases have been developed. A recent advance in generatioin of a new class of pluripotent stem cells, induced pluripotent stem cells (iPSCs), derived from patients' own skin fibroblasts, opens doors for a totally new field of personalized medicine. Transplantation of NSCs, neurons or glia generated from stem cells in animal models of neurodegenrative diseases, including PD, HD, ALS and AD, demonstrates clinical improvement and also life extension of these animals. Additional therapeutic benefits in these animals can be provided by stem cell-mediated gene transfer of therapeutic genes such as neurotrophic factors and enzymes. Although further research is still needed, cell and gene therapy based on stem cells, particularly using neurons and glia derived from iPSCs, ESCs or NSCs, will become a routine treatment for patients suffering from neurodegenerative diseases and also stroke and spinal cord injury.
Stem cell therapy has long been a popular method of treatment for Parkinson's disease currently being researched in both preclinical and clinical settings. While early clinical results are based upon fetal tissue transplants rather than stem cell transplants, the lack of successful integration in some patients and gradual loss of effect in others suggests a more robust protocol is needed. We propose a two-front approach, one where transplants are directly stimulated in coordination with host activity elicited by behavioral tasks, which we refer to as behavioral context. After a pilot with unilateral 6-OHDA rats transplanted with dopaminergic cells differentiated from mesenchymal stem cells that were optogenetically stimulated during a swim task, we discovered that early stimulation predicted lasting reduction of motor deficits, even in the absence of later stimulation. This led to a follow-up with n = 21 rats split into three groups: one stimulated while performing a swim task (Stim-Swim; St-Sw), one not stimulated while swimming (NoStim-Swim; NSt-Sw), and one stimulated while stationary in a bowl (Stim-NoSwim; St-NSw). After initial stimulation (or lack thereof), all rats were retested two and seven days later with the swim task in the absence of stimulation. The St-Sw group gradually achieved and maintained symmetrical limb use, whereas the NSt-Sw group showed persistent asymmetry and the St-NSw group showed mixed results. This supports the notion that stem cell therapy should integrate targeted stimulation of the transplant with behavioral stimulation of the host tissue to encourage proper functional integration of the graft.
Huntington's disease (HD) is a hereditary, neurodegenerative disorder characterized by a triad of symptoms: motor, cognitive and psychiatric. HD is caused by a genetic mutation, expansion of the CAG repeat in the huntingtin gene, which results in loss of medium spiny neurons (MSNs) of the striatum. Cell replacement therapy (CRT) has emerged as a possible therapy for HD, aiming to replace those cells lost to the disease process and alleviate its symptoms. Initial pre-clinical studies used primary fetal striatal cells to provide proof-of-principal that CRT can bring about functional recovery on some behavioral tasks following transplantation into HD models. Alternative donor cell sources are required if CRT is to become a viable therapeutic option and human pluripotent stem cell (hPSC) sources, which have undergone differentiation toward the MSNs lost to the disease process, have proved to be strong candidates. The focus of this chapter is to review work conducted on the functional assessment of animals following transplantation of hPSC-derived MSNs. We discuss different ways that graft function has been assessed, and the results that have been achieved to date. In addition, this chapter presents and discusses challenges that remain in this field.
Parkinson’s disease is a neurodegenerative condition characterized by motor impairments caused by the selective loss of dopaminergic neurons in the substantia nigra. Levodopa is an effective and well-tolerated dopamine replacement agent. However, levodopa provides only symptomatic improvements, without affecting the underlying pathology, and is associated with side effects after long-term use. Cell-based replacement is a promising strategy that offers the possibility to replace lost neurons in Parkinson’s disease treatment. Clinical studies of transplantation of human fetal ventral mesencephalic tissue have provided evidence that the grafted dopaminergic neurons can reinnervate the striatum, release dopamine, integrate into the host neural circuits, and improve motor functions. One of the limiting factors for cell therapy in Parkinson’s disease is the low survival rate of grafted dopaminergic cells. Different factors could cause cell death of dopaminergic neurons after grafting such as mechanical trauma, growth factor deprivation, hypoxia, and neuroinflammation. Neurotrophic factors play an essential role in the survival of grafted cells. However, direct, timely, and controllable delivery of neurotrophic factors into the brain faces important limitations. Different types of cells secrete neurotrophic factors constitutively and co-transplantation of these cells with dopaminergic neurons represents a feasible strategy to increase neuronal survival. In this review, we provide a general overview of the pioneering studies on cell transplantation developed in patients and animal models of Parkinson’s disease, with a focus on neurotrophic factor-secreting cells, with a particular interest in mesenchymal stromal cells; that co-implanted with dopaminergic neurons would serve as a strategy to increase cell survival and improve graft outcomes.
In future, neurodegenerative diseases will take over cancer's place and become the major cause of death in the world, especially in developed countries. Advancements in the medical field and its facilities have led to an increase in the old age population, and thus contributing to the increase in number of people suffering from neurodegenerative diseases. Economically it is of a great burden to society and the affected family. No current treatment aims to replace, protect, and regenerate lost neurons; instead, it alleviates the symptoms, extends the life span by a few months and creates severe side effects. Moreover, people who are affected are physically dependent for performing their basic activities, which makes their life miserable. There is an urgent need for therapy that could be able to overcome the deficits of conventional therapy for neurodegenerative diseases. Stem cells, the unspecialized cells with the properties of self-renewing and potency to differentiate into various cells types can become a potent therapeutic option for neurodegenerative diseases. Stem cells have been widely used in clinical trials to evaluate their potential in curing different types of ailments. In this review, we discuss the various types of stem cells and their potential use in the treatment of neurodegenerative disease based on published preclinical and clinical studies.
Glioblastoma, the deadliest form of primary brain tumor, remains a disease without cure. Treatment resistance is in large part attributed to limitations in the delivery and distribution of therapeutic agents. Over the last 20 years, numerous preclinical studies have demonstrated the feasibility and efficacy of stem cells as antiglioma agents, leading to the development of trials to test these therapies in the clinic. In this review we present and analyze these studies, discuss mechanisms underlying their beneficial effect and highlight experimental progress, limitations and the emergence of promising new therapeutic avenues. We hope to increase awareness of the advantages brought by stem cells for the treatment of glioblastoma and inspire further studies that will lead to accelerated implementation of effective therapies.
The potential of Neural Stem Cells (NSCs) to provide therapeutic benefit for a variety of neurologicaldisorders, including brain malignancies, has been long recognized and has inspired many scientists to design,test and successfully demonstrate that NSCs are efficient and effective therapeutic agents. Glioblastoma, thedeadliest form of primary brain tumor, despite extensive and sustained efforts to find better therapies, remainsa disease without cure, with a median survival after diagnosis of less than two years. Treatment resistance inglioblastoma is in large part attributed to limitations in the delivery and distribution of therapeutic agentsadministered either systemically or directly into the tumor due to the highly invasive nature of this cancer andits abnormal intratumoral vasculature. Stem Cells (SCs) have an innate tumor-tropic migratory behavior, canbe modified to deliver a variety of therapeutic agents and efficiently distribute their cargo into brain tumors,pursuing invading streams of tumor cells, deep into the brain parenchyma. Over the last twenty years,numerous preclinical trials have demonstrated the feasibility and efficacy of SCs as antiglioma agents, leadingto the development of trials to test these therapies in the clinic. In this review we present and analyze thesestudies and discuss mechanisms underlying their beneficial effect, highlighting experimental progress,limitations and the emergence of promising new therapeutic avenues. We hope to increase awareness of theadvantages of using SCs for the treatment of glioblastoma and inspire further studies that will lead toaccelerated implementation of effective therapies.
