Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process

Background aimsWe have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models.MethodsIn this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model.ResultsWe showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)⁺ dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH⁺ fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH⁺ neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products.ConclusionsDopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model.     

Enhanced differentiation of human dopaminergic neuronal cell model for preclinical translational research in Parkinson's disease

Human-derived neuronal cell lines are progressively being utilized in understanding neurobiology and preclinical translational research as they are biologically more relevant than rodent-derived cells lines. The Lund human mesencephalic (LUHMES) cell line comprises human neuronal cells that can be differentiated to post-mitotic neurons and is increasingly being used as an in vitro model for various neurodegenerative diseases. A previously published 2-step differentiation procedure leads to the generation of post-mitotic neurons within 5-days, but only a small proportion (10%) of the total cell population tests positive for tyrosine hydroxylase (TH). Here we report on a novel differentiation protocol that we optimized by using a cocktail of neurotrophic factors, pleiotropic cytokines, and antioxidants to effectively generate proportionately more dopaminergic neurons within the same time period. Visualization and quantification of TH-positive cells revealed that under our new protocol, 25% of the total cell population expressed markers of dopaminergic neurons with the TH-positive neuron count peaking on day 5. These neurons showed spontaneous electrical activity and responded to known Parkinsonian toxins as expected by showing decreased cell viability and dopamine uptake and a concomitant increase in apoptotic cell death. Together, our results outline an improved method for generating a higher proportion of dopaminergic neurons, thus making these cells an ideal neuronal culture model of Parkinson's disease (PD) for translational research.     

Immunomodulation by placenta-derived decidua stromal cells. Role of histocompatibility,accessory cells and freeze–thawing

Background aimsHuman placenta-derived decidua stromal cells (DSCs) are newly introduced stromal cells that have successfully been used in several clinical trials for the treatment of acute inflammatory diseases. Despite published data about DSCs, deeper exploration of mechanisms of action and crosstalk with other immune cells need to be explored.MethodsIn mixed lymphocyte culture (MLC), the splenocytes from Balb/c or B6 mice were stimulated using mitogen (concanavalin A), allogeneic (B6 or Balb/c splenocytes) or xenogeneic activation with human peripheral blood mononuclear cells.ResultsWhen 10% of the mouse bone marrow-derived–MSC, being autologous, allogeneic or haploidentical (from F1), was added, >95% inhibition was seen. Using human (h)-DSCs, the inhibitory capacity was a median 68% as a xenogeneic immunomodulatory cell when used in mitogen and allogeneic setting in mice MLC. However, when human peripheral blood mononuclear cells were used as stimulator for mouse splenocyte (xenogeneic MLC), hDSC showed a median inhibition of 88%. We explored the presence and function of monocytes in the immunomodulatory function of stromal cells. CD14+ monocyte cells reduced the immunosuppressive effect by hDSC. hDSCs did not show any inhibitory effect on natural killer cell activation and proliferation by interleukin-2. In contrast DSCs increased natural killer proliferation by a median of 58%. Fresh or frozen–thawed hDSCs had similar inhibitory effects on human T-cell proliferation (both allo-stimulation and mitogen stimulation) in vitro. Cell viability at room temperature during 24 h was similar using fresh or freeze–thawed DSCs.ConclusionsTo conclude, histocompatibility and CD14+ monocyte cells had an impact on hDSC immunomodulation but frozen–thawed or freshly prepared cells did not.     

Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells

Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multi-passaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Over-expression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers.     

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.     

Xeno-Free Defined Conditions for Culture of Human Embryonic Stem Cells,Neural Stem Cells and Dopaminergic Neurons Derived from Them

Background

Human embryonic stem cells (hESCs) may provide an invaluable resource for regenerative medicine. To move hESCs towards the clinic it is important that cells with therapeutic potential be reproducibly generated under completely defined conditions.

Methodology/Principal Findings

Here we report a four-step scalable process that is readily transferable to a Good Manufacture Practice (GMP) facility for the production of functional dopaminergic neurons from hESCs for potential clinical uses. We show that each of the steps (propagation of ESC→generation of neural stem cells (NSC)→induction of dopaminergic precursors→maturation of dopaminergic neurons) could utilize xeno-free defined media and substrate, and that cells could be stored at intermediate stages in the process without losing their functional ability. Neurons generated by this process expressed midbrain and A9 dopaminergic markers and could be transplanted at an appropriate time point in development to survive after transplant.

