| 1. |
- Ahlenius, Henrik, et al.
(författare)
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Isolation and generation of neurosphere cultures from embryonic and adult mouse brain.
- 2010
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 633, s. 241-252
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Tidskriftsartikel (refereegranskat)abstract
- Neural stem cells are defined as cells that either gives rise to or derives from the cells of the central nervous system and have the unique properties of stem cells, i.e. self-renewal and multipotentiality. One of the widely used methods of expanding neural stem cells under culture conditions is based on the capacity of these cells to divide continuously when cultured in serum-free medium supplemented with various growth factors. One common method used is to grow neural stem cells as free-floating aggregates of cells called neurospheres. Neurospheres can be generated from several structures of the embryonic and adult mammalian brain. Although viable lines can be generated from crude extracts of brain, a precise dissection is crucial to get a pure population of cells. Here we describe methods for dissection, isolation and generation of neurospheres from embryonic ganglionic eminences and adult subventricular zone of mice and rats.
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| 2. |
- Canals, Isaac, et al.
(författare)
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CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders
- 2021
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Ingår i: Neural Reprogramming : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616017 - 9781071616000 ; 2352, s. 237-251
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Bokkapitel (refereegranskat)abstract
- Recent advances in genome editing have brought new hopes for personalized and precision medicine but have also dramatically facilitated disease modeling studies. Combined with reprogramming approaches, stem cells and differentiation toward neural lineages, genome engineering holds great potential for regenerative approaches and to model neurological disorders. The use of patient-specific induced pluripotent stem cells combined with neural differentiation allows studying the effect of specific mutations in different brain cells. New genome editing tools such as CRISPR/Cas9 represent a step further by facilitating the introduction or correction of specific mutations within the same cell line, thus eliminating variability due to differences in the genetic background. Here, we present a step-by-step protocol from design to generation of human pluripotent stem cell lines with specific mutations introduced or corrected with CRISPR/Cas9 gene editing that can be used in combination with transcription factor-based protocols to dissect underlying mechanisms of neurological disorders.
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| 3. |
- Hansen, Marita Grønning, et al.
(författare)
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In Vitro Functional Characterization of Human Neurons and Astrocytes Using Calcium Imaging and Electrophysiology
- 2019
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer New York. - 1940-6029. ; 1919, s. 73-88
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Tidskriftsartikel