Fetal Mesencephalic Cells Research Articles

Panning for gold: Purifying mesencephalic dopaminergic progenitors differentiated from human pluripotent stem cells.

In a recently published study, Xu et al. used two surface markers, CLSTN2 and PTPRO, to generate highly purified donor dopaminergic neurons and achieved stable and predictable therapeutic outcomes by transplantation into the brain of PD animal models (Xu et al., 2022).

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

Clinical Trial for Parkinson's Disease Gets a Green Light in the US.

Cell-based therapy is expected as an alternative treatment for Parkinson's disease. In this issue of Cell Stem Cell, two accompanying papers (Kim etal., 2021; Piao etal., 2021) report the induction of clinically applicable dopaminergic neurons from human embryonic stem cells and the results of pre-clinical study toward a clinical trial.

Metabolic studies of synaptamide in an immortalized dopaminergic cell line

IntroductionSynaptamide, the N-acylethanolamine of docosahexaenoic acid (DHA), is structurally similar to the endocannabinoid N-arachidonoylethanolamine, anandamide. It is an endogenous ligand at the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), and induces neuritogenesis and synaptogenesis in hippocampal and cortical neurons, as well as neuronal differentiation in neural stem cells. PurposeOur goal was to characterize the metabolic fate (synthesis and metabolism) of synaptamide in a dopaminergic cell line using immortalized fetal mesencephalic cells (N27 cells). Both undifferentiated and differentiating N27 cells were used in this study in an effort to understand synaptamide synthesis and metabolism in developing and adult cells. MethodsRadiotracer uptake and hydrolysis assays were conducted in N27 cells incubated with [1-14C]DHA or with one of two radioisotopomers of synaptamide: [α,β-14C2]synaptamide and [1-14C-DHA]synaptamide. ResultsNeither differentiated nor undifferentiated N27 cells synthesized synaptamide from radioactive DHA, but both rapidly incorporated radioactivity from exogenous synaptamide into membrane phospholipids, regardless of which isotopomer was used. Pharmacological inhibition of fatty acid amide hydrolase (FAAH) reduced formation of labeled phospholipids in undifferentiated but not differentiated cells. ConclusionsIn undifferentiated cells, synaptamide uptake and metabolism is driven by its enzymatic hydrolysis (fatty acid amide hydrolase; FAAH), but in differentiating cells, the process seems to be FAAH independent. We conclude that differentiated and undifferentiated N27 cells utilize synaptamide via different mechanisms. This observation could be extrapolated to how different mechanisms may be in place for synaptamide uptake and metabolism in developing and adult dopaminergic cells.

Repairing the Brain: Cell Replacement Using Stem Cell-Based Technologies.

Current approaches to cell replacement therapy in Parkinson’s disease are strongly focused on the dopamine system, with the view that restoring dopaminergic inputs in a localized and physiologic manner will provide superior benefits in terms of effect and longevity compared with oral medication. Experience using transplants of fetal tissue containing dopaminergic cell precursors has provided valuable proof that the approach is feasible, and that engrafted cells can survive and function over many years. However, multiple drawbacks and procedural complications are recognized in using fetal cells. Recent strides in stem cell technology now make it possible to overcome some of the barriers associated with fetal tissue. In particular the generation of high numbers of specific cell types, such as dopaminergic neurons, from stem cells means that quality, consistency, activity, and safety can be more thoroughly determined prior to transplantation, thus providing hope for more robust outcomes. These cells are also predicted to provide benefit without leading to the graft-induced dyskinesia that led to morbidity in a subset of individuals who underwent fetal mesencephalic cell and tissue grafting in the 1990s. In thinking about developing such novel therapeutics, the choice of starting material has also expanded, with the availability of multiple human embryonic stem cell lines, as well as the possibilities for producing induced pluripotent cells, or neuronal cells from a patient’s own tissue. In this article, we speculate on how rapidly expanding knowledge and technical possibilities may impact on stem cell-based therapies for cell replacement in Parkinson’s disease over the next two decades.