Wound healing is a multifaceted biological process involving the replacement of damaged tissues and cellular structures, restoring the skin barrier's function, and maintaining internal homeostasis. Over the past two decades, numerous approaches are undertaken to improve the quality and healing rate of complex acute and chronic wounds, including synthetic and natural polymeric scaffolds, skin grafts, and supramolecular hydrogels. In this context, this review assesses the advantages and drawbacks of various types of supramolecular hydrogels including both polymeric and peptide‐based hydrogels for wound healing applications. The molecular design features of natural and synthetic polymers are examined, as well as therapeutic‐based and drug‐free peptide hydrogels, and the strategies for each system are analyzed to integrate key elements such as biocompatibility, bioactivity, stimuli‐responsiveness, site specificity, biodegradability, and clearance. Supramolecular polymeric and peptidic hydrogels present effective alternatives for the treatment of complex acute and chronic wounds. Nonhealing wounds of this nature require constant medical intervention, placing a tremendous burden on patients. However, the tunability of hydrogels makes them conducive to wound healing by incorporating growth factors, cytokines, and therapeutic bioactive molecules in their design.
Blastocyst complementation is an emerging methodology in which human stem cells are transferred into genetically engineered preimplantation animal embryos eventually giving rise to fully developed human tissues and organs within the animal host for use in regenerative medicine. The ethical issues surrounding this method have caused the National Institutes of Health to issue a moratorium on funding for blastocyst complementation citing the potential for human cells to substantially contribute to the brain of the chimeric animal. To address this concern, we performed an in-depth review of the neural transplantation literature to determine how the integration of human cells into the nonhuman neural circuitry has altered the behavior of the host. Despite reports of widespread integration of human cell transplants, our review of 150 transplantation studies found no evidence suggestive of humanization of the animal host, and we thus conclude that, at present, concerns over humanization should not prevent research on blastocyst complementation to continue. We suggest proceeding in a controlled and transparent manner, however, and include recommendations for future research with careful consideration for how human cells may contribute to the animal host nervous system.
Neural stem cells isolated from the developing and adult brain are an ideal source of cells for use in clinical applications such as cell replacement therapy. The clear advantage of these cells over the more commonly utilised embryonic and pluripotent stem cells is that they are already neurally committed. Of particular importance is the fact that these cells don’t require the same level of in vitro culture that can be cost and labour intensive. Foetal neural stem cells can be readily derived from the foetal brain and expand in culture over time. Similarly, adult stem cells have been explored for their potential in vitro and in vivo animal models. In this chapter we identify the progress made in developing these cells as well as the advantages of taking them forward for clinical use.
The combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease(HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stemcells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors,lipid nanocapsules and solid SPAN nanoparticles,carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD.
One in four people worldwide suffer some form of neurodegenerative disorder. The World Health Organization estimates that there are currently four million people worldwide with Parkinson’s disease (PD), 37 million with Alzheimer’s disease (AD) (4.5 million in the United States), and 5.5 million die each year as a result of cerebrovascular events. In the United States, more than 50 million people are affected by various central nervous system (CNS) diseases. Each year, 11,000 people sustain spinal cord injury, adding to the 400,000 or so already affected. Two million people have been disabled by head injuries with 1.5 million people a year suffering traumatic brain injury (TBI), adding to the 5.3 million already living with disabilities resulting from TBI (http://www.who.int). With an aging global population, the number of people with neurodegenerative and cerebrovascular conditions continues to grow, as does the cost to the health service.
Human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple sclerosis (MS), stroke and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. I review here notable experimental and pre-clinical studies previously published involving stem cell-based cell- and gene-therapies for PD, HD, ALS, AD, MS and stroke, and discuss for future prospect for the stem cell therapy of neurological disorders in clinical setting. There are still many obstacles to be overcome before clinical application of cell- and gene-therapy in neurological disease patients is adopted: (i) it is still uncertain how to generate specific cell types of neurons or glia suitable for cellular grafts in great quantity, (ii) it is required to abate safety concern related to tumor formation following NSC transplantation, and (iii) it needs to be better understood by what mechanism transplantation of NSCs leads to an enhanced functional recovery. Steady and stepwise progress in stem cell research in both basic and pre-clinical settings should support the hope for development of stem cell-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells particularly NSCs as substrates for structural and functional repair of the diseased or injured brain.
Neural stem cells (NSC) exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. A great deal of literature has addressed the identification, screening and isolation of neural stem cells. The NSC in these regards have been reviewed (Gage, 1994; Gage et al., 1995b; Weiss et al., 1996b; McKay et al., 1997; Vescovi & Snyder, 1999; Gage, 2000). The mammalian adult brain reserves a limited pool of neural stem cells, which can differentiate and incorporate into the mature brain. In this chapter, the regulation of stem cells in the adult brain and spinal cord is discussed. The potency and neurogenesis of endogenous NSC in a genetically predetermined state are frequently affected by environmental factors, such as enriched environment, excercise, stress, or stroke. Neural replenishment, a process preserved in lower animals, is now being assessed in the repair of the mammalian nervous system. In addition, NSCcan also be recruited from non-neural origins. The mechanisms that convert early stem cells or non-NSCinto NSCremain elusive, but are beginning to be unveiled. Potential uses of neural stem cells, including transplantation to repair missing cells and activation of endogenous cells to promote “self-repair” are also reviewed in this chapter.
The ability to isolate neural stem and precursor cells and expand them in culture has provided researchers a new tool, not only assisting studies of neural development but also providing a new source of defined and expandable cells for in vivo studies using transplantation. The purposes of this chapter are, first, to review available protocols for in vitro expansion of neural precursor cells, either epigenetically using growth factors or genetically by inserting immortalizing genes; and, second, to discuss the in vivo properties of in vitro-propagated neural stem and progenitor cells, as assessed by grafting to the developing or adult rodent brain. This discussion will focus on our own recent studies using growth factor-expanded neurosphere cells of mouse and human origin and a particularly interesting, conditionally immortalized neural cell line, RN33B.
Although complex circuit reconstruction in the brain or spinal cord still presents many formidable challenges (1), an extensive body of embryonic central nervous system (CNS) transplantation research has established a compelling proof of principle for cell replacement therapies in animal models of Parkinson’ s disease (PD) (2) and other intractable neurological disorders (3,4). With the advent of stem/progenitor cell biology, focus has shifted from primary fetal CNS tissue to alternative cell sources, and many of these options offer promise for therapeutic neuronal replacement in the brain and spinal cord. The interest this has generated also has contributed to a greater focus on the progression toward clinical trials within the academic and commercial biotechnology sectors, as well as among patient advocacy groups. However, despite exciting recent advances, many intersecting scientific issues and principles of translational bench-to-bedside neurobiology remain to be resolved before the high expectations of stem or progenitor-cell-based therapies can be fully realized.
Human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple sclerosis (MS), stroke and spinal cord injury are caused by a loss of neurons and glial cells in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. I review here notable experimental and pre-clinical studies previously published involving stem cell-based cell- and gene-therapies for PD, HD, ALS, AD, MS and stroke, and discuss for future prospect for the stem cell therapy of neurological disorders in clinical setting. There are still many obstacles to be overcome before clinical application of cell- and gene-therapy in neurological disease patients is adopted: (i) it is still uncertain how to generate specific cell types of neurons or glia suitable for cellular grafts in great quantity, (ii) it is required to abate safety concern related to tumor formation following NSC transplantation, and (iii) it needs to be better understood by what mechanism transplantation of NSCs leads to an enhanced functional recovery. Steady and stepwise progress in stem cell research in both basic and pre-clinical settings should support the hope for development of stem cell-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells particularly NSCs as substrates for structural and functional repair of the diseased or injured brain.
