Generation Of Functional Dopaminergic Neurons Research Articles

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.

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 Functional Dopaminergic Neurons from Human Spermatogonial Stem Cells to Rescue Parkinsonian Phenotypes

Background: Recent progress in the induced generation of dopaminergic neurons (DANs) 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 treatment of human neurological disorders. Additionally, the current protocols for in vitro differentiation or cell reprogramming to generate human DANs are laborious, time-consuming and expensive, and efficient conversion of human spermatogonial stem cells (hSSCs) to functional DANs has not yet been achieved. Methods: Primary hSSCs from 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 DAN 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 DANs and genuine ones. Concomitantly, the functional properties of converted DANs 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 MTPT-induced PD animals. Findings: The hSSCs-derived neurons not only acquired neuronal morphological features but also expressed various phenotypic genes and proteins characteristic of DANs and several effectors critical for pro-DA neurogenesis. Strikingly, as the period of induction was prolonged, expression of the critical molecules for DAN epigenetic status gradually increased while SSC-specific markers sharply decreased. After 3 weeks of induction, the transdifferentiation efficiency reached 21%. In addition, hierarchical clustering analysis showed that the differentiated DANs closely resembled genuine ones. Furthermore, the hSSC-derived neurons gained sophisticated functional properties of wild-type DANs, and pro-induced hSSCs efficiently survived, migrated, and differentiated into DANs without tumorigenesis after transplantation into mouse striatum, leading to improvement of functional deficits in PD animals. Interpretation: Neurons-derived from hSSCs acquired DAN morphological features and functional properties, and rescued Parkinsonian phenotypes. Our strategy for the conversion of hSSCs into DANs is very efficient and thus may provide an alternative approach suitable for clinical cell therapy to treat neurodegenerative diseases including PD. Funding Statement: This work was supported by the Natural Science Foundation of China (grant nos. 81371411, 81571208, 81472098 and 81772357), the Natural Science Foundation of Shaanxi province (2017JM8012), Shanghai Jiaotong University Med-X Fund (No.YG2014MS45), and a key fund of Honghui Hospital (YJ2017001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Human testicular tissues were obtained from clinically obstructive azoospermic (OA) patients who had normal spermatogenesis and all corpse subjects for autopsy and histopathological assessment in accordance with local and Nation Ethics committee approved. All animal procedures were performed in agreement with the National Institutes of Health guidelines and were approved by the Institutional Ethical Review Committee of Hong Hui Hospital affiliated by Xi’an Jiaotong University.

Generation of Functional Dopaminergic Neurons from Reprogramming Fibroblasts by Nonviral-based Mesoporous Silica Nanoparticles

Direct-lineage conversion of the somatic cell by reprogramming, in which mature cells were fully converted into a variety of other cell types bypassing an intermediate pluripotent state, is a promising regenerative medicine approach. Due to the risk of tumorigenesis by viral methods, a non-viral carrier for the delivery of reprogramming factors is very desirable. This study utilized the mesoporous silica nanoparticles (MSNs) as a non-viral delivery system for transduction of the three key factors to achieve conversion of mouse fibroblasts (MFs) into functional dopaminergic neuron-like cells (denoted as fDA-neurons). At the same time, a neurogenesis inducer, ISX-9, was co-delivered with the MSNs to promote the direct conversion of neuron-like cells. Good transfection efficiency of plasmid@MSN allowed repeated dosing to maintain high exogenous gene expression analyzed by qPCR and the changes in neural function markers were monitored. To further validate the dopaminergic function and the electrophysiological properties of fDA-neurons, the results of ELISA assay showed the high levels of secreted-dopamine in the conditional medium and rich Na+/K+-channels were observed in the fDA-neurons on Day 22. The results demonstrated that MSN nanocarrier is effective in delivering the reprogramming factors for the conversion of functional dopaminergic neurons from adult somatic cells.

Synergistic Effect of BDNF and FGF2 in Efficient Generation of Functional Dopaminergic Neurons from human Mesenchymal Stem Cells

To understand the process of neurogenesis, generation of functional dopaminergic (DAergic) neurons from human mesenchymal stem cells (hMSCs) is important. BDNF has been reported to be responsible for inducing neuronal maturation and functionality. Previously, we have reported the efficient generation of neurons from human bone marrow derived MSCs using FGF2 alone. We hypothesize that hMSCs from various tissues [(bone marrow (BM), adipose tissue (AD) and dental pulp (DP)], if treated with BDNF on 9th day of induction, alongwith FGF2 will generate functional DAergic neurons. Hence, cells were characterized at morphometric, transcription and translational levels for various markers like MAP2, TH, NGN2, PITX3, DAT, synaptophysin, Kv4.2 and SCN5A. Functionality of in vitro generated neurons was studied by calcium ion imaging. Result analysis depicted that BDNF has effect on expression of dopaminergic neuronal markers at gene and protein levels and functionality of neurons. Among these hMSCs, DP-MSC showed significantly better neuronal characteristics in terms of morphology, expression of neuronal markers and foremost, functionality of neurons. From the present study, therefore, we concluded that i) BDNF has additive effect on neuronal characteristics and functionality ii) DP-MSC are better MSC candidate to study DAergic neurogenesis and perform future studies.

