| 1. |
- Aldrin-Kirk, Patrick, et al.
(författare)
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A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:1, s. 159-172
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.
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| 2. |
- Drouin-Ouellet, Janelle, et al.
(författare)
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Age-related pathological impairments in directly reprogrammed dopaminergic neurons derived from patients with idiopathic Parkinson's disease
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:10, s. 2203-2219
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Tidskriftsartikel (refereegranskat)abstract
- We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
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| 3. |
- Johansson, Pia A, et al.
(författare)
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A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development
- 2022
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Ingår i: Cell Stem Cell. - : Elsevier BV. - 1934-5909 .- 1875-9777. ; 29:1, s. 8-69
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Tidskriftsartikel (refereegranskat)abstract
- The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.
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| 4. |
- Jönsson, Marie, et al.
(författare)
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Comprehensive analysis of microRNA expression in regionalized human neural progenitor cells reveals microRNA-10 as a caudalizing factor.
- 2015
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Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 142:18, s. 3166-3177
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) have been implicated in regulating multiple processes during brain development in various species. However, the function of miRNAs in human brain development remains largely unexplored. Here, we provide a comprehensive analysis of miRNA expression of regionalized neural progenitor cells derived from human embryonic stem cells and human foetal brain. We found miR-92b-3p and miR-130b-5p to be specifically associated with neural progenitors and several miRNAs that display both age-specific and region-specific expression patterns. Among these miRNAs, we identified miR-10 to be specifically expressed in the human hindbrain and spinal cord, while being absent from rostral regions. We found that miR-10 regulates a large number of genes enriched for functions including transcription, actin cytoskeleton and ephrin receptor signalling. When overexpressed, miR-10 influences caudalization of human neural progenitor cells. Together, these data confirm a role for miRNAs in establishing different human neural progenitor populations. This dataset also provides a comprehensive resource for future studies investigating the functional role of different miRNAs in human brain development.
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| 5. |
- Adler, Andrew F., et al.
(författare)
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hESC-Derived Dopaminergic Transplants Integrate into Basal Ganglia Circuitry in a Preclinical Model of Parkinson's Disease
- 2019
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 28:13, s. 5-3473
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Tidskriftsartikel (refereegranskat)abstract
- Cell replacement is currently being explored as a therapeutic approach for neurodegenerative disease. Using stem cells as a source, transplantable progenitors can now be generated under conditions compliant with clinical application in patients. In this study, we elucidate factors controlling target-appropriate innervation and circuitry integration of human embryonic stem cell (hESC)-derived grafts after transplantation to the adult brain. We show that cell-intrinsic factors determine graft-derived axonal innervation, whereas synaptic inputs from host neurons primarily reflect the graft location. Furthermore, we provide evidence that hESC-derived dopaminergic grafts transplanted in a long-term preclinical rat model of Parkinson's disease (PD) receive synaptic input from subtypes of host cortical, striatal, and pallidal neurons that are known to regulate the function of endogenous nigral dopamine neurons. This refined understanding of how graft neurons integrate with host circuitry will be important for the design of clinical stem-cell-based replacement therapies for PD, as well as for other neurodegenerative diseases. Adler et al. graft hESC-derived dopaminergic progenitors into a rat model of Parkinson's disease. They find grafts correctly innervate host targets and receive appropriate synaptic input after intranigral and intrastriatal placement. Furthermore, the same host neurons projecting toward endogenous dopamine neurons are found to also connect to the grafts.
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| 6. |
- Adler, Andrew, et al.