Human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple sclerosis (MS), stroke and spinal cord injury (SCI) are caused by loss of neurons and glia in the brain or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glia have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. I review here notable experimental and pre-clinical studies previously published involving stem cell-based cell- and gene-therapies for PD, HD, ALS, AD, MS, stroke and SCI, and discuss for future prospect for the stem cell therapy of neurological disorders in clinical setting. There are still many obstacles to be overcome before clinical application of cell- and gene-therapy in neurological disease patients is adopted: (i) it is still uncertain how to generate specific cell types of neurons or glia suitable for cellular grafts in great quantity, (ii) it is required to abate safety concern related to tumor formation following NSC transplantation, and (iii) it needs to be better understood by what mechanism transplantation of NSCs leads to an enhanced functional recovery. Steady and stepwise progress in stem cell research in both basic and pre-clinical settings should support the hope for development of stem cell-based therapies for neurodegenerative diseases. This review focuses on the utility of stem cells particularly NSCs as substrates for structural and functional repair of the diseased or injured brain.
Stem cell therapies have been explored as a new avenue for the treatment of neurologic disease and damage within the CNS in part due to their native ability to mimic repair mechanisms in the brain. Mesenchymal stem cells have been of particular clinical interest due to their ability to release beneficial neurotrophic factors and their ability to foster a neuroprotective microenviroment. While early stem cell transplantation therapies have been fraught with technical and political concerns as well as limited clinical benefits, mesenchymal stem cell therapies have been shown to be clinically beneficial and derivable from nonembryonic, adult sources. The focus of this review will be on emerging and extant stem cell therapies for juvenile and adult-onset Huntington's disease.
Huntingtons disease is a dominantly inherited neurodegenerative disorder, usually starting in mid-life and leading to progressive disability and early death. There are currently no disease-modifying treatments available. Cell transplantation is being considered as a potential therapy, following proof of principle that cell transplantation can improve outcomes in another basal ganglia disorder, namely Parkinsons disease. The principle aim is to replace the striatal medium spiny neurons lost in Huntingtons disease with new cells that are able to take over their function and reconnect the circuitry. This article reviews the experimental background and evidence from clinical studies that suggest that cell transplantation may improve function in Huntingtons disease, reviews the current status of the field and considers the current challenges to taking this experimental strategy forward to becoming a reliable therapeutic option.
Huntington's disease is currently incurable, but cell therapy is seen as a promising alternative treatment. We analyze the safety and efficacy of the intrastriatal transplantation of human fetal neuroblasts from ganglionic eminences in patients with Huntington's disease. A few rare surgical incidents were reported, but the main difficulty associated with this therapeutic approach is the occurrence of recipient alloimmunization against the graft and the lack of availability, standardization and quality control for the fetus-derived products required for cell therapy. Some patients showed sustained cognitive improvement over periods of more than six years, and motor improvements for more than four years. Grafting outcomes are variable even within individual transplantation centers. The reasons for this variability are poorly understood, highlighting the need for further research in this specific area. With the perspective of additional trials in the future, we review here the development of human pluripotent stem cell-derived cell therapy products for HD, and their advantages and disadvantages with respect to fetal cells.
Copyright © 2014. Published by Elsevier Masson SAS.
Neural stem cells have the
potential to migrate to areas
of pathology in the CNS
making them ideal agents for
the treatment of various CNS
pathologies, especially brain
tumours. In this present issue of
infocus the current literatures
related with stem cells are
reviewed and the possibility of
the neural cell therapy for several
neurosurgical disorders are
discussed in detail.The potential targets of stem cell therapy reveals, offer so much hope for revolutionary advances in medicine.
There is lot of interest has developed among the medical researcher to understand the biology and therapeutic
potential of human stem cells. Stem cells are emerging as one of the promising areas of neuroscience, not only
to reveal insights into normal development, but also as a therapeutic modality for a spectrum of neurological
disorders. Because of involvement of human lives involved in stem cell research was tightly linked to human
lives; it is not surprising that stem cell research is not free from controversies. It is believed that stem cells will
not only be used as part to cure the diseases but also will be in applied to understand the disease processes.
The polyglutamine (polyQ) diseases are a group of neurodegenerative disorders caused by expanded cytosine-adenine-guanine (CAG) repeats encoding a long polyQ tract in the respective proteins. To date, a total of nine polyQ disorders have been described: six spinocerebellar ataxias (SCA) types 1, 2, 6, 7, 17; Machado-Joseph disease (MJD/SCA3); Huntington's disease (HD); dentatorubral pallidoluysian atrophy (DRPLA); and spinal and bulbar muscular atrophy, X-linked 1 (SMAX1/SBMA). PolyQ diseases are characterized by the pathological expansion of CAG trinucleotide repeat in the translated region of unrelated genes. The translated polyQ is aggregated in the degenerated neurons leading to the dysfunction and degeneration of specific neuronal subpopulations. Although animal models of polyQ disease for understanding human pathology and accessing disease-modifying therapies in neurodegenerative diseases are available, there is neither a cure nor prevention for these diseases, and only symptomatic treatments for polyQ diseases currently exist. Long-term pharmacological treatment is so far disappointing, probably due to unwanted complications and decreasing drug efficacy. Cellular transplantation of stem cells may provide promising therapeutic avenues for restoration of the functions of degenerative and/or damaged neurons in polyQ diseases.
This chapter focuses on the clinical features and care of Huntington disease (HD). The HD gene is highly penetrant, such that clinical features emerge eventually in HD gene carriers who live long enough to manifest illness. Clinical expressivity varies, mainly related to age at clinical onset, which is heavily influenced by the extent of cytosine-adenine-guanine repeat (CAGn) expansion. The clinical characteristics of HD have traditionally been categorized as motor, cognitive, and behavioral, but these domains are highly inter-related and stem from a common process of selective neuronal degeneration and resulting gliosis and atrophy. The identification of the HD gene, the rapid pace of scientific discovery, and the expanding knowledge about the clinical and biological features of premanifest and manifest HD hold great promise for substantive therapeutic advances. The family approach to care is becoming increasingly applicable in the age of molecular genetics, and DNA predictive testing remains challenging because of often complicated and unique family dynamics and interactions. Direct prenatal DNA testing, involving detection of the actual CAG expansion in fetal cells, is accurate and also reveals the HD gene-carrier status of the at-risk parent. Reasonably effective pharmacotherapy has been developed for temporary treatment of the motor, cognitive, and behavioral symptoms of HD notwithstanding the progressive underlying neurodegeneration. The chapter also discusses the treatment of movement disorder, experimental treatments for HD, and restorative strategies.
Alterations in signal transduction pathways involving neurotransmitters, trophic factors, and cytokines that are involved in regulating neuronal excitability and plasticity are also subject to modification by aging. The cellular and molecular changes that occur in the nervous system during “usual” aging may predispose neurons to degeneration in disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Decreases in synapse numbers in some regions of the nervous system may occur during aging, but may be offset by increases in the synaptic size. These changes are often associated with alterations in cytoskeletal proteins and the deposition of insoluble proteins such as amyloid in the extracellular space. Several different alterations that occur during usual aging of the nervous system are greatly enhanced in neurodegenerative disorders. For example, levels of lipid peroxidation products such as lipid peroxides and the aldehyde 4-hydroxnonenal are significantly elevated in vulnerable brain regions of AD and PD patients, and in spinal cords of ALS patients.
