| 1. |
- Andersson, My, et al.
(författare)
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Optogenetic control of human neurons in organotypic brain cultures
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies.
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| 2. |
- Avaliani, Natalia
(författare)
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Chemogenetics, Induced Neurons and Pluripotent Stem Cells: Towards Advanced Gene and Cell Therapies Targeting Epilepsy
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The complexity of the central nervous system and existence of the blood-brain barrier often causes difficulties for traditional pharmacological treatments of neurological diseases. This thesis explores the feasibility and potential for novel gene and cell therapy approaches, which hold better promise for neurological disorders, while particularly targeting epilepsy. Epilepsy is a multifactorial neurological disorder affecting 1% of the population. Available pharmacological therapies are merely symptomatic, and have severe side effects, while failing to adequately control seizures in one third of the patients, predominately in those with temporal lobe epilepsy (TLE). Targeted silencing of the pathological circuits by expressing therapeutic genes, or increasing the inhibition by introducing new populations of GABA-releasing neurons, might prove therapeutic for epilepsy, by counteracting seizures and even modifying the disease. Gene therapy offers localized, cell-type specific alteration of neuronal excitability, but on-demand seizure suppression can only be achieved by tools allowing external temporal control. One such recently developed chemogenetic technology is based on viral expression of modified muscarinic G-protein coupled receptors, specifically activated by otherwise inert clozapine-N-oxide (CNO). In paper I, we explored if such modified receptor, hM4Di, which selectively activates Gi pathway, thereby causing neuronal inhibition, could suppress epileptiform activity upon CNO application. This approach proved effective for localized suppression of neuronal excitability and seizure-like events in an in vitro model of TLE, organotypic hippocampal slice cultures (OHSC), without altering intrinsic properties of the hM4Di-expressing neurons, demonstrating the therapeutic potential of this technology. In papers II and III we characterized two different cell sources with the prospect of cell replacement therapy: induced Pluripotent Stem cells (iPS) and induced Neurons (iN). These two patient specific alternative cell sources offer a solution to ethical and immunogenicity issues, related to embryonic stem cell use. Already six weeks after grafting in OHSCs, iPS-derived neuroepitelial-like stem cells (lt-NES), predominately differentiating to GABA-ergic neurons, displayed functional neuronal properties and certain rate of synaptic integration. In vivo studies showed that longer differentiation time (up to 24 weeks) was needed for the grafts to fully mature and extensively integrate into the host synaptic network. The grafted cells still retained some of the distinct electrophysiological features, however, such as high input resistance. Next, we studied long-term survival of human foetal fibroblast-derived induced neurons (iN) in rodent hippocampus. Human iNs survive and maintain neuronal profile up to six months post-grafting, developing more elaborate neuronal morphology and complex dendritic arborisation over time. Graft-derived neurons with mature neuronal properties could be observed at six months, although a portion of non-converted fibroblasts, as well as asynchronous neuronal conversion was apparent among the grafts. Improvements in conversion, survival and integration rate of iN cells are required before these cells can offer a better alternative to iPS or stem cells. While showing potential as candidates for cell replacement therapy, both characterised cell types have to be further tested in relevant epilepsy model systems to demonstrate their therapeutic effect. In summary, this thesis adds new knowledge and experimental basis for development of gene- and cell-based therapies for neurological disorders.
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| 3. |
- 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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| 4. |
- Avaliani, Natalia, et al.
(författare)
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Optogenetics reveal delayed afferent synaptogenesis on grafted human induced pluripotent stem cell-derived neural progenitors.
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:12, s. 3088-3098
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Tidskriftsartikel (refereegranskat)abstract
- Reprogramming of somatic cells into pluripotency stem cell state have opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSC), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for six weeks, lt-NES cell-derived neurons displayed neuronal properties such as TTX-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the It-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time-point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, e.g. such as epilepsy. Stem Cells 2014.
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| 5. |
- Canals, Isaac, et al.
(författare)
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Astrocyte dysfunction and neuronal network hyperactivity in a CRISPR engineered pluripotent stem cell model of frontotemporal dementia
- 2023
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Ingår i: Brain Communications. - 2632-1297. ; 5:3, s. 1-16
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Tidskriftsartikel (refereegranskat)abstract
- Frontotemporal dementia (FTD) is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro FTD disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of FTD astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development.
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| 6. |
- Martinez-Curiel, Raquel, et al.
(författare)
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Oligodendrocytes in human induced pluripotent stem cell-derived cortical grafts remyelinate adult rat and human cortical neurons
- 2023
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Ingår i: Stem Cell Reports. - 2213-6711. ; 18:8, s. 1643-1656
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal loss and axonal demyelination underlie long-term functional impairments in patients affected by brain disorders such as ischemic stroke. Stem cell-based approaches reconstructing and remyelinating brain neural circuitry, leading to recovery, are highly warranted. Here, we demonstrate the in vitro and in vivo production of myelinating oligodendrocytes from a human induced pluripotent stem cell (iPSC)-derived long-term neuroepithelial stem (lt-NES) cell line, which also gives rise to neurons with the capacity to integrate into stroke-injured, adult rat cortical networks. Most importantly, the generated oligodendrocytes survive and form myelin-ensheathing human axons in the host tissue after grafting onto adult human cortical organotypic cultures. This lt-NES cell line is the first human stem cell source that, after intracerebral delivery, can repair both injured neural circuitries and demyelinated axons. Our findings provide supportive evidence for the potential future use of human iPSC-derived cell lines to promote effective clinical recovery following brain injuries.
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| 7. |
- Martínez-Serrano, Alberto, et al.
(författare)
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Short-term grafting of human neural stem cells : Electrophysiological properties and motor behavioral amelioration in experimental Parkinson’s disease
- 2016
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Ingår i: Cell Transplantation. - 0963-6897. ; 25:12, s. 2083-2097
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Tidskriftsartikel (refereegranskat)abstract
- Cell replacement therapy in Parkinson’s disease (PD) still lacks a study addressing the acquisition of electrophysiological properties of human grafted neural stem cells and their relation with the emergence of behavioral recovery after transplantation in the short term. Here we study the electrophysiological and biochemical profiles of two ventral mesencephalic human neural stem cell (NSC) clonal lines (C30-Bcl-XL and C32-Bcl-XL) that express high levels of Bcl-XL to enhance their neurogenic capacity, after grafting in an in vitro parkinsonian model. Electrophysiological recordings show that the majority of the cells derived from the transplants are not mature at 6 weeks after grafting, but 6.7% of the studied cells showed mature electrophysiological profiles. Nevertheless, parallel in vivo behavioral studies showed a significant motor improvement at 7 weeks postgrafting in the animals receiving C30-Bcl-XL, the cell line producing the highest amount of TH+ cells. Present results show that, at this postgrafting time point, behavioral amelioration highly correlates with the spatial dispersion of the TH+ grafted cells in the caudate putamen. The spatial dispersion, along with a high number of dopaminergic-derived cells, is crucial for behavioral improvements. Our findings have implications for long-term standardization of stem cell-based approaches in Parkinson’s disease.
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| 8. |
- Palma-Tortosa, Sara, et al.
(författare)
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Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation
- 2022
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2022:190
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Tidskriftsartikel (refereegranskat)abstract
- Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.
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| 9. |
- Quist, Ella, et al.
(författare)
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Transcription factor-based direct conversion of human fibroblasts to functional astrocytes
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:7, s. 1620-1635
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Tidskriftsartikel (refereegranskat)abstract
- Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.
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