(författare)
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Transsynaptic tracing and its emerging use to assess graftreconstructed neural circuits
- 2020
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 38:6, s. 716-726
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Tidskriftsartikel (refereegranskat)abstract
- Fetal neural progenitor grafts have been evaluated in preclinical animal models of spinal cord injury and Parkinson’s disease for decades, but the initial reliance on primary tissue as a cell source limited the scale of their clinical translatability. With the development of robust methods to differentiate human pluripotent stem cells to specific neural subtypes, cell replacement therapy holds renewed promise to treat a variety of neurodegenerative diseases and injuries at scale. As these cell sources are evaluated in preclinical models, new transsynaptic tracing methods are making it possible to study the connectivity between host and graft neurons with greater speed and detail than was previously possible. To date, these studies have revealed that widespread, long-lasting, and anatomically-appropriate synaptic contacts are established between host and graft neurons, as well as new aspects of host-graft connectivity which may be relevant to clinical cell replacement therapy. It is not yet clear, however, whether the synaptic connectivity between graft and host neurons is as celltype specific as it is in the endogenous nervous system, or whether that connectivity is responsible for the functional efficacy of cell replacement therapy. Here, we review evidence suggesting that the new contacts established between host and graft neuronsmay indeed be cell-type specific, and how transsynaptic tracing can be used inthe future to further elucidate the mechanisms of graft-mediated functional recovery in spinal cord injury and Parkinson’s disease.
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| 7. |
- Aldrin-Kirk, Patrick, et al.
(författare)
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DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor
- 2016
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Ingår i: Neuron. - : Elsevier BV. - 0896-6273. ; 90:5, s. 955-968
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson's disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.
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| 8. |
- Alekseenko, Zhanna, et al.
(författare)
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Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery
- 2022
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.
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| 9. |
- Andersson, Elin, et al.
(författare)
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Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2.
- 2006
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Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 133:3, s. 507-516
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Tidskriftsartikel (refereegranskat)abstract
- Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. We show here that lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons, as the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain.
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| 10. |
- Andersson, Elin, et al.
(författare)
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Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells.
- 2007
-
Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 313:6, s. 1172-1180
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Tidskriftsartikel (refereegranskat)abstract
- 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 multipassaged 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. Overexpression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers. (c) 2006 Elsevier Inc. All rights reserved.
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| 11. |
- Avaliani, Natalia, et al.
(författare)
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Directly Converted Human Fibroblasts Mature to Neurons and Show Long-Term Survival in Adult Rodent Hippocampus
- 2017
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Ingår i: Stem Cells International. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2017
-
Tidskriftsartikel (refereegranskat)abstract
- Direct conversion of human somatic cells to induced neurons (iNs), using lineage-specific transcription factors has opened new opportunities for cell therapy in a number of neurological diseases, including epilepsy. In most severe cases of epilepsy, seizures often originate in the hippocampus, where populations of inhibitory interneurons degenerate. Thus, iNs could be of potential use to replace these lost interneurons. It is not known, however, if iNs survive and maintain functional neuronal properties for prolonged time periods in in vivo. We transplanted human fibroblast-derived iNs into the adult rat hippocampus and observed a progressive morphological differentiation, with more developed dendritic arborisation at six months as compared to one month. This was accompanied by mature electrophysiological properties and fast high amplitude action potentials at six months after transplantation. This proof-of-principle study suggests that human iNs can be developed as a candidate source for cell replacement therapy in temporal lobe epilepsy.
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| 12. |
- Barker, Roger A, et al.
(författare)
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Are Stem Cell-Based Therapies for Parkinson's Disease Ready for the Clinic in 2016?
- 2016
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Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 6:1, s. 57-63
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Tidskriftsartikel (refereegranskat)abstract
- Recent news of an impending clinical cell transplantation trial in Parkinson's disease using parthenogenetic stem cells as a source of donor tissue have raised hopes in the patient community and sparked discussion in the research community. Based on discussions held by a global collaborative initiative on translation of stem cell therapy in Parkinson's disease, we have identified a set of key questions that we believe should be addressed ahead of every clinical stem cell-based transplantation trial in this disorder. In this article, we first provide a short history of cell therapy in Parkinson's disease and briefly describe the current state-of-art regarding human stem cell-derived dopamine neurons for use in any patient trial. With this background information as a foundation, we then discuss each of the key questions in relation to the upcoming therapeutic trial and critically assess if the time is ripe for clinical translation of parthenogenetic stem cell technology in Parkinson's disease.
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| 13. |
- Barker, Roger A., et al.