Human neurological diseases such as Parkinson disease, Huntington disease, amyotrophic lateral sclerosis (ALS), Alzheimer
disease, multiple sclerosis (MS), stroke and spinal cord injury are caused by a loss of neurons and glial cells in the brain
or spinal cord. Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the
development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However,
the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered
the development of this promising therapeutic approach. In recent years, neurons and glial cells have successfully been generated
from stem cells such as embryonic stem cells, mesenchymal stem cells and neural stem cells, and extensive efforts by investigators
to develop stem cell-based brain transplantation therapies have been carried out. I review here notable experimental and pre-clinical
studies previously published involving stem cell-based cell- and gene-therapies for Parkinson disease, Huntington disease,
ALS, Alzheimer disease, MS, stroke, spinal cord injury, brain tumor and lysosomal storage diseases, and discuss for future
prospect for the stem cell therapy of neurological disorders in clinical setting. There are still many obstacles to be overcome
before clinical application of cell therapy in neurological disease patients is adopted: (1) it is still uncertain what kind
of stem cells would be an ideal source for cellular grafts, and (2) it needs to be better understood by what mechanism transplantation
of stem cells leads to an enhanced functional recovery and structural reorganization. Steady and solid progress in stem cell
research in both basic and pre-clinical settings should support the hope for development of stem cell-based cell therapies
for neurological diseases.
Mesenchymal stromal cells have the ability to self renew for a limited amount of divisions, and at the very least to differentiate towards bone, cartilage and fat lineages.Their homing potential towards an injured tissue and their low immunogenicity making possible their use in allogeneic therapy explain the interest these cells have aroused in the field of regenerative medicine for two decades, further increased by the lack of ethical issues about their way of collection.Recently, the notions underlying the supportive role MSC played in the hematopoietic stem cell niche have been extended to a wide array of more mature cells. Clinical benefits formerly linked to mesenchymal stromal cells differentiation were then explained by their supporting capacity towards neighbouring cells, opening a whole new field of clinical applications for MSCs.Along with the first clinical trials based on MSCs, more than a decade ago, the vessel embolisation risk with clots of injected cells, and more disquieting, the genomic instability possibly increased by culture conditions have drawn a lot of attention, without the benefit/risk ratio weighing against clinical use of MSCs.In this short review we will sum up the characteristics allowing a cell population to be termed MSC, the organ locations where they can be found, in particular for a clinical use, the clinical trials under way as well as their predictable close future uses.
Disease of the nervous system causes considerable suffering, places a large economic burden on society and, to date, remains relatively untapped by pharmaceutical and biotechnology companies. The burgeoning field of stem cell biology promises to revolutionise and expand the use of cell therapy in the treatment of nervous system disease. By virtue of their multipotentiality stem cells are ideally suited to replace diverse neural and glial populations lost to disease or injury, and thereby functionally reconstruct neural circuits. The ease with which they can be genetically modified also makes them appealing vectors for ex vivo gene therapy in a multitude of conditions. This article reviews the approaches used to obtain large numbers of stem cells for such applications, including stem cells derived from neural tissue and propagated using either genetic or epigenetic means, totipotential embryonic stem cells and stem cells isolated from other tissues. The evidence for the efficacy of neural stem cell therapy in prototypical animal models of disease is then reviewed.
STROKE mortality has declined over recent decades, prompting a demand for the development of effective rehabilitative therapies for stroke survivors. This effort has been facilitated by significant progress in replicating the behavioral and neuropathological changes of authentic human cerebral ischemia using relevant animal models. Since the rodent model of middle cerebral artery occlusion mimics several motor abnormalities seen in clinical cerebral ischemia, we have utilized this model to investigate treatment strategies for stroke. The present study explored the potential benefits of neural transplantation of fetal rat striatal cells or human neurons derived from a clonal embryonal carcinoma cell line to correct the abnormalities associated with cerebral ischemia. We report here that ischemia-induced behavioral dysfunctions were ameliorated by the neural grafts as early as 1 month post-transplantation. Of note, transplantation of human neurons induced a significantly more robust recovery than fetal rat striatal grafts. Thus, the logistical and ethical concerns about the use of fetal striatal cells for transplantation therapy can be eliminated by exploiting cell line-derived human neurons as alternative graft sources. Transplantation of human neurons has a therapeutic potential for treatment of behavioral deficits associated with cerebral ischemia.
Neurogenesis in the mammalian central nervous system is believed to end in the period just after birth; in the mouse striatum
no new neurons are produced after the first few days after birth. In this study, cells isolated from the striatum of the adult
mouse brain were induced to proliferate in vitro by epidermal growth factor. The proliferating cells initially expressed nestin,
an intermediate filament found in neuroepithelial stem cells, and subsequently developed the morphology and antigenic properties
of neurons and astrocytes. Newly generated cells with neuronal morphology were immunoreactive for gamma-aminobutyric acid
and substance P, two neurotransmitters of the adult striatum in vivo. Thus, cells of the adult mouse striatum have the capacity
to divide and differentiate into neurons and astrocytes.
Human fetal mesencephalic tissue obtained from elective first-trimester abortions was grafted to 6-hydroxydopamine-denervated striatum of athymic (nude) rats. After 3-6 months, the transplants were evaluated by light and electron microscopy using antibodies against tryosine hydroxylase (TH), human specific Thy-1 (Thy-1), 5-hydroxytryptamine (5-HT), and laminin. In vivo chronoamperometric studies of K+-induced release of electroactive species were done prior to the histochemical evaluations. At the light microscopical level, Thy-1-immunoreactivity was evenly distributed throughout the entire transplants. Thy-1-immunoreactive nerve fibers were observed radiating from the graft into the host striatum. In sections that were double-stained with antibodies against Thy-1 and TH, such nerve fibers contained both markers. Also 5-HT-immunoreactive cells were found in the grafts with processes both in the grafts and radiating into host neuropil. Laminin immunohistochemistry showed an even distribution of capillaries in the graft with less density than in host brain, suggesting immaturity of graft tissue. At the ultrastructural level, TH-immunoreactive axons made symmetric contacts with unlabeled dendritic shafts and dendritic spines within the host brain. A few asymmetric contacts with TH-immunoreactive axons were seen. 5-HT-immunoreactive terminals made both symmetric and asymmetric contacts with unlabeled dendritic shafts and spines. In vivo chronoamperometry using local application of K+ revealed average signals that were lower on the transplanted side than in control striatum. However, close to the grafts significant amounts of the K+-evoked signal amplitudes were as large as 1.3 microM, and the ratio of the reduction to oxidation currents suggested release of a mixture of dopamine and 5-HT. Taken together, this study shows that human fetal mesencephalic tissue pieces survive grafting into nude rats, develop normal vascularization, and express coexistence of TH- and Thy-1-immunoreactivity. Human TH- and 5-HT-immunoreactive nerve fibers form synapses in host striatum and release monoamine neurotransmitters.
We have used a rat model of Parkinson's disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD. Human mesencephalic DA neurons, obtained from 6.5-8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosuppressed with Cyclosporin A. The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway. Grafts from an 11.5-week old donor exhibited a lower survival rate and smaller functional effects. As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels. The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system. DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue. Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves. In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression. In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells. This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection. Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient.
Human neural transplants are being developed to treat Parkinson's disease. Previous characterization of human transplants focused on neuronal development, while little is known of the interaction between the transplant and its environment, among which blood is of prime importance. We evaluated here the formation of blood vessels in human neural xenografts placed into the brain of rats immunosuppressed with cyclosporin A. Using capillary wall markers, we found that human transplants remain virtually nonvascularized for more than 1 month. Angiogenesis takes place very slowly and the density of blood vessels is still quite poor after 3 months, the fine structure of these capillaries, when they form, is apparently normal. Functional studies indicate that the vascular network formed in the transplant allows blood circulation and exhibits a working barrier to macromolecules. Glucose uptake and consumption and cytochrome oxidase activity are almost undetectable up to 3 months after grafting. These results demonstrate that vascularization is much delayed in human xenografts into the rat brain. This delay is likely to be dependent on the maturation of the transplanted tissue. A dedifferentiation of human endothelial cells cotransplanted with neural cells occurs since histochemical and immunocytochemical markers revealing endothelial cells in the human fetus are not present up to 1 month in the transplant. The origin of this phenomenon is a matter of speculation. How neural cells survive and mature in such conditions are issues of prime interest for the future of human neural grafting.