Paraquat-induced metabolic stress signature in human foetal mesencephalic cells

Paraquat-induced metabolic stress signature in human foetal mesencephalic cells

Valproic acid in normal therapeutic concentration has no neuroprotective or differentiation influencing effects on long term expanded murine neural stem cells

The antiepileptic drug valproic acid (VPA) has shown neuroprotective effects in different cell types including mesencephalic neural primary cultures. Furthermore, an influence on neural differentiation and neurite outgrowth has been described. Nevertheless, results in this regard are contradictory and data on long term expanded neural stem cells are missing. This is why we investigated possible neuroprotective effects of VPA on fetal mesencephalic neural stem cells (fmNSCs) in vitro, using the neurotoxic agent 1-methyl-4-phenyl-pyridin (MPP+). We also examined potential VPA effects on cell expansion and differentiation and the underlying signaling pathways. In our study, we could exclude any relevant toxic effects of 100 μg/ml and 200 μg/ml VPA on fmNSCs during expansion and differentiation for up to 96 h. MPP+ treatment in concentrations of 30 and 60 μM MPP+ significantly decreased the survival rate of fmNSCs during expansion and differentiation. In all used concentrations, VPA did neither reverse these MPP+ effects when applied simultaneously with MPP+ nor after pre-treatment with VPA for 24 h. In contrast, MPP+ effects were emphasized by VPA pretreatment for 24h when applied during cell expansion. Concerning the self-renewing capacity of fmNSCs, measured by BrdU and Ki67 staining, we did not find any significant influence of VPA. Additionally there was no significant influence of therapeutic VPA dosages on astroglial (GFAP), oligodendroglial (GalC) and neuronal (MAP2) differentiation, measured by immunostaining after 10 days of differentiation. Summing up, we did not find any neuroprotective effects of VPA on fmNSCs in vitro, neither during expansion nor during cell differentiation. Also the self-renewing and differentiation potential of the used fmNSCs was not altered. These findings have implications for the large community of patients having to take VPA on a chronic base, especially in the light of knowledge that a regular cell replacement out of hippocampal adult stem cells is mandatory for the maintenance of normal cognition through adulthood.

Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons.

Small-conductance Ca2+-activated K+ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson's disease, where dopaminergic cell loss is associated with progression of the disease.

Treating Parkinson’s disease: preserve the spines! (Commentary on Soderstrom et al.)

Loss of dopaminergic neurons in Parkinson’s disease (PD) and PD animal models has been extensively documented to cause global changes in electrophysiological activity throughout the cortico-basal ganglia network. However, such loss is also associated with a range of morphological alterations of neurons forming this network, most notably the medium spiny neurons (MSNs) that are the main output neurons of the striatum. MSNs are the primary synaptic target of both nigrostriatal dopaminergic and cortico-striatal glutamatergic afferents. Both afferents converge onto dendritic spines, the critical site for synaptic integration in MSNs. In advanced PD there is a marked atrophy of dendrites and spines in these neurons, indicative of dysfunctional signal integration in the striatofugal pathway. Similar pathology, triggered by a dysregulation of intraspine Cav1.3 L-type Ca channels (Day et al., 2006), has been observed in rodent and primate models of PD (Day et al., 2006; Neely et al., 2007; Scholz et al., 2008). The significance of such dendritic atrophy and spine pruning for the symptoms and the treatment of PD has remained poorly understood. However, there is increasing awareness that these morphological alterations represent a major obstacle for therapeutic approaches to enhance striatal function (Schuster et al., 2009). Most notably, the efficacy of dopamine cell replacement strategies, designed to restore nigrostriatal connectivity, may be hampered by striatal dendritic and spine atrophy. In order for grafted dopamine neurons to re-establish functional connections, the morphological target of such reinnervation would need to be preserved or reestablished. In this issue of EJN, Soderstrom et al. (2010) report the results of a study on the impact of dendritic spine preservation in MSNs upon both antiparkinsonian and prodyskinetic effect of fetal mesencephalic cell grafts. The authors elegantly and convincingly show that administration of the L-type Ca channel blocker nimodipine prevented loss of spines in MSNs in unilaterally lesioned rats that were grafted with embryonic ventral midbrain cells. Nimodipine treatment also resulted in improved therapeutic benefit and reduced graft-induced behavioral abnormalities of these hemi-parkinsonian rats. Specifically, the results indicate that graft-mediated anti-parkinsonian efficacy was not potentiated by the prevention of spine loss; however, the impact of the graftand levodopa-induced side-effects was greatly diminished by nimodipine treatment. Interestingly, these effects were not due to increased survival of grafted cells but correlated with a greater reinnervation of the affected striatum. These results underscore the importance of prevention (or reversal) of spine loss in striatofugal neurons for effective therapy based on dopamine cell replacement. They extend a previous report of reduced levodopa-induced dyskinesia by prior treatment with L-type Ca channel antagonists (Schuster et al., 2009). The results described in Soderstrom et al. (2010) suggest that unless MSN spine loss and dendritic atrophy are reversed by appropriate pharmacological treatment, therapeutic interventions may be of limited efficacy or even cause unwarranted outcome. The findings and conclusions from the study by Soderstrom et al. (2010) need to be taken into consideration when designing dopamine cell replacement therapies in PD, using embryonic or stem cell-derived neurons in combination with appropriate drug cocktails to block MSN atrophy.