(författare)
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Designing stem-cell-based dopamine cell replacement trials for Parkinson’s disease
- 2019
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 25, s. 1045-1053
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Tidskriftsartikel (refereegranskat)abstract
- Clinical studies of Parkinson’s disease (PD) using a dopamine cell replacment strategy have been tried for more than 30 years. The outcomes following transplantation of human fetal ventral mesencephalic tissue (hfVM) have been variable, with some patients coming off their anti-PD treatment for many years and others not responding and/or developing significant side effects, including graft-induced dyskinesia. This led to a re-appraisal of the best way to do such trials, which resulted in a new European-Union-funded allograft trial with fetal dopamine cells across several centers in Europe. This new trial, TRANSEURO (NCT01898390), is an open-label study in which some individuals in a large observational cohort of patients with mild PD who were undergoing identical assessments were randomly selected to receive transplants of hfVM. The TRANSEURO trial is currently ongoing as researchers have completed both recruitment into a large multicenter observational study of younger onset early-stage PD and transplantation of hfVM in 11 patients. While completion of TRANSEURO is not expected until 2021, we feel that sharing the rationale for the design of TRANSEURO, along with the lessons we have learned along the way, can help inform researchers and facilitate planning of transplants of dopamine-producing cells derived from human pluripotent stem cells for future clinical trials.
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| 14. |
- Barker, Roger A., et al.
(författare)
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Human Trials of Stem Cell-Derived Dopamine Neurons for Parkinson's Disease : Dawn of a New Era
- 2017
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Ingår i: Cell Stem Cell. - : Elsevier BV. - 1934-5909. ; 21:5, s. 569-573
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell-based therapies for Parkinson's disease are moving into a new and exciting era, with several groups pursuing clinical trials with pluripotent stem cell (PSC)-derived dopamine neurons. As many groups have ongoing or completed GMP-level cell manufacturing, we highlight key clinical translation considerations from our recent fourth GForce-PD meeting. Stem cell-based therapies for Parkinson's disease are moving into a new and exciting era, with several groups pursuing clinical trials with pluripotent stem cell (PSC)-derived dopamine neurons. As many groups have ongoing or completed GMP-level cell manufacturing, we highlight key clinical translation considerations from our recent fourth GForce-PD meeting.
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| 15. |
- Barker, Roger A, et al.
(författare)
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New approaches for brain repair-from rescue to reprogramming
- 2018
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 557:7705, s. 329-334
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Forskningsöversikt (refereegranskat)abstract
- The ability to repair or promote regeneration within the adult human brain has been envisioned for decades. Until recently, such efforts mainly involved delivery of growth factors and cell transplants designed to rescue or replace a specific population of neurons, and the results have largely been disappointing. New approaches using stem-cell-derived cell products and direct cell reprogramming have opened up the possibility of reconstructing neural circuits and achieving better repair. In this Review we briefly summarize the history of neural repair and then discuss these new therapeutic approaches, especially with respect to chronic neurodegenerative disorders.
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| 16. |
- Barker, Roger, et al.
(författare)
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Cell-based therapies for Parkinson disease-past insights and future potential.
- 2015
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Ingår i: Nature Reviews Neurology. - : Springer Science and Business Media LLC. - 1759-4766 .- 1759-4758. ; 11:9, s. 492-503
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Forskningsöversikt (refereegranskat)abstract
- Parkinson disease (PD) is characterized by loss of the A9 nigral neurons that provide dopaminergic innervation to the striatum. This discovery led to the successful instigation of dopaminergic drug treatments in the 1960s, although these drugs were soon recognized to lose some of their efficacy and generate their own adverse effects over time. Despite the fact that PD is now known to have extensive non-nigral pathology with a wide range of clinical features, dopaminergic drug therapies are still the mainstay of therapy, and work well for many years. Given the success of pharmacological dopamine replacement, pursuit of cell-based dopamine replacement strategies seemed to be the next logical step, and studies were initiated over 30 years ago to explore the possibility of dopaminergic cell transplantation. In this Review, we outline the history of this therapeutic approach to PD and highlight the lessons that we have learned en route. We discuss how the best clinical outcomes have been obtained with fetal ventral mesencephalic allografts, while acknowledging inconsistencies in the results owing to problems in trial design, patient selection, tissue preparation, and immunotherapy used post-grafting. We conclude by discussing the challenges of bringing the new generation of stem cell-derived dopamine cells to the clinic.