The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.
Central nervous system neurons and glia arise from undifferentiated embryonic neuroepithelial cells. Such progenitor cells from the human fetal forebrain can be propagated in vitro for extended periods, when grown on non-adhesive substrates in medium containing epidermal growth factor and insulin-like growth factor-1. These actively-dividing cells can be induced to differentiate into a variety of histochemically-characterized neurons and glia consistent with their forebrain origin. Electrophysiological recording indicates that differentiated neurons derived from these progenitors mature slowly, and display a range of glutamate- and GABA-mediated conductances characteristic of normal mammalian forebrain neurons. Our observations support a role for these trophic factors in normal development of the human brain. The methods described here may provide abundant normal, untransformed human forebrain neurons and glia for research and therapeutic applications.
Huntington's disease is an autosomal dominant, inherited disorder that results in progressive degeneration of the basal ganglia (especially the neostriatal caudate nucleus and putamen) and other forebrain structures and is associated with a clinical profile of movement, cognitive and psychiatric impairments for which there is at present no effective therapy. Neuropathological, neurochemical and behavioral features of the disease can all be reproduced in experimental animals by local injection of excitotoxic or metabolic toxins into the neostriatum. All these features of the disease can be alleviated, at least in rats, by transplantation of embryonic striatal tissue into the degenerated striatum, which was the basis for commencing the first clinical trials of striatal transplantation in Huntington's patients. However, although rat striatal xenografts may temporarily reduce apomorphine-induced dyskinesias in monkeys, there has been no demonstration that allograft techniques that work well in rats translate effectively to the much larger differentiated striatum of primates. Here we demonstrate good survival, differentiation and integration of striatal allografts in the primate neostriatum, and recovery in a test of skilled motor performance. Long-term graft survival in primates indicates probable success for clinical transplants in Huntington's disease; in addition, our data suggest that graft placement has a direct influence on the pattern and extent of functional recovery.
Axonal growth from cortically placed fetal neural transplants to subcortical targets in adult hosts has been difficult to demonstrate and is assumed to be minimal; however, experiments using xenogeneic neural grafts of either human or porcine fetal tissues into the adult rat striatum, mesencephalon, and spinal cord have demonstrated the capability for long-distance axonal growth. This study reports similar results for porcine cortical xenografts placed in the adult rat cerebral cortex and compares these findings with results from cortical allografts. Adult rats that previously received unilateral cortical lesions by an oblique intracortical stereotaxic injection of quinolinic acid, were implanted with suspensions of either E14 rat or E38 xenogeneic porcine fetal cortical cells. Xenografted rats were immunosuppressed by cyclosporin A. The corpus callosum was intact in all cases and grafts were confined to the overlying cortex. After a 31-34 wk posttransplant survival period, acetylcholinesterase (AChE) staining and tyrosine hydroxylase (TH) immunocytochemistry revealed that both allo- and xenografts received host afferents. Retrograde tracer injections into the ipsilateral striatum and cerebral peduncle in allografted animals failed to show any axonal growth to either subcortical target. Using a porcine-specific axonal marker in xenografted animals, we found graft axons in white matter tracts (corpus callosum, internal capsule, cingulum bundle, and medial forebrain bundle) and within the caudate-putamen and both the ipsilateral and contralateral cerebral cortex. Graft axons were not found in the thalamus, midbrain, or spinal cord. In addition, using an antibody to porcine glial fibers, we observed more extensive graft glial fiber growth into the same host fiber tracts, as far caudally as the cerebral peduncle, but not into gray matter targets outside the cortex. These results demonstrate that porcine cortical xenograft axons and glia can extend from lesioned cerebral cortex to cortical and subcortical targets in the adult rat brain. These findings are relevant for prospects of repairing cortical damage and obtaining functional recovery.
We investigated the 3-nitropropionic acid (3-NP)–induced hypoactive model of Huntington's disease (HD) to demonstrate whether fetal tissue transplantation can ameliorate behavioral deficits associated with a more advanced stage of HD. Twelve-week-old Sprague–Dawley rats were introduced to the 3-NP dosing regimen (10 mg/kg, i.p., once every 4 days for 28 consecutive days), and were then tested for general spontaneous locomotor activity in the Digiscan locomotor apparatus. All rats displayed significant hypoactivity compared to their pre-3-NP injection locomotor activity. Randomly selected rats then received bilateral intrastriatal solid grafts of fetal striatal (lateral ganglionic eminence, LGE) tissues from embryonic day 14 rat fetuses. Approximately 1/3 of each LGE in hibernation medium was infused into each lesioned host striatum. In control rats, medium alone was infused intrastriatally. A 3-mo posttransplant maturation period was allowed prior to locomotor activity testing. Animals receiving fetal LGE grafts exhibited a significant increase in locomotor activity compared to their post-3-NP injection activity or to the controls’ posttransplant activity. Surviving striatal grafts were noted in functionally recovered animals. This observation supports the use of fetal striatal transplants to correct the akinetic stage of HD. To the best of our knowledge, this is the first study that has investigated the effects of fetal striatal transplantation in a hypoactive model of HD.
Precise determination of donor age in human embryonic and fetal tissue is crucial for cell transplantation due to the existence of distinct time windows within which successful grafting is possible. This study demonstrates that between 4-12 wk postconception embryonic and fetal age can be estimated based on various morphometric parameters measured on a routine basis in suction abortion material. The greatest length, the neck-rump length, the foot length, and the proximal and distal arm and leg length were correlated with the anamnestic and ultra-sonographically estimated age. Multivariate regression analyses showed a linear correlation between age and the logarithmic value of the various morphometric parameters. The best correlation was found for a mathematical model combining the limb parameters (r = 0.904; p < 0.001; n −37). A prospective follow-up study (n = 40) was carried out to test the validity of the mathematical model. A high correlation was found between the calculated age and the estimated age based on anamnestic data (r = 0.749, p < 0.001). Outliers due to errors in the anamnestic data were readily identified by comparing anamnestic with calculated age. This method allows determination of embryonic and fetal age within and beyond the age group of the Carnegie classification and may, therefore, be useful for the needs of experimental and clinical cell transplantation.
Neurotransplantation has been proposed as a potential treatment for the neurodegenerative disorder of Huntington's disease (HD), which currently has no effective therapy. While patients with Parkinson's disease have received neurotransplantation, until recently no HD patients have undergone transplantation for HD with standardized evaluations of their progress following surgery. The current report presents the cognitive changes in three patients with HD who underwent bilateral transplantation of human fetal striatal tissue. As part of the pre- and postsurgical evaluation, all three patients were administered a neuropsychological battery sensitive to the cognitive effects of HD within 2 mo prior to surgery and at 4-6 mo following transplantation. Four to 6 mo subsequent to surgery, all patients demonstrated increased scores on some measures of cognitive functioning. However, the pattern of changes was not uniform across subjects. These findings suggest that fetal striatal transplantation may improve some of the cognitive symptoms associated with HD in the three reported patients.
In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.
In vitro expansion of central nervous system (CNS) precursors might overcome the limited availability of dopaminergic neurons in transplantation for Parkinson's disease, but generating dopaminergic neurons from in vitro dividing precursors has proven difficult. Here a three-dimensional cell differentiation system was used to convert precursor cells derived from El2 rat ventral mesencephalon into dopaminergic neurons. We demonstrate that CNS precursor cell populations expanded in vitro can efficiently differentiate into dopaminergic neurons, survive intrastriatal transplantation and induce functional recovery in hemiparkinsonian rats. The numerical expansion of primary CNS precursor cells is a new approach that could improve both the ethical and the technical outlook for the use of human fetal tissue in clinical transplantation.