Frontiers in the surgical treatment of Parkinson’s disease

Despite the continued refinement of medical and surgical therapies, the treatment of Parkinson’s disease (PD) remains challenging. Current treatment strategies are largely focused on managing the motor symptoms of the disease, either by dopamine-based medications or, in advanced stages, by the application of deep brain stimulation to more stably alter the function of the basal ganglia. Important advances have been made in the last decade, but unfortunately a number of the motor symptoms of late-stage PD remain poorly treated, and while currently available therapies address the symptoms of the disease, they fail to alter the course of the disease itself. This has spurred basic and clinical exploration on a number of fronts. Several centers have examined novel stimulation targets to treat refractory symptoms of gait difficulty and axial imbalance. Basic and clinical researchers are examining whether the use of deep brain stimulation might slow the progress of the disease and thus be a useful neuroprotective therapy if initiated earlier in the progression of the disease. An expanded understanding of the genetic and cellular events that underlie PD has led some researchers to explore the use of neurotrophic factors or genetic restoration to preserve threatened neuronal populations. Finally, there has been much research on the use of fetal mesencephalic or stem cell populations to restore dopaminergic function. In this report, we will examine each of these potential new surgical therapies and the promise they may hold for the future treatment of PD.

Dexamethasone blocks astroglial differentiation from neural precursor cells

In previous studies, we demonstrated functional neuronal and dopaminergic differentiation of fetal mesencephalic neural precursor cells. The major factors for orienting their progeny towards a dopaminergic phenotype are forskolin and interleukin-1beta. Here, we investigated the effects of dexamethasone (10 microM) on neuronal and glial differentiation. Exposure of mesencephalic neural precursor cells to dexamethasone significantly reduces the amount of glial fibrillary acidic protein astroglia, but not of galactocerebrosidase C oligodendroglia, MAP2ab neurons and tyrosine hydroxylase dopaminergic cells. Presuming a possible involvement of the nuclear factor-kappaB pathway, we examined the effects of wortmannin (phosphatidylinositol 3'-kinase inhibitor) and SN50 (nuclear factor-kappaB inhibitor) on gliogenesis. Both wortmannin and SN50 mimicked the effects of dexamethasone suggesting that dexamethasone specifically blocks astroglial differentiation from mesencephalic neural precursor cells most likely via inhibition of the nuclear factor-kappaB pathway.

Dendritic cell recruitment following xenografting of pig fetal mesencephalic cells into the rat brain

Following transplantation into the rat brain, porcine neuroblasts differentiate and integrate host tissue, but due to their xenogeneic nature, these cells are generally rejected within several weeks. This rejection is accompanied by infiltration of the graft by macrophages and αβT lymphocytes, but so far nothing is known about the potential role of dendritic cells (DCs) in this process. DCs are professional antigen presenting cells that have the unique ability to prime naive T cells, thereby initiating an antigen-directed immune response. Here, we provide evidence for DC recruitment following the transplantation of pig mesencephalic neural cells into the striatum of LEW.1A rats, as indicated by the high number of OX62+ cells in the rejecting graft and the absence of V65 staining. DCs were found as early as 3 and 8 days postimplantation together with ED1+ and OX42+ cells. This early recruitment, which is probably due to the surgical procedure, might be a critical step in the rejection process, enabling DCs to be loaded with xenoantigens. The number of intracerebral DCs subsequently decreased, being barely detectable in older non-infiltrated xenografts. However, DCs re-appeared as they were observed in grafts infiltrated by macrophages and T cells, a phenomenon that usually precedes graft rejection. Interestingly, we observed a tight correlation between the number of DCs and that of R7.3+ T cells infiltrating the graft. In addition, DCs were often found in close proximity to αβT cells and most expressed MHCII. Taken together, these findings give credence to a role for infiltrating DCs in the mediation of T cell responses to intracerebral xenografting.