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| 17. |
- Barraud, Perrine, et al.
(författare)
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In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133.
- 2007
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 85, s. 250-259
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Tidskriftsartikel (refereegranskat)abstract
- The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133+), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133+/SSEA4+ and CD133+/CD15+ isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.
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| 18. |
- Barraud, Perrine, et al.
(författare)
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Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse.
- 2005
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Ingår i: European Journal of Neuroscience. - : Wiley. - 1460-9568 .- 0953-816X. ; 22:7, s. 1555-1569
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Tidskriftsartikel (refereegranskat)abstract
- We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1–GFP-expressing population. Thus, the Sox1–GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1–GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1–GFP precursors were derived. On the other hand, Sox1–GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1–GFP cells possess the same capacity for neuronal differentiation in vivo.
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| 19. |
- Besusso, Dario, et al.
(författare)
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Stem Cell-Derived Human Striatal Progenitors Innervate Striatal Targets and Alleviate Sensorimotor Deficit in a Rat Model of Huntington Disease
- 2020
-
Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 14:5, s. 876-891
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Tidskriftsartikel (refereegranskat)abstract
- Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach.
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| 20. |
- Birtele, Marcella, et al.
(författare)
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Dual modulation of neuron-specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons
- 2019
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 593:23, s. 3370-3380
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Tidskriftsartikel (refereegranskat)abstract
- Direct neuronal reprogramming can be achieved using different approaches: by expressing neuronal transcription factors or microRNAs; and by knocking down neuronal repressive elements. However, there still exists a high variability in terms of the quality and maturity of the induced neurons obtained, depending on the reprogramming strategy employed. Here, we evaluate different long-term culture conditions and study the effect of expressing the neuronal-specific microRNAs, miR124 and miR9/9*, while reprogramming with forced expression of the transcription factors Ascl1, Brn2, and knockdown of the neuronal repressor REST. We show that the addition of microRNAs supports neuronal maturation in terms of gene and protein expression, as well as in terms of electrophysiological properties.
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| 21. |
- Birtele, Marcella, et al.
(författare)
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Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain
- 2022
-
Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 149:23
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Tidskriftsartikel (refereegranskat)abstract
- Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells (PSCs) for application in disease modeling, diagnostics, drug screening, and cell-based therapies for Parkinson's Disease (PD). An increased understanding of the timing and molecular mechanisms promoting the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from PSCs. Here, we used droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach revealed that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons, which can be used to guide stem cell-based therapies and disease modeling approaches in PD.
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| 22. |
- Björklund, Anders, et al.
(författare)
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A Combined α-Synuclein/Fibril (SynFib) Model of Parkinson-Like Synucleinopathy Targeting the Nigrostriatal Dopamine System
- 2022
-
Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 12:8, s. 2307-2320
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Forskningsöversikt (refereegranskat)abstract
- Injections of pre-formed α-synuclein fibrils (PFFs) or overexpression of α-synuclein using AAV vectors are commonly used as models of Parkinson-like synucleinopathy in rats and mice. In the modified method reviewed here, the "SynFib" model, the PFFs and the AAV vector are administered together unilaterally into the substantia nigra. This approach combines the key features of these two models, i.e., the generation of toxic α-synuclein aggregates and Lewy body-like inclusions, in combination with the increased vulnerability caused by increased cellular levels of α-synuclein. The combined AAV/PFF delivery offers several advantages over the standard PFF model due to the enhanced and accelerated α-synuclein pathology and microglial response induced by the PFF seeds in the presence of an elevated α-synuclein level. Injection of the AAV/PFF mixture into the substantia nigra makes it possible to target a larger proportion of the nigral dopamine neurons and obtain a level of dopamine cell loss (>60%) needed to induce significant impairments in drug-induced and spontaneous motor tests. The SynFib model shares attractive features of the standard 6-OHDA lesion model: a single unilateral stereotaxic intervention; pathology and cell loss developing over a short time span; and the possibility to monitor the degenerative changes using tests of motor behavior.
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| 23. |
- Björklund, Anders, et al.