In vitro generation of functional dopaminergic neurons from full-term amniotic fluid stem cells

Event Abstract Back to Event In vitro generation of functional dopaminergic neurons from full-term amniotic fluid stem cells Siti Nurussaadah Hamzah1, 2, Hoo Mun-Fun1, 2, Sharmili Vidyadaran3 and Norshariza Nordin1, 2* 1 Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Genetics and Regenerative Medicine Research Centre, Malaysia 2 Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Department of Biomedical Science, Malaysia 3 Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Department of Pathology, Malaysia Stem cell replacement therapy has been seen as a promising alternative treatment for Parkinson’s disease. Unfortunately, the efficiency in generating functional dopaminergic (DA) neurons from stem cells remains a great challenge. Different stem cell types have different dopaminergic potential that require different differentiation protocols. Amniotic fluid stem cells (AFSCs) are among the prominent stem cell sources in regenerative medicine that have broad differentiation capacity with less safety and ethical concerns. Here, we aimed to explore the differentiation potential of our newly established full-term rat AFSCs in generating DA neurons and evaluate their functional properties and maturity in-vitro. Two methods; adherent monoculture and multicellular aggregation (embryoid bodies, EBs) with addition of all-trans retinoic acid (4-/4+) protocols, were used to differentiate the AFSCs into neurons. Both methods clearly showed AFSCs to be highly neurogenic and successfully generated cells with good neuron-like morphology expressing neural precursor marker (Nestin), post-mitotic and mature neuronal markers (Class III β-tubulin and Microtubule-associated protein 2; MAP2) and DA marker (Tyrosine hydroxylase; TH). We further examined the effects of patterning factors; fibroblast growth factor 8b (Fgf8b) and sonic hedgehog (Shh) using the adherent monoculture method in generating DA neurons. The differentiated cells expressed DA neuronal markers MAP2 and TH, midbrain DA marker (Pituitary homeobox 3; Pitx3), neuronal maturity (G-protein-regulated inward-rectifier potassium (K+) channel 2; GIRK2) and functionality markers (Vesicular monoamine transporter 2; VMAT2 and Synaptophysin). Interestingly, no significant difference was observed in expression of these markers by the cells generated with and without the addition of Fgf8b and Shh. The neurons were also observed to release dopamine in the absence of these factors. These results highly suggest that full-term AFSCs as a highly potential source for stem cell-derived DA neurons thus offer as a valuable tool in the endeavour towards PD therapy. Acknowledgements This project was supported by Research University Grant Scheme (RUGS): project number 04-02-12-1758RU, from Universiti Putra Malaysia awarded to N Nordin, and Special Graduate Research Award (SGRA) from Universiti Putra Malaysia awarded to SN Hamzah. Keywords: Amniotic Fluid, Dopamine, Stem Cells, in vitro, neurone Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Hamzah S, Mun-Fun H, Vidyadaran S and Nordin N (2016). In vitro generation of functional dopaminergic neurons from full-term amniotic fluid stem cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00185 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Norshariza Nordin, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Genetics and Regenerative Medicine Research Centre, Serdang, Selangor, Malaysia, shariza@upm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Siti Nurussaadah Hamzah Hoo Mun-Fun Sharmili Vidyadaran Norshariza Nordin Google Siti Nurussaadah Hamzah Hoo Mun-Fun Sharmili Vidyadaran Norshariza Nordin Google Scholar Siti Nurussaadah Hamzah Hoo Mun-Fun Sharmili Vidyadaran Norshariza Nordin PubMed Siti Nurussaadah Hamzah Hoo Mun-Fun Sharmili Vidyadaran Norshariza Nordin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs. Stem Cells 2010;28:1893–1904

One year survival and significant reversal of motor deficits in parkinsonian rats transplanted with hESC derived dopaminergic neurons

We report the generation of functional dopaminergic neurons from human embryonic stem cells (hESCs) using a growth factor mediated multistep EB protocol and its therapeutic effects in vivo. Embryoid bodies (EBs) were cultured in insulin–transferrin–selenium fibronectin (ITSFn) media for the selection of neural precursor cells (NPC). The selected cells on exposure to N2 media supplemented with EGF, bFGF initially aggregated to generate spontaneous free floating neurospheres and on exposure to signaling molecules Shh and FGF-8 differentiated into dopaminergic neurons (40% TH+ cells/total neurons). The differentiated NPC expressed dopaminergic specific markers both at cellular and molecular levels. They secreted detectable levels of dopamine into the culture supernatant. The most unique feature of our protocol is the generation of free floating neurospheres which can be expanded for a longer period without losing their capability to differentiate into DA neurons. Further, transplantation of NPCs into the substantia nigra of 6-OHDA lesioned rat model of Parkinson’s disease elicited significant reversal of lesion induced motor deficits which was sustained upto the end of 1 year long study period. Immunohistochemical studies of the grafted area one year post transplantation revealed that transplanted hESC derived neural precursor cells survived, integrated in vivo and differentiated into dopaminergic neurons without teratoma formation. In summary, our results encourage the potential use of hESC derived dopaminergic neurons for future clinical application in Parkinson’s disease.

Derivation of Functional Dopamine Neurons from Embryonic Stem Cells

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra of the midbrain. Pharmacological treatment of PD has been a prevailing strategy. However, it has some limitations because its effectiveness gradually decreases and side effects develop. As an alternative, cell transplantation therapy has been tried. Although transplantation of fetal ventral mesencephalic cells looks promising for the treatment of PD in some cases, ethical and technical problems in obtaining large numbers of human fetal brain tissues also lead to difficulty in its clinical application. Our recent studies showed that a high yield of DA neurons could be derived from embryonic stem (ES) cells and they efficiently induced behavioral recovery in a PD animal model. Here we summarize methods for generation of functional DA neurons from ES cells for application to PD models.