Conclusions/Significance

hESCs and NSCs can be maintained in xeno-free defined media for a prolonged period of time while retaining their ability to differentiate into authentic dopaminergic neurons. Our defined medium system provides a path to a scalable GMP-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential screening applications.     

Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH⁺ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.     

Impaired neurite development associated with mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of children with autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restrictive interests, and repetitive stereotypic behaviors. Among the various mechanisms underlying the pathogenesis of ASD, dysfunctions of dopaminergic signaling and mitochondria have been hypothesized to explain the core symptoms of children with ASD. However, only a few studies focusing on the pathological association between dopaminergic neurons (DN) and mitochondria in ASD have been performed using patient-derived stem cells and in vitro differentiated neurons. Stem cells from human exfoliated deciduous teeth (SHED) are neural crest-derived mesenchymal stem cells present in the dental pulp of exfoliated deciduous teeth; these cells can differentiate into dopaminergic neurons (DN) in vitro. This study aimed to investigate the pathological association between development of DN and mitochondria in ASD by using SHED as a disease- or patient-specific cellular model. The SHED obtained from three children with ASD and three typically developing children were differentiated into DN, and the neurobiology of these cells was examined. The DN derived from children with ASD showed impaired neurite outgrowth and branching, associated with decreased mitochondrial membrane potential, ATP production, number of mitochondria within the neurites, amount of mitochondria per cell area and intracellular calcium level. In addition, impaired neurite outgrowth and branching of ASD-derived DN were not improved by brain-derived neurotrophic factor (BDNF), suggesting impairment of the BDNF signaling pathway in ASD. These results imply that intracerebral dopamine production may have decreased in these children. The earliest age at which deciduous teeth spontaneously exfoliate in humans, and SHED can be noninvasively collected, is approximately 6 years. Our results suggest that in vitro analysis of SHED-derived DN obtained from children with ASD provides neurobiological information that may be useful in determining treatment strategies in the early stages of ASD.     

H2O2- or l-DOPA-injured dopaminergic neurons trigger the release of soluble mediators that up-regulate striatal GDNF through different signalling pathways

Glial cell line-derived neurotrophic factor (GDNF) is a potent neuroprotective molecule for dopaminergic neurons of the nigrostriatal pathway that degenerate in Parkinson's disease. We have previously shown that H₂O₂- or l-3,4-dihydroxyphenylalanine (l-DOPA)-challenged dopaminergic neurons trigger the release of soluble factors that signal ventral midbrain astrocytes to increase GDNF expression. In the present work, we evaluated whether the factors released by ventral midbrain-challenged cells were able to alter GDNF expression in striatal cells, the targets of dopaminergic neurons projecting from the substantia nigra, and investigated the signalling pathways involved. Our data showed that soluble mediators released upon H₂O₂- or l-DOPA-induced dopaminergic injury up-regulated GDNF in striatal cells, with different temporal patterns depending on the oxidative agent used. Conditioned media from H₂O₂- or l-DOPA-challenged midbrain astrocyte cultures failed to up-regulate GDNF in striatal cultures. Likewise, there was no direct effect of H₂O₂ or l-DOPA on striatal GDNF levels suggesting that GDNF up-regulation was mediated by soluble factors released in the presence of failing dopaminergic neurons. Both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways were involved in striatal GDNF up-regulation triggered by H₂O₂-induced dopaminergic injury, while diffusible factors released in the presence of l-DOPA-challenged dopaminergic neurons induced GDNF expression in striatal cells through the activation of the MAPK pathway. These soluble mediators may constitute, in the future, important targets for the control of endogenous GDNF expression enabling the development of new and, hopefully, more efficient neuroprotective/neurorestorative strategies for the treatment of Parkinson's disease.     

Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required.     