(författare)
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Dopamine Cell Therapy : From Cell Replacement to Circuitry Repair
- 2021
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Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 11:s2, s. 159-165
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Forskningsöversikt (refereegranskat)abstract
- Cell therapy for Parkinson's disease (PD) is aimed to replace the degenerated midbrain dopamine (mDA) neurons and restore DA neurotransmission in the denervated forebrain targets. A limitation of the intrastriatal grafting approach, which is currently used in clinical trials, is that the mDA neurons are implanted into the target area, in most cases the putamen, and not in the ventral midbrain where they normally reside. This ectopic location of the cells may limit their functionality due to the lack of appropriate afferent regulation from the host. Homotopic transplantation, into the substantia nigra, is now being pursued in rodent PD models as a way to achieve more complete circuitry repair. Intranigral grafts of mDA neurons, derived from human embryonic stem cells, have the capacity to re-establish the nigrostriatal and mesolimbic pathways in their entirety and restore dense functional innervations in striatal, limbic and cortical areas. Tracing of host afferent inputs using the rabies tracing technique shows that the afferent connectivity of grafts implanted in the nigra matches closely that of the intrinsic mDA system, suggesting a degree of circuitry reconstruction that exceeds what has been achieved before. This approach holds great promise, but to match the larger size of the human brain, and the 10 times greater distance between substantia nigra and its forebrain targets, it may be necessary to find ways to improve the growth capacity of the grafted mDA neurons, pointing to a combined approach where growth promoting factors are used to enhance the performance of mDA neuron grafts.
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| 24. |
- Björklund, Anders, et al.
(författare)
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Neuronal Replacement as a Tool for Basal Ganglia Circuitry Repair : 40 Years in Perspective
- 2020
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Ingår i: Frontiers in Cellular Neuroscience. - : Frontiers Media SA. - 1662-5102. ; 14, s. 146-146
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Forskningsöversikt (refereegranskat)abstract
- The ability of new neurons to promote repair of brain circuitry depends on their capacity to re-establish afferent and efferent connections with the host. In this review article, we give an overview of past and current efforts to restore damaged connectivity in the adult mammalian brain using implants of fetal neuroblasts or stem cell-derived neuronal precursors, with a focus on strategies aimed to repair damaged basal ganglia circuitry induced by lesions that mimic the pathology seen in humans affected by Parkinson's or Huntington's disease. Early work performed in rodents showed that neuroblasts obtained from striatal primordia or fetal ventral mesencephalon can become anatomically and functionally integrated into lesioned striatal and nigral circuitry, establish afferent and efferent connections with the lesioned host, and reverse the lesion-induced behavioral impairments. Recent progress in the generation of striatal and nigral progenitors from pluripotent stem cells have provided compelling evidence that they can survive and mature in the lesioned brain and re-establish afferent and efferent axonal connectivity with a remarkable degree of specificity. The studies of cell-based circuitry repair are now entering a new phase. The introduction of genetic and virus-based techniques for brain connectomics has opened entirely new possibilities for studies of graft-host integration and connectivity, and the access to more refined experimental techniques, such as chemo- and optogenetics, has provided new powerful tools to study the capacity of grafted neurons to impact the function of the host brain. Progress in this field will help to guide the efforts to develop therapeutic strategies for cell-based repair in Huntington's and Parkinson's disease and other neurodegenerative conditions involving damage to basal ganglia circuitry.
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| 25. |
- Brambach, Max, et al.
(författare)
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Neural tube patterning : from a minimal model for rostrocaudal patterning toward an integrated 3D model
- 2021
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 24:6
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Tidskriftsartikel (refereegranskat)abstract
- Rostrocaudal patterning of the neural tube is a defining event in vertebrate brain development. This process is driven by morphogen gradients which specify the fate of neural progenitor cells, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose tristable topology was determined in a data-driven way. Using this model, we identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. Furthermore, we combined our model with an established minimal model for dorsoventral patterning on a realistic 3D neural tube and found that key features of neural tube patterning could be recapitulated. Doing so, we demonstrate how data and models from different sources can be combined to simulate complex in vivo processes.
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