Optimization of the procedures for neural grafting is a timely issue, as this technique has proven beneficial for a few patients with late-stage Parkinson's disease in pilot studies and therefore may expand to become a more widely available therapeutic. In this research, one major issue is that of the placement of the cell deposits in the right target areas within the striatum. Although it is widely accepted that these suitable regions are the sensorimotor regions of the putamen, reliable delineation of these areas using classical stereotactical mapping techniques remains difficult. Along the course of a 5-year-long clinical transplantation program, we have developed an original procedure based on magnetic resonance imaging of the striatum on parasagittal views. This technique allowed us to identify precisely, and reproducibly in each patient, three subregions of the putamen (precommissural, commissural, and postcommissural) to be implanted. On the basis of the literature defining the sensorimotor putaminal regions in nonhuman primates, it was subsequently possible to extrapolate and localize these regions in each patient, thus providing a basis for the placement of cell deposits. Examples taken from our series of grafted patients demonstrate the value of this procedure that, in addition, minimizes interference of interindividual variability in the interpretation of clinical results.
This study was designed to explore the efficacy of a human clone cell line as an alternative neural graft source and to validate the practice of cryopreservation and xenografting as logistical approaches toward conducting neural transplantation. We investigated the biological effects of transplanting cultured human neurons (NT2N cells) derived from a well-characterized embryonal carcinoma cell line into the brains of rats subjected to transient, focal cerebral ischemia induced by embolic occlusion of the middle cerebral artery. At 1 month and extending throughout the 6-month posttransplantation test period, ischemic animals that were transplanted with NT2N cells and treated with an immunosuppressive drug displayed a significant improvement in a passive avoidance task as well as a normalization of asymmetrical motor behavior compared to ischemic animals that received rat fetal cerebellar cell grafts or vehicle alone. Remarkably, cryopreserved NT2N cell grafts compared with fresh NT2N cell grafts, remained viable in the immunosuppressed rat brain and effective in producing behavioral recovery in immunosuppressed ischemic animals. The long-term viability of cryopreserved NT2N cell xenografts in vivo and their sustained effectiveness in promoting behavioral recovery suggest potential utilization of xenografting and cryopreservation as useful protocols for establishing clone cell lines as graft source in neural transplantation therapies for central nervous system disorders.
Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.
The failure of lesioned axons to regenerate over long distances in the mammalian central nervous system (CNS) is not due to an inability of central neurons to regenerate, but rather to the non-permissive nature of the CNS tissue environment. Regenerating CNS axons, which grow well within a peripheral nerve, for example, fail to penetrate mature CNS tissue by more than about 1 mm. Recent evidence indicates that this may be due to inhibitory membrane proteins associated with CNS oligodendrocytes and myelin. We report here that human telencephalic neuroblasts implanted into the excitotoxically lesioned striatum of adult rats can escape or neutralize this inhibitory influence of the adult CNS environment and extend axons along major myelinated fibre tracts for distances of up to approximately 20 mm. The axons were seen to elongate along the paths of the striato-nigral and cortico-spinal tracts to reach the substantia nigra, the pontine nuclei and the cervical spinal cord, which are the normal targets for the striatal and cortical projection neurons likely to be present in these implants.
The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14-15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats. The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immunoreactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry. During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters. Grafts implanted into ibotenic acid-lesioned striatum grew rapidly (about five-fold) in volume over the first week. The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e. were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e. non-striatum-like) tissue. Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts. By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches. Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions. Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts. Grafts implanted into dopamine-denervated hosts showed a normal morphological maturation of both DARPP-32-positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts. Grafts implanted into non-lesioned striata did not grow beyond their initial size. The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders. DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions.(ABSTRACT TRUNCATED AT 400 WORDS)
Fetal striatal grafts display a striking modularity of composition. With acetylcholinesterase (AChE) histochemistry, the tissue of such grafts can be divided into regions with strong AChE staining of the neuropil and regions in which AChE staining of the neuropil is weak. In the experiments reported here, we reexamined the nature of this modularity. Striatal grafts were made by injecting dissociated cells of E15 ganglionic eminence into the striatum of adult rats, which 7 days before had recived intrastriatal deposits of ibotenic acid. Some donors had been exposed to 3H-thymidine at E11-E15. After 9-17 month survivals, the anatomical organization of the grafts was studied by histochemistry, immunohistochemistry, and autoradiography. In every graft, the AChE-rich regions formed patches (P regions) in a larger AChE-poor surround (NP regions). Neurons labeled with 3H-thymidine appeared in both P and NP regions, suggesting that donor cells were distributed in each type of region and that neither type of tissue, P or NP, was composed exclusively of host tissue. In the AChE-rich P regions, markers characteristic of normal perinatal and mature rat striatum were expressed by medium-sized cells: calcium-binding protein (calbindin D28k) immunostaining, metenkephalin (mENK) immunostaining, and, more rarely, somatostatin (SOM) immunostaining. In the NP regions, however, medium-sized cells expressing calbindin and mENK immunostaining were very rare, and there was an abundance of neuronal types not found in normal mature striatal tissue. These included (1) large, multipolar, calbindin-positive neurons with well-ramified, densely stained dendrites, (2) large, SOM-positive neurons with prominent dendritic trees, and (3) mENK-positive cells smaller than typical striatal, medium-sized, mENK-immunoreactive neurons. In Nissl stains, the AChE-rich P regions resembled the normal striatum of mature animals, whereas the AChE-poor NP regions did not. These findings suggest that the P regions of fetal striatal grafts achieve a phenotypy similar to that of normal striatum at maturity and during much of postnatal development. The dominant expression of perikaryal calbindin-like immunoreactivity in the P regions further suggests that these zones have a high proportion of tissue resembling striatal matrix. By contrast, expression of marker antigens in the NP zones of the grafts suggests that these zones are predominantly composed of nonstriatal tissue or that they have the phenotypy of immature striatum intermixed with some nonstriatal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Previous reports from this laboratory have indicated that fetal rat striatal grafts have the major types of neuronal and glial components known to be involved in Huntington's chorea. In this study a number of major afferent and efferent innervations seen in normal striatum were examined in the striatal grafts and were compared with embryonic striatal afferents. First, using immunocytochemistry and histochemistry, the host serotonergic (5-HT), dopaminergic (DA, stained with anti-tyrosine hydroxylase (TH) antiserum), and acetylcholinesterase (AChE) fibers exhibited vigorous growth into the grafts implanted in neostriatum, lateral ventricle, globus pallidus or substantia nigra within a period of 6 and 10 weeks. Individual characteristic terminal patterns formed in striatal grafts: 5-HT fibers were diffused; TH fibers became heavily packed, and AChE fibers were patchy. This peculiar patternization of 5-HT and TH growth into striatal graft appeared to be a recapitulation of the normal 5-HT and TH ingrowth into striatum in the embryonic stage. However, a significantly slow (6 week) onset of adult 5-HT and TH ingrowth into the fetal graft was noted, as compared with that of normal embryonic development (5-6 days from the appearance of 5-HT and TH neurons). With the anterograde-transport marker Phaseolus vulgaris agglutinin leuca method, host cortical neurons also projected to the graft, but in limited numbers. Finally, with the retrograde-transport marker (horseradish peroxidase method, the grafts implanted in neostriatum were found incapable of sending fibers to a major, distal target, substantia nigra. In a current evaluation of striatal transplants, it is shown that major input to the graft can be achieved over time, but output to the distal nigra seems an unrealistic expectation. These data suggest that: (1) the fetal brain tissue was found to be a strong stimulant for sprouting or regeneration of adult nerve fibers; (2) a number of functional recoveries reported on the tested behavior paradigm in this grafting model could be due to the survival of striatal graft and the establishment of input circuitries; further, (3) the data illustrate the necessity of seeking a bridge from the striatal transplant to the host nigra. If a proper functional recovery in Huntington's chorea requires complete striatonigral circuitry, then such a bridge is worthy of a major investigation.
