| 1. |
- Pfisterer, Ulrich, et al.
(författare)
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Direct conversion of human fibroblasts to dopaminergic neurons.
- 2011
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 108:25, s. 10343-10348
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Tidskriftsartikel (refereegranskat)abstract
- Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy.
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| 2. |
- 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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| 3. |
- 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
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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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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| 4. |
- 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
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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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| 5. |
- Brattås, Per Ludvik, et al.
(författare)
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TRIM28 Controls a Gene Regulatory Network Based on Endogenous Retroviruses in Human Neural Progenitor Cells
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 18:1, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- Endogenous retroviruses (ERVs), which make up 8% of the human genome, have been proposed to participate in the control of gene regulatory networks. In this study, we find a region- and developmental stage-specific expression pattern of ERVs in the developing human brain, which is linked to a transcriptional network based on ERVs. We demonstrate that almost 10,000, primarily primate-specific, ERVs act as docking platforms for the co-repressor protein TRIM28 in human neural progenitor cells, which results in the establishment of local heterochromatin. Thereby, TRIM28 represses ERVs and consequently regulates the expression of neighboring genes. These results uncover a gene regulatory network based on ERVs that participates in control of gene expression of protein-coding transcripts important for brain development.
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| 6. |
- Lehnen, Daniela, et al.
(författare)
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IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells
- 2017
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 9:4, s. 1207-1220
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Tidskriftsartikel (refereegranskat)abstract
- Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. In this article, Knöbel and colleagues identified IAP as a cell surface marker for mesDA progenitor cells. Immunomagnetic sorting for IAP+ led to reproducible and homogeneous cell compositions. Intrastriatal transplantation of sorted cells at day 16 of differentiation in a PD rat model resulted in functional recovery, and grafts were more homogeneous in size and DA neuron density than unsorted cells.
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| 7. |
- Nilsson, Fredrik, et al.
(författare)
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Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation
- 2021
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson's disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.
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| 8. |
- Nolbrant, Sara, et al.
(författare)
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Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation
- 2017
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1750-2799 .- 1754-2189. ; 12:9, s. 1962-1979
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Tidskriftsartikel (refereegranskat)abstract
- Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans. We show how to precisely set the balance of patterning factors to obtain specifically the caudal VM progenitors that give rise to DA-rich grafts. By specifying how to perform quality control (QC), troubleshooting and adaptation of the procedure, this protocol will facilitate implementation in different laboratories and with a variety of hPSC lines. To facilitate reproducibility of experiments and enable shipping of cells between centers, we present a method for cryopreservation of the progenitors for subsequent direct transplantation or terminal differentiation into DA neurons. This protocol is free of xeno-derived products and can be performed under good manufacturing practice (GMP) conditions.
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| 9. |
- Parmar, Malin, et al.
(författare)
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GFORCE-PD still going strong in 2016
- 2017
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Ingår i: npj Parkinson's Disease. - : Springer Science and Business Media LLC. - 2373-8057. ; 3
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Forskningsöversikt (refereegranskat)abstract
- In 2014, a new initiative was undertaken by groups working on plans for the transplantation of stem-cell-based derived dopaminergic neurons for treating Parkinson’s disease patients. This GForce-PD group held its annual meeting on 18–19 April 2016 in Chicago at Rush University to discuss their progress and the challenges that the translation of this experimental therapy still faces. Over 2 days, the key issues were discussed around the cell lines that will be used, the differentiation protocols, preclinical testing, GMP-adaptation, and cell manufacturing to allow first in human clinical trials, which are anticipated to start in 2017–2018. GForce-PD members also discussed how they can improve outreach and be of better service to the Parkinson's disease (PD) community and help them to make the best possible decisions when pursuing stem cell treatments.
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| 10. |
- Pircs, Karolina, et al.
(författare)
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Huntingtin Aggregation Impairs Autophagy, Leading to Argonaute-2 Accumulation and Global MicroRNA Dysregulation
- 2018
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 24:6, s. 1397-1406
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Tidskriftsartikel (refereegranskat)abstract
- Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates, resulting in alterations in autophagy. However, the consequences of impaired autophagy for neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation as well as post-mortem material from patients with Huntington's disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNA-induced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together, these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation but also directly contributes to global alterations of intracellular post-transcriptional networks. Pircs et al. report that aggregation of the mutant huntingtin protein, a hallmark of Huntington's disease proteinopathy, impairs macroautophagy, leading to Argonaute-2 accumulation and global dysregulation of microRNAs. These results indicate that autophagy not only influences protein aggregation but also directly contributes to the global alterations of post-transcriptional networks in Huntington's disease.
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