Functional Dopaminergic Neurons Research Articles

Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/β-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of β-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/β-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

Potential Novel Treatments for Parkinson’s Disease

This review aims to evaluate novel developments in treatments for Parkinson’s disease that focus on slowing, stopping, and/or reversing the neurodegeneration associated with the disease. Parkinson’s disease (PD) is a neurodegenerative disease that affects dopaminergic neurons in the substantia nigra, located in the basal ganglia of the brain. This region also contains high levels of alpha synuclein, a protein found abundantly in the brain due to its important role in neurotransmitter release. α-synuclein has been found to be misfolded and overexpressed in people with PD, which leads to fibril formation and aggregation, which directly interfere with basic cellular processes and results in neurodegeneration. In PD patients, dopaminergic neurons are most affected; therefore, an artificial supply of dopamine must be provided to them via dopamine precursors that can cross the blood-brain barrier. However, these medications fail to slow, stop, or reverse the progression of PD and merely pose as treatments for physical symptoms, such as tremors. Researchers are now focusing on 2 major avenues for new PD treatments: treatments that target α-synuclein aggregates/Lewy body assemblies and treatments that focus on replacement of lost dopaminergic neurons in the brain. The use of antibodies (immunotherapy) to target and clear αsynuclein aggregates or the upregulation of genes that encode autophagic mechanisms to destroy dysfunctional proteins were shown to reduce α-synuclein aggregation and behavioral deficits in mice. Results from clinical trials have shown a 96.5% reduction in the concentration of extracellular α-synuclein aggregates, and doses were well tolerated with no serious side effects. In order to rebuild the neurons that were lost to PD neurodegeneration, a renewable source of dopamine-producing cells that can integrate into the host brain and survive for years is required. More specifically, the reprogramming of astrocytes can be used to develop functional dopaminergic neurons. To reprogram the astrocytes and fibroblasts, the RNA binding protein PTB was suppressed; PTB suppresses numerous neuronal genes required for neuronal maturation, thus, downregulation of PTB generates functional, mature neurons. When in vivo astrocyte reprogramming was performed in a mouse model of PD, researchers found new, successful dopaminergic neurons in the substantia nigra and increased dopamine levels. Although both avenues for treatment require further clinical trials and testing in human subjects, they both hold significant promise in the future.

Graphene nanofiber composites for enhanced neuronal differentiation of human mesenchymal stem cells.

Aim: To differentiate mesenchymal stem cells into functional dopaminergic neurons using an electrospun polycaprolactone (PCL) and graphene (G) nanocomposite. Methods: A one-step approach was used to electrospin the PCL nanocomposite, with varying G concentrations, followed by evaluating their biocompatibility and neuronal differentiation. Results: PCL with exiguous graphene demonstrated an ideal nanotopography with an unprecedented combination of guidance stimuli and substratecues, aiding the enhanced differentiation of mesenchymal stem cells into dopaminergic neurons. These newly differentiated neurons were seen to exhibit unique neuronal arborization, enhanced intracellular Ca2+ influxand dopamine secretion. Conclusion: Having cost-effective fabrication and room-temperature storage, the PCL-G nanocomposites could pave the way for enhanced neuronal differentiation, thereby opening a new horizon for an array of applications in neural regenerative medicine.

Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson’s disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.

Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics.

SummaryThe differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We found that the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction.

Treating Parkinson's disease by astrocyte reprogramming: Progress and challenges.

Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is typified by both motor and nonmotor symptoms. The current medications provide symptomatic relief but do not stimulate the production of new dopaminergic neurons in the substantia nigra. Astrocyte reprogramming has recently received much attention as an avenue for increasing functional dopaminergic neurons in the mouse PD brain. By targeting a microRNA (miRNA) loop, astrocytes in the mouse brain could be reprogrammed into functional dopaminergic neurons. Such in vivo astrocyte reprogramming in the mouse model of PD has successfully added new dopaminergic neurons to the substantia nigra and increased dopamine levels associated with axonal projections into the striatum. This review deliberates the astrocyte reprogramming methods using specific transcription factors and mRNAs and the progress in generating dopaminergic neurons in vivo. In addition, the translational potential, challenges, and potential risks of astrocyte reprogramming for an enduring alleviation of parkinsonian symptoms are conferred.

Generation of human midbrain organoids from induced pluripotent stem cells

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.