Grafts of fetal striatum were implanted in the form of a cell suspension into the brains of rats with prior ibotenic acid lesions of the caudate-putamen. The grafts were placed in three different sites: the lesioned caudate-putamen, or the denervated (but otherwise undamaged) globus pallidus and substantia nigra. After 3–6 months survival the grafts were investigated by means of immunohistochemistry and receptor autoradiography in combination with routine histology and acetylcholinesterase histochemistry. The grafts placed within the lesioned caudate-putamen were at least 10-fold larger larger than those placed in the substantia nigra region, with the grafts placed in the globus pallidus being of intermediate size. In all locations the acetylcholinesterase staining had an uneven, patchy distribution, which was most pronounced in the grafts located within the caudate-putamen. These patches did not bear any obvious relationship to variations in density of the neuronal perikarya within the grafted tissue.
Future advances in neuro-oncology will increasingly rely on an understanding of the molecular biology of brain tumors. Recent laboratory work, including the identification of oncogenes and tumor suppressor genes, has elucidated many of the molecular events contributing to oncogenesis. In particular, the signaling pathways for the growth factors have been implicated in the genesis and the maintenance of several human tumors, including neoplasms of the central nervous system (CNS). Growth factor autocrine and paracrine stimulatory loops promote tumor proliferation and angiogenesis. A family of structurally related growth factor receptors, the receptor tyrosine kinases, are particularly relevant to tumors of the CNS. This large family includes the receptors for the epidermal growth factor, the platelet-derived growth factor, the fibroblast growth factor, the insulin-like growth factor, the neurotrophins related to the nerve growth factor, and the vascular endothelial growth factor, as well as several receptors for which no growth factor ligand has been identified. Several of these receptor molecules and their growth factor ligands are preferentially expressed in the embryonic brain and are thought to play a central role in regulating the determination of the cell fate during the development of the CNS. Moreover, the overexpression or the mutation of genes encoding these receptors can be oncogenic. Researchers think that some receptors in this family (i.e., those that have been shown to be overexpressed or mutated in human brain tumors) contribute to brain tumor oncogenesis. This article will focus on recent experimental work and will discuss the classification and the biology of the receptor tyrosine kinases, as well as their roles in the development of the CNS and in tumorigenesis.
Previous studies suggest that a population of precursor cells from the developing and adult mouse striatum can be expanded in culture using serum-free, N2-supplemented medium and mitogenic factors such as epidermal growth factor (EGF). Here we show that EGF-responsive precursor cells from embryonic rat striatum and mesencephalon can also be expanded in culture, incorporate bromodeoxy uridine (BrDU) and develop into spheres that either adhere to the surface of the culture dish or float freely in the medium. Addition of B27, a medium supplement that increases neuronal survival in primary CNS cultures, resulted in a tenfold increase in the number of proliferating cells in vitro over the first week. The effects of B27-supplemented medium on precursor cell survival were only seen when primary cultures were used, such that dividing cells grown in B27 for 1 week could then be transferred to either B27 or N2 medium and show similar survival and division rates in response to EGF. After 1, 2 or 4 weeks of growth in B27-supplemented medium, dissociated precursor cells from either striatal or mesencephalic cultures could be differentiated when exposed to a poly-l-lysine-coated substrate in serum and EGF-free medium supplemented with B27. These cells then matured into a mixed culture containing neurons (approximately 35% of cells), astrocytes (approximately 44% of cells), and oligodendrocytes (approximately 10% of cells), based on immunocytochemical staining with microtuble-associated protein (MAP2), glial fibriallary acidic protein and galactocerebrosidase. When whole spheres of precursor cells were allowed to differentiate, every one examined was found to generate neurons, astrocytes and oligodendrocytes in similar proportions.(ABSTRACT TRUNCATED AT 250 WORDS)
Intrastriatal grafts of nigral and adrenal tissues have been found to be effective in alleviating many of the simple motor and sensorimotor deficits associated with lesions of the nigrostriatal dopamine system. However, the mechanisms by which such grafts exert their effects may be less specific than originally conceived, and both pharmacological and trophic actions play an essential role. Damage to intrinsic cortico-striatal circuits are unlikely to prove similarly amenable to such diffuse mechanisms of repair. Nevertheless, striatal grafts have been found to alleviate cognitive and motor deficits after excitotoxic lesions of the neostriatum. Accumulating evidence suggests that in this particular case many aspects of functional recovery may indeed be attributable to the striatal grafts providing an effective functional reconstruction of damaged neuronal circuits within the host brain.
Transplantations for neurological disorders are limited by the supply of human fetal tissue. To generate larger numbers of cells of appropriate phenotype, we investigated whether human neural progenitors expanded in vitro could be modified with recombinant adenoviruses. Strong expression of beta-galactosidase was obtained in vitro. Two or three weeks after transplantation of engineered cells to the rat brain, we observed a small percentage of surviving neuroblasts strongly expressing beta-galactosidase in four out of 13 rats. Thus human precursor cells that have been genetically modified using adenoviruses are a promising tool for ex vivo gene therapy of neurodegenerative diseases.
Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.
Axonal growth from cortically placed fetal neural transplants to subcortical targets in adult hosts has been difficult to demonstrate and is assumed to be minimal; however, experiments using xenogeneic neural grafts of either human or porcine fetal tissues into the adult rat striatum, mesencephalon, and spinal cord have demonstrated the capability for long-distance axonal growth. This study reports similar results for porcine cortical xenografts placed in the adult rat cerebral cortex and compares these findings with results from cortical allografts. Adult rats that previously received unilateral cortical lesions by an oblique intracortical stereotaxic injection of quinolinic acid, were implanted with suspensions of either E14 rat or E38 xenogeneic porcine fetal cortical cells. Xenografted rats were immunosuppressed by cyclosporin A. The corpus callosum was intact in all cases and grafts were confined to the overlying cortex. After a 31-34 wk posttransplant survival period, acetylcholinesterase (AChE) staining and tyrosine hydroxylase (TH) immunocytochemistry revealed that both allo- and xenografts received host afferents. Retrograde tracer injections into the ipsilateral striatum and cerebral peduncle in allografted animals failed to show any axonal growth to either subcortical target. Using a porcine-specific axonal marker in xenografted animals, we found graft axons in white matter tracts (corpus callosum, internal capsule, cingulum bundle, and medial forebrain bundle) and within the caudate-putamen and both the ipsilateral and contralateral cerebral cortex. Graft axons were not found in the thalamus, midbrain, or spinal cord. In addition, using an antibody to porcine glial fibers, we observed more extensive graft glial fiber growth into the same host fiber tracts, as far caudally as the cerebral peduncle, but not into gray matter targets outside the cortex. These results demonstrate that porcine cortical xenograft axons and glia can extend from lesioned cerebral cortex to cortical and subcortical targets in the adult rat brain. These findings are relevant for prospects of repairing cortical damage and obtaining functional recovery.