Injection of frozen-thawed porcine first polar bodies into enucleated oocytes results in fertilization and embryonic development

The objective was to determine whether enucleated oocytes injected with frozen porcine first polar bodies (pPB1s) could be fertilized and developed into viable embryos in vitro. Metaphase II (MII) oocytes with pPB1s were frozen (vitrified) and stored for 2 mo. The pPB1s were isolated from thawed MII oocytes and injected into enucleated recipient oocytes by micromanipulation. All recipients injected with thawed pPB1s were fertilized by intracytoplasmic sperm injection (ICSI), and the resulting recombinant zygotes were incubated to assess their developmental competence in vitro. Furthermore, double-antibody immunohistochemistry was used to verify that the nucleus of the pPB1 participated in fertilization and supported embryonic development. Porcine embryos (2- to 8-cell stage) were obtained from the recombinants. The average in vitro cleavage rate of 2-, 4-, and 8-cell stage recombinant embryos was 25.3, 17.7, and 9.3% (P < 0.05), respectively. Chromosomes in the labeled pPB1 participated in the formation of the two blastomere nuclei of 2-cell stage embryos derived from recombinant oocytes. In conclusion, nuclear materials of frozen-thawed pPB1 supported oocyte fertilization and subsequent embryonic development, thereby providing a new way to use frozen PB1s for preservation and reproduction of mammals.     

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ_m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H₂O₂-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ_m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ_m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.     

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease

The adoptive transfer of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.     

In situ analysis of acupuncture protecting dopaminergic neurons from lipid peroxidative damage in mice of Parkinson's disease

ObjectivesAcupuncture stimulation has proven to protect dopaminergic neurons from oxidative damage in animal models of Parkinson''s disease (PD), but it remains unclear about the in situ information of biochemical components in dopaminergic neurons. Here, we aimed to analyse in situ changes of biochemical components and lipid peroxidation levels in dopaminergic neurons in PD mice treated with acupuncture by synchrotron FTIR micro‐spectroscopy technique.Materials and MethodsAbout 9–10‐week‐old C57BL/6 mice were used to establish PD model by intraperitoneal injection of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP, 30 mg/kg for 5 days). Acupuncture stimulation was performed once a day for 12 days. Behaviour test was determined using the rotarod instrument. Biochemical compositions of dopaminergic neurons in substantia nigra pars compacta were analysed by synchrotron FTIR micro‐spectroscopy technique. The number and ultrastructure of dopaminergic neurons were respectively observed by immunofluorescence and transmission electron microscopy (TEM).ResultsWe found that the number and protein expression of dopaminergic neurons in MPTP‐treated mice were reduced by about half, while that in the mice treated by acupuncture were significantly restored. Acupuncture treatment also restored the motor ability of PD mice. The results of single cell imaging with synchrotron FTIR micro‐spectroscopy technique showed that the proportion of lipid in MPTP treated mice increased significantly. Especially the ratio of CH₂ asymmetric stretching and CH₃ asymmetric stretching increased significantly, suggesting that MPTP induced lipid peroxidation damage of dopaminergic neurons. It is also supported by the result of TEM, such as mitochondrial swelling or atrophy, loss of mitochondrial crests and mitochondrial vacuolization. Compared with MPTP treated mice, the proportion of lipid in acupuncture treated mice decreased and the mitochondrial structure was restored.ConclusionsAcupuncture can inhibit the level of lipid peroxides in dopaminergic neurons and protect neurons from oxidative damage. The study provides a promising method for in situ analysis of biochemical compositions in PD mice and reveals the mechanism of acupuncture in treating neurodegenerative diseases.

Synchrotron FTIR micro‐spectroscopy technique was used to analyse in situ changes of biochemical components and lipid peroxidation levels in dopaminergic neurons in Parkinson''s disease (PD) mice treated with acupuncture. Results showed that the lipid proportion increased significantly in MPTP induced PD mice, including the ratio of lipid to protein, the ratio of lipid to nucleic acid and the ratio of CH₂ asym str to CH₃ asym str. In particular, the increase of the ratio of CH₂ asym str to CH₃ asym str implies the higher level of lipid peroxidation. The acupuncture stimulation at Yanglingquan (GB34) acupoints lowered the lipid proportion in dopaminergic neurons of PD mice brain. Our study successfully assesses acupuncture inhibiting oxidative stress damage in dopaminergic cells by in situ analysis of FTIR micro‐spectroscopy.     

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron‐like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule‐associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β‐tubulin III). It also increased expression of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine‐beta‐hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.