Temporal changes in key developmental transcription factors in dopamine neurons during MPP+ induced injury and recovery in zebrafish brain

Poor regeneration of functional dopaminergic neurons (DA) is one of the most common healthcare challenges for Parkinson’s Disease (PD) patients. In contrast to mammals, zebrafish has an amazing potential to repair their dopaminergic neurons (DA) after injury. However, the molecular mechanisms that regulate these reparative events remain to be determined. To address this, we compared the temporal changes in key transcription factors (TFs), which regulate the developmental trajectories of DA neurons during injury and regeneration in zebrafish. MPP+ was exposed to zebrafish embryos between 18 Hpf and 96 Hpf to create a model of DA injury and regeneration after injury (1day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days and 87 days post DA injury). During time series of MPP+ exposure, we found temporal alterations in the expression patterns of TFs; Nurr1, Foxa1, Lmx1a/b and En1/2 using WISH and RT-PCR. This turning point in expression dynamics coincided with a DA phenotypic turning point, as shown by 50% decline in TH+/DAT+ neurons and locomotor activity in the days following the MPP+ exposure. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional DA phenotype after 50% DA ablation. Following acute DA injury, mRNA levels of most TFs started to increase between 3-8 days after injury which was significantly elevated to normal levels in adult zebrafish brain i.e. at 87th day after injury. Remarkably, the changes in mRNA expression of TFs temporally correlate with corresponding increase in TH/DAT expression and functional recovery. Taken together, this study showed a highly relevant role of TFs for dopamine producing neurons during regeneration of DA neurons following ablation with restoration of normal behavior. This study implies that TFs as potential therapeutic targets for enhancing regeneration of DA neurons in mammalsobserved over.

N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms in neurons remains difficult due to the limited access to human tissue. Using human induced pluripotent stem cells (hiPSCs), we generated functional dopaminergic (DA) neurons showing positive expression of dopaminergic markers TH and DAT, mature neuronal marker MAP2 and functional synaptic markers synaptophysin and PSD-95. These DA neurons were electrophysiologically characterized, and exhibited inward Na + currents, overshooting action potentials and spontaneous postsynaptic currents (sPSCs). POLG patient-specific DA neurons (POLG-DA neurons) manifested a phenotype that replicated the molecular and biochemical changes found in patient post-mortem brain samples namely loss of complex I and depletion of mtDNA. Compared to disease-free hiPSC-derived DA neurons, POLG-DA neurons exhibited loss of mitochondrial membrane potential, loss of complex I and loss of mtDNA and TFAM expression. POLG driven mitochondrial dysfunction also led to neuronal ROS overproduction and increased cellular senescence. This deficit was selectively rescued by treatment with N-acetylcysteine amide (NACA). In conclusion, our study illustrates the promise of hiPSC technology for assessing pathogenetic mechanisms associated with POLG disease, and that NACA can be a promising potential therapy for mitochondrial diseases such as those caused by POLG mutation.

Evaluation of Priming Efficiency of Forskolin in Tissue-Specific Human Mesenchymal Stem Cells into Dopaminergic Neurons: An In Vitro Comparative Study

Background: Human mesenchymal stem cells (hMSC) can be derived from various tissue sources and differentiated into dopaminergic (DAergic) neurons using various types of inducers. There are several strategies that have been reported to generate functional dopaminergic neuronal cells from hMSCs in the most efficient manner possible. However, this area is still under extensive research. In this study, we aim to compare hMSCs derived from bone marrow (BM), adipose tissue (AD) and dental pulp (DP) to generate functional dopaminergic neurons, using FGF2 and forskolin. Post-differentiation, multiple factors were used to characterize the cells at morphological, morphometric, ultra-structural, mRNA and protein levels for various markers (Nestin, NF, MAP2, Tuj1, TH, DAT, PitX3, Ngn2, Kv4.2, SCN5A). Cells’ functionality was studied by calcium ion imaging, along with the amount of dopamine secreted by the cells in the culture medium. Results: Data analysis revealed that forskolin has comparable effect on BM- and AD-derived MSC (28.43% and 29.46% DAergic neurons, respectively), whereas DP-MSC (42.78 ± 1.248% DAergic neurons) show better outcome in terms of efficient generation of DAergic neuronal cells, expression of neuronal associated markers, dopamine release and calcium ion efflux. Ultra-structural studies by SEM and TEM also revealed a substantial change in both cellular morphology and composition of cellular organelles. It was observed that AD-MSCs showed the best neuronal features, at morphological, gene, and protein levels upon induction with the above-mentioned induction cocktail. Conclusion: It may be concluded that a combination of FGF2 and forskolin yields functionally active dopaminergic neuronal cells in vitro, with highest percentage of the same from AD-MSCs, as compared to that in BM-MSCs and DP-MSCs. The outcomes and comparative evaluation provide a substantial platform for further studies on molecular pathways involved in the process of DAergic neurogenesis in individual cases.