Quebrachitol (2- O-methyl- l-inositol) attenuates 6-hydroxydopamine-induced cytotoxicity in rat fetal mesencephalic cell cultures

Naturally occurring plant substances have the potential to prevent oxidative damage in various pathophysiological conditions including neurodegenerative disorders. Recent findings indicate that impaired energy metabolism plays a prominent role in neurodegeneration. The present study investigated whether quebrachitol (2- O-methyl- l-inositol) (QCT), a sugar like natural compound that was suggested to have both antioxidant and membrane stabilization activity prevents the cytotoxic effect of 6-hydroxydopamine (6-OHDA, 200 μM) on cultured rat fetal mesencephalic cells. While QCT (0.1–100 μg/ml) produced no effect per se on cell viability as measured in the 3[4,5-dimethylthiazole-2il]-2,5-diphenyltetrazolium bromide (MTT) test, it offered concentration-related protection against cell death induced by 6-OHDA. In addition, QCT demonstrated an antioxidant activity against 6-OHDA-induced oxidative stress as evidenced by reduced formation of nitrite–nitrate and thiobarbituric acid-related substances. Fluorescence microscopy using acridine orange/ethidium bromide double staining further affirmed the absence of 6-OHDA (200 μM)-induced morphological changes characteristic of apoptosis/necrosis in cultures pretreated with QCT (100 μg/ml). Also, results of tyrosine hydroxylase immunoreactivity indicated that 6-OHDA induces cell death in mesencephalic cultures affecting both TH + positive and TH − negative (TH + and TH −, respectively) and QCT pretreatment protects them from cell death, in a non-specific manner. Our data indicate that QCT has a cytoprotective role due, at least in part, to an antioxidant and free radical scavenging mechanism. Furthermore, the study suggests that inositol compounds might serve as leads in developing drugs for the treatment of various neurodegenerative disorders.

Fetal mesencephalic cells exposed to MDMA (ecstasy) enhance dopamine cell survival and neurite outgrowth in vitro

Fetal mesencephalic cells exposed to MDMA (ecstasy) enhance dopamine cell survival and neurite outgrowth in vitro

Fetal mesencephalic cells exposed to MDMA (Ecstasy) enhance dopamine cell survival and neurite outgrowth in vitro

Fetal mesencephalic cells exposed to MDMA (Ecstasy) enhance dopamine cell survival and neurite outgrowth in vitro

In Vivo PET Measurements with [11C]PE2I to Evaluate Fetal Mesencephalic Transplantations to Unilateral 6-OHDA-Lesioned Rats

Positron emission tomography (PET) is a useful tool to assess and visualize neurotransmissions in vivo. In this study, we performed repeated PET scans with [11C]PE2I, a tracer of the dopamine transporter, to evaluate the alteration of the expression of dopamine (DA) transmission component after a fetal mesencephalic transplantation. The fetal mesencephalic cells were transplanted into the striatum of unilateral 6-OHDA-lesioned rats. PET scans with [11C]PE2I were performed to evaluate the DA transporter before and 2 and 4 weeks after the transplantation. Rotation behavior tests, in vitro autoradiography, measurements of DA contents in the striatum by high-performance liquid chromatography (HPLC), and tyrosine hydroxylase (TH) immunohistological examinations were performed at the same time points and examined for their relationship to changes in the dopamine transporter. The number of ipsilateral rotations induced by methamphetamine injections decreased. DA contents in the striatum measured with HPLC significantly increased. In the PET study, the binding potential of [11C]PE2I increased at 4 weeks. The results of the in vitro autoradiography study corresponded with those of the PET study. The degrees of the change in the binding potentials correlated with those of the numbers of rotations in the behavioral study and the DA contents in the striatum. In the histological examination, TH-positive cells with axons were observed at 2 and 4 weeks after the transplantation. As the dopamine transporter exists only in the axon terminal of DA neurons, these results suggested that PET measurements of [11C]PE2I binding indicated not only survival, but maturity and functioning of the transplanted cells. Repeated PET measurements of DA transporters are a useful tool in assessing the effectiveness of neural transplantations.