After a number of reports indicating positive clinical outcome of intrastriatal transplantation of fetal ventral mesencephalic tissue into patients with Parkinson's disease, the time may have come to consider the possibility of using this technique to treat patients with Huntington's disease. On the basis of the available literature, the Network of European CNS Transplantation and Restoration has established a program aiming at defining the optimal conditions for such clinical trials. The present study, conducted within this framework, pursued the goal of providing information concerning the period of striatal neuronal ontogeny in humans, taking into account the technical and legal requirements imposed by the clinical procedure of neural transplantation using human tissue. On this basis, it aimed at establishing a reliable dissecting method for the intrastriatal grafting of human fetal striatal neurons. The ontogeny of medium-spiny neurons within the developing striatum was first studied in a series of human fetal brains, 5 to 10 weeks postconception, using immunocytochemical detection of DARPP-32. Immunoreactive neurons were observed in fetuses at 7 weeks of age and older. They were mostly localized in clusters, packed in the lateral ganglionic eminence. Over a 2-week-long period, DARPP-32 neurons increased in number. Their morphology remained poorly differentiated, however, with small cell bodies, few branched dendrites, and variable intensity of immunostaining. Based on these findings, selective dissection of the lateral ganglionic eminence was carried out. This tissue was stereotaxically implanted into the striatum of immunosuppressed adult rats previously lesioned. Two months postgrafting, DARPP-32 neurons were observed as discrete patches, embedded within areas of essentially DARPP-32-negative tissue. Up to 2 months after grafting, neurons remained poorly differentiated in general, with only a few neurons exhibiting a dense immunoreactivity and long processes. These results indicate that striatal DARPP-32-immunoreactive neurons are present in the lateral ganglionic eminence in fetuses as soon as 7 weeks postconception. The striatal tissue can be dissected out and successfully transplanted. Within the grafts, neuronal differentiation appears to be a very long process, suggesting that many months might be necessary for these neurons to become functionally integrated into an adult host brain.
In cultures of embryonic striatum, we previously reported that EGF induces the proliferation of single precursor cells, which give rise to spheres of undifferentiated cells that can generate neurons and glia. We report here that, in vitro, these embryonic precursor cells exhibit properties and satisfy criteria representative of stem cells. The EGF-responsive cell was able to generate the three major phenotypes of the mammalian CNS--neurons, astrocytes, and oligodendrocytes. Approximately 90% of both primary spheres and secondary expanded clones, derived from the primary spheres, contained all three cell types. The increase in frequency of EGF-generated spheres, from 1% in primary culture to close to 20% in secondary culture, and the large number of clonally derived secondary spheres that could be generated from a single primary sphere indicate that EGF induces both renewal and expansion of the precursor cell itself. In population studies, the EGF-responsive cells were carried through 10 passages, resulting in a 10(7)-fold increase in cell number, without losing their proliferative and multilineage potential. Thus, this study describes the first demonstration, through clonal and population analyses in vitro, of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS.
Neurotransplantation of human fetal dopamine (DA) neurons is currently being investigated as a therapeutic modality for Parkinson's disease (PD). However, the practical limitations of human fetal transplantation indicate a need for alternative methodologies. Using the 6-hydroxydopamine rat model of PD, we transplanted dopaminergic neurons derived from Embryonic Day 27 porcine fetuses into the denervated striatum of cyclosporine-A (CyA)-treated or non-CyA-treated rats. Functional recovery was assessed by amphetamine-induced rotation, and graft survival and morphology were analyzed using neuronal and glial immunostaining as well as in situ hybridization with a porcine repeat element DNA probe. A significant, sustained reduction in amphetamine-induced rotational asymmetry was present in the CyA-treated rats whereas the non-CyA-treated rats showed a transient behavioral recovery. The degree of rotational recovery was highly correlated to the number of surviving transplanted porcine dopaminergic neurons. TH+ neuronal survival and graft volume were significantly greater in the CyA-treated group as compared to the non-CyA group. By donor-specific neuronal and glial immunostaining as well as donor-specific DNA labeling, we demonstrate that porcine fetal neuroblasts are able to survive in the adult brain of immunosuppressed rats, mediate functional recovery, and extensively reinnervate the host striatum. These findings suggest that porcine DA neurons may be a suitable alternative to the use of human fetal tissue in neurotransplantation for PD.
The intracerebral transplantation of freshly dissected fetal tissue containing cholinergic neurons of the developing basal forebrain has been reported to reverse lesion-induced or age-related cognitive deficits in animal models of cholinergic neuronal degeneration. Grafts of cultured fetal neurons, however, have generally shown poor cellular survival and limited therapeutic benefit. We tested the hypothesis that recent advances in the identification of growth factors that promote the survival and propagation of fetal precursor cells in vitro would improve the long-term survival of cultured neurons following intracerebral implantation. Dissociated cells from gestational Day 14 rodent basal forebrain were grown in chemically defined media supplemented with 20 ng/ml basic fibroblast growth factor. Two weeks postplating, numerous cells were present in the cultures and showed immunoreactive labeling for a variety of markers, including glutamic acid decarboxylase, neuron-specific enolase, neurofilament proteins, glial fibrillary acidic protein and, occasionally, choline acetyltransferase. To determine if cultured basal forebrain cells would survive intracerebral implantation, the cells were implanted homotypically into the nucleus basalis magnocellularis. To enhance the potential for graft survival in vivo, cells were also implanted into the nucleus basalis magnocellularis following an ibotenic acid lesion and into the denervated frontal cortex. Animals sacrificed between 2 weeks and 7 months following transplantation showed good and comparable graft survival in all sites. Immunocytochemical analysis revealed that representative populations of cells observed in vitro survived for prolonged periods in vivo, even in sites distal from their normal cellular targets. Thus, neuronal populations expanded in vitro can successfully survive and maintain cellular phenotypes post-transplantation. These results suggest a potential for isolating and growing specific neuronal populations in vitro for intracerebral transplantation.
Precise determination of donor age in human embryonic and fetal tissue is crucial for cell transplantation due to the existence of distinct time windows within which successful grafting is possible. This study demonstrates that between 4-12 wk postconception embryonic and fetal age can be estimated based on various morphometric parameters measured on a routine basis in suction abortion material. The greatest length, the neck-rump length, the foot length, and the proximal and distal arm and leg length were correlated with the anamnestic and ultrasonographically estimated age. Multivariate regression analyses showed a linear correlation between age and the logarithmic value of the various morphometric parameters. The best correlation was found for a mathematical model combining the limb parameters (r = 0.904; p < 0.001; n = 37). A prospective follow-up study (n = 40) was carried out to test the validity of the mathematical model. A high correlation was found between the calculated age and the estimated age based on anamnestic data (r = 0.749, p < 0.001). Outliers due to errors in the anamnestic data were readily identified by comparing anamnestic with calculated age. This method allows determination of embryonic and fetal age within and beyond the age group of the Carnegie classification and may, therefore, be useful for the needs of experimental and clinical cell transplantation.
Neurogenesis continues throughout adulthood in discrete regions. Proliferative zones include the subependymal zone, from where progenitors migrate along the rostral migratory pathway to differentiate into neurons in the olfactory bulb, and the hippocampal subgranular zone, where they migrate and differentiate into granule neurons. Progenitors isolated from adult subependymal zone exhibit in vitro neurogenesis when stimulated with epidermal or fibroblast growth factor. Cultured adult rat hippocampal progenitors (AHPs) grafted to adult rat hippocampus show site-specific neuronal differentiation. Here we investigate determinants of multipotentiality in the adult central nervous system, by grafting AHPs into homotypic (hippocampus) or heterotypic (the rostral migratory pathway) neurogenic sites or a heterotypic, non-neurogenic site (the cerebellum). We found that grafts into neurogenic, but not nonneurogenic sites, showed neuronal differentiation. Furthermore, AHPs grafted in the rostral migratory pathway migrated into the olfactory bulb, differentiating into tyrosine-hydroxylase-positive neurons, a non-hippocampus phenotype. These results reveal that AHP populations can respond to persistent neuronal differentiation cues in the adult central nervous system.