Dopamine transporter neuroimaging accurately assesses the maturation of dopamine neurons in a preclinical model of Parkinson\u2019s disease

BackgroundSignificant developments in stem cell therapy for Parkinson’s disease (PD) have already been achieved; however, methods for reliable assessment of dopamine neuron maturation in vivo are lacking. Establishing the efficacy of new cellular therapies using non-invasive methodologies will be critical for future regulatory approval and application. The current study examines the utility of neuroimaging to characterise the in vivo maturation, innervation and functional dopamine release of transplanted human embryonic stem cell-derived midbrain dopaminergic neurons (hESC-mDAs) in a preclinical model of PD.MethodsFemale NIH RNu rats received a unilateral stereotaxic injection of 6-OHDA into the left medial forebrain bundle to create the PD lesion. hESC-mDA cell and sham transplantations were carried out 1 month post-lesion, with treated animals receiving approximately 4 × 105 cells per transplantation. Behavioural analysis, [18F]FBCTT and [18F]fallypride microPET/CT, was conducted at 1, 3 and 6 months post-transplantation and compared with histological characterisation at 6 months.ResultsPET imaging revealed transplant survival and maturation into functional dopaminergic neurons. [18F]FBCTT-PET/CT dopamine transporter (DAT) imaging demonstrated pre-synaptic restoration and [18F]fallypride-PET/CT indicated functional dopamine release, whilst amphetamine-induced rotation showed significant behavioural recovery. Moreover, histology revealed that the grafted cells matured differently in vivo producing high- and low-tyrosine hydroxylase (TH) expressing cohorts, and only [18F]FBCTT uptake was well correlated with differentiation.ConclusionsThis study provides further evidence for the value of in vivo functional imaging for the assessment of cell therapies and highlights the utility of DAT imaging for the determination of early post-transplant cell maturation and differentiation of hESC-mDAs.

Mitochondrial Homeostasis and Signaling in Parkinson’s Disease

The loss of dopaminergic (DA) neurons in the substantia nigra leads to a progressive, long-term decline of movement and other non-motor deficits. The symptoms of Parkinson’s disease (PD) often appear later in the course of the disease, when most of the functional dopaminergic neurons have been lost. The late onset of the disease, the severity of the illness, and its impact on the global health system demand earlier diagnosis and better targeted therapy. PD etiology and pathogenesis are largely unknown. There are mutations in genes that have been linked to PD and, from these complex phenotypes, mitochondrial dysfunction emerged as central in the pathogenesis and evolution of PD. In fact, several PD-associated genes negatively impact on mitochondria physiology, supporting the notion that dysregulation of mitochondrial signaling and homeostasis is pathogenically relevant. Derangement of mitochondrial homeostatic controls can lead to oxidative stress and neuronal cell death. Restoring deranged signaling cascades to and from mitochondria in PD neurons may then represent a viable opportunity to reset energy metabolism and delay the death of dopaminergic neurons. Here, we will highlight the relevance of dysfunctional mitochondrial homeostasis and signaling in PD, the molecular mechanisms involved, and potential therapeutic approaches to restore mitochondrial activities in damaged neurons.

Exosomes from Human Periapical Cyst-MSCs: Theranostic Application in Parkinson's Disease.

The scientific community continuously strives to get new disease models, to discover early markers or novel therapeutic approaches, improving the diagnosis and prognosis of several human pathologies. Parkinson's Disease (PD) is characterized by a long asymptomatic phase, characterized by a selective loss of dopaminergic neurons. Recently, the human Periapical Cyst-Mesenchymal Stem Cells (hPCy-MSCs) have been differentiated in functional dopaminergic neurons: such oral-derived MSCs and the hPCy-MSCs-derived exosomes may represent a strategic and useful in vitro study-model, as well as intriguing therapeutic carriers. Circadian rhythm (CR) alteration variously impacts on PD pathways: an interesting research target is represented by the analysis of the exosomes released by dopaminergic neurons, derived from neural-differentiated hPCy-MSCs, after having reproduced in-vitro PD-like conditions. This review aims to describe the crosstalk among some aspects of circadian rhythm related to the onset of PD and the exosomes released by cells of PD patients. More in detail: the first part of this article will describe the main characteristics of circadian rhythm and the involvement of the exosomes found to be effective in the pathogenesis of PD. Finally, the authors will suggest how those exosomes derived from dopaminergic neurons, obtained by oral-derived stem cells (hPCy-MSCs) may represent a smart model for the in vitro research on PD, to find new biomarkers, to test new drugs or, fatally, to find new pathways applicable in future therapeutic approaches.