Restaurative therapy for Parkinson’s disease

In Parkinson’s disease (PD) the transplantation of fetal mesencephalic cells into the striatum has been intensively studied in animals. Clinical studies with human fetal mesencephalis cells have been performed in about 300 patients. Limited access, shifted the focus towards embryonic stem (ES) cells. Recent clinical studies showed an improvement of motor function over time, but revealed new side effects as non-pharmacologically induced dyskinesias (Freed et al 2001, Olanow et al 2002). Experimental studies to test the site of transplantation, number of grafted cells and the optimal circumstances of cell survival are needed in the light of the current clinical results. The group of Ron McKay at the NIH published positive behavioural data of unilaterally lesioned rats, that have received a graft of dopaminergic neurons derived from murine embryonic stem cells. Long-term results are still lacking. Further approaches of restaurative therapy are ongoing for treatment of multiple system atrophy (MSA) and Huntington’s chorea (HD).

Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson’s disease

Neural progenitor cells have shown the effectiveness in the treatment of Parkinson’s disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson’s disease. We found that a combination of 50ng/ml fibroblast growth factor 8, 10ng/ml glial cell line-derived neurotrophic factor and 10μM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site.These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson’s disease.

Behavioral changes in unilaterally 6-hydroxy-dopamine lesioned rats after transplantation of differentiated mouse embryonic stem cells without morphological integration.

Transplantation of fetal mesencephalic cells into the striatum has been performed in about 350 patients with Parkinson's disease and has been intensively studied in rat models of Parkinson's disease. Limited access to this material has shifted the focus toward embryonic stem (ES) cells. The grafting of undifferentiated ES cells to 6-hydroxy-dopamine (6-OHDA)-lesioned rats leads to behavioral improvements but may induce teratoma-like structures. This risk might be avoided by using more differentiated ES cells. In this study, we aimed to investigate differentiated mouse ES cells regarding their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced after transplantation. Mouse ES cells were differentiated on PA6 feeder cells for 14 days before grafting. Twenty to twenty-five percent of the neurons obtained were positive for tyrosine-hydroxylase (TH). PKH26-labeled cells were transplanted in the striata of unilaterally 6-OHDA-lesioned rats. Direct PKH26 fluorescence visualization and TH staining proved the existence of cell deposits in the striata of all grafted animals, indicating cell survival for at least 5 weeks posttransplantation. There was no evidence of tumor formation. Immunocytochemical staining showed glial immunoreactivity surrounding the grafted cell deposits, probably inhibiting axonal outgrowth into the surrounding host tissue. There was a significant reduction in amphetamine-induced rotational behavior seen in grafted animals, which was not observed in sham-operated animals. The findings of this study suggest that the amphetamine-induced rotational behavioral test without histological confirmation is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation.

Forskolin cooperating with growth factor on generation of dopaminergic neurons from human fetal mesencephalic neural progenitor cells

Forskolin was tested for its co-activating ability to enhance the function of fibroblast growth factor (FGF) 8 on dopaminergic (DAergic) differentiation from human fetal mesencephalic neural progenitor cells (NPCs). When NPCs were treated with FGF8 alone, the DAergic phenotype was expressed lightly. The addition of 10 μM forskolin increased the number of DAergic neurons, cooperating with 50 ng/ml FGF8. These cells produced neurotransmitter DA, which was measured by high-performance liquid chromatography. Reverse transcriptase-polymerase chain reaction analysis demonstrated that differentiated cells expressed DAergic development-relative genes tyrosine hydroxylase (TH), nuclear receptor-related factor 1 (Nurr1) and D2 receptor (D2R), indicating that matured DAergic neurons could be obtained under these present conditions. The results suggest that forskolin plus FGF8 may contribute to more efficient production of DAergic neurons from human-derived NPCs for therapy of neurodegenerative diseases.