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

SummaryParkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Dopaminergic induction of human dental pulp stem cells by photobiomodulation: comparison of 660nm laser light and polychromatic light in the nir

Human dental pulp stem cells (hDPSCs) are able to differentiate into dopaminergic neurons and help the maintenance of partially degenerated neurons, which makes them as an alternative cell source for treatment of Parkinsons' disease (PD) patients. Here, the effect of photobiomodulation with polychromatic light source in the near infrared (NIR) range (600–1200 nm) or low level 660 nm diode laser light on hDPSCs during dopaminergic induction was investigated. Real time RT-qPCR analysis indicated that expressions of brain derived neurotrophic factor (BDNF), glial cell line derived neurotropic factor (GNDF), matrix associated protein 2 (MAP2), nuclear receptor related 1 protein (NURR1) and dopamine transporter (DAT) were increased, especially in the first 7 days of dopaminergic induction when 660 nm laser light was applied with a total energy density of 1.6 J/cm2. The activity of polychromatic light on hDPSCs depended on the differentiation media and protein type. BDNF, GDNF, NURR-1 and MAP2 expressions were increased in the presence of pre-induction factors, and decreased when the post-induction factors were added into the culture medium. In contrast with all these promising results, the dopaminergically induced hDPSCs did not show any functional characteristics of dopaminergic neurons and died after they were transferred to a new laminin coated culture plates. In conclusion, the expression of dopaminergic neuron protective protein mRNAs in hDPSCs was increased by photobiomodulation in defined conditions. However, the cells were not able to differentiate into functional dopaminergic neurons either in control or in photobiomodulated groups that are prone to cell death and exhibit immature dopaminergic neuron characteristics.

Functional dopaminergic neurons derived from human chorionic mesenchymal stem cells ameliorate striatal atrophy and improve behavioral deficits in Parkinsonian rat model.

Human chorionic mesenchymal stem cells (HCMSCs) have been recognized as a desirable choice for cell therapy in neurological disorders such as Parkinson's disease (PD). Due to invaluable features of HCMSCs including their immunomodulatory and immunosuppressive properties, easily accessible and less differentiated compared to other types of MSCs, HCMSCs provide a great hope for regenerative medicine. Thus, the purpose of this study was to determine the in vitro and in vivo efficacy of HCMSCs-derived dopaminergic (DA) neuron-like cells with regard to PD. Initially, HCMSCs were isolated and underwent a 2-week DA differentiation, followed by in vitro assessments, using quantitative real-time polymerase chain reaction, immunocytochemistry, patch clamp recording, and high-performance liquid chromatography. In addition, the effects of implanted HCMSCs-derived DA neuron-like cells on the motor coordination along with stereological alterations in the striatum of rat models of PD were investigated. Our results showed that under neuronal induction, HCMSCs revealed neuron-like morphology, and expressed neuronal and DA-specific genes, together with DA release. Furthermore, transplantation of HCMSCs-derived DA neurons into the striatum of rat models of PD, augmented performance. Besides, it prevented reduction of striatal volume, dendritic length, and the total number of neurons, coupled with a diminished level of cleaved caspase-3. Altogether, these findings suggest that HCMSCs could be considered as an attractive strategy for cell-based therapies in PD.

Rapid and Efficient Conversion of Human Fibroblasts into Functional Neurons by Small Molecules.

SummaryRecent studies have demonstrated that human astrocytes and fibroblasts can be directly converted into functional neurons by small molecules. However, fibroblasts, as a potentially better cell resource for transplantation, are not as easy to reprogram as astrocytes regarding their fate to neurons, and chemically induced neurons (iNs) with low efficiency from fibroblasts resulted in limited application for the treatment of neurological disorders, including depression. Here, we report that human fibroblasts can be efficiently and directly reprogrammed into glutamatergic neuron-like cells by serially exposing cells to a combination of small molecules. These iNs displayed neuronal transcriptional networks, and also exhibited mature firing patterns and formed functional synapses. Importantly, iNs could integrate into local circuits after transplantation into postnatal mouse brain. Our study provides a rapid and efficient transgene-free approach for chemically generating neuron-like cells from human fibroblasts. Furthermore, our approach offers strategies for disease modeling and drug discovery in central nervous system disorders.

Generation of functional dopaminergic neurons from human spermatogonial stem cells to rescue parkinsonian phenotypes

BackgroundRecent progress in the induced generation of dopaminergic (DA) neurons from different types of stem cells or reprogrammed somatic cells holds tremendous potential for the treatment of Parkinson’s disease (PD). However, the lack of a reliable source for cell replacement therapy remains a major limitation in the treatment of human neurological disorders. Additionally, the current protocols for in vitro differentiation or cell reprogramming to generate human DA neurons are laborious, time-consuming, and expensive, and efficient conversion of human spermatogonial stem cells (hSSCs) to functional DA neurons has not yet been achieved.MethodsPrimary hSSCs from testicular tissues of patients were exposed to an improved induction system, which consisted mainly of olfactory ensheathing cell conditioned culture medium (OECCM) and a set of defined cell-extrinsic factors and small molecules. Morphological changes were assessed, along with the expression of various DA neuron phenotypic markers (e.g., Tuj-1, TH, Nurr1, DAT) and several critical pro-DA neurogenesis effectors (e.g., EN-1, Pitx3, Foxa2, Lmx1a, Lmx1b, and OTX2). In addition, transcriptome analysis was used to further evaluate the genetic similarity between the artificially differentiated DA neurons and genuine ones. Concomitantly, the functional properties of converted DA neurons including synapse formation, dopamine release, electrophysiological activity, and neuron-specific Ca2+ signaling images were determined. Finally, hSSCs in the early stage of induction were evaluated for survival, differentiation, migration, tumorigenicity in the mouse striatum, and improvement of functional deficits in MPTP-induced PD animals.ResultsThe hSSC-derived neurons not only acquired neuronal morphological features but also expressed various phenotypic genes and protein characteristic of DA neurons and several effectors critical for pro-DA neurogenesis. Strikingly, as the period of induction was prolonged, expression of the critical molecules for DA neuron epigenetic status gradually increased while hSSC-specific markers sharply decreased. After 3 weeks of induction, the transdifferentiation efficiency reached 21%. In addition, hierarchical clustering analysis showed that the differentiated DA neurons closely resembled genuine ones. Furthermore, the hSSC-derived neurons gained sophisticated functional properties of wild-type DA neurons, and pro-induced hSSCs efficiently survived, migrated, and differentiated into DA neurons without tumorigenesis after transplantation into mouse striatum, leading to improvement of functional deficits in PD animals.ConclusionsThe results showed that, using the present improved straightforward approach, hSSCs could acquire DA neuron morphological features and functional properties and rescue parkinsonian phenotypes. Our strategy for the conversion of hSSCs into DA neurons is very efficient and thus may provide an alternative approach suitable for clinical cell therapy to treat neurodegenerative diseases including PD.

Generation of human midbrain organoids from induced pluripotent stem cells

The development of brain organoids represents a major technological advance in the stem cell field, a novel bridge between traditional 2D cultures and in vivo animal models. In particular, the development of midbrain organoids containing functional dopaminergic neurons producing neuromelanin granules, a by-product of dopamine synthesis, represents a potential new model for Parkinson’s disease. To generate human midbrain organoids, we introduce specific inductive cues, at defined timepoints, during the 3D culture process to drive the stem cells towards a midbrain fate. In this method paper, we describe a standardized protocol to generate human midbrain organoids (hMOs) from induced pluripotent stem cells (iPSCs). This protocol was developed to demonstrate how human iPSCs can be successfully differentiated into numerous, high quality midbrain organoids in one batch. We also describe adaptations for cryosectioning of fixed organoids for subsequent histological analysis.

Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells.

We have previously shown that human parthenogenetic stem cells (hpSC) can be chemically directed to differentiate into a homogeneous population of multipotent neural stem cells (hpNSC) that are scalable, cryopreservable, express all the appropriate neural markers, and can be further differentiated into functional dopaminergic neurons. Differentiation of hpSC into hpNSC provides a platform to study the molecular basis of human neural differentiation, to develop cell culture models of neural disease, and to provide neural stem cells for the treatment of neurodegenerative diseases. Additionally, the hpNSC that are generated could serve asa platform for drug discovery and the determination of pharmaceutical-induced neural toxicity. Here, we describe in detail the stepwise protocol that was developed in our laboratory that facilitates the highly efficient and reproducible differentiation of hpSC into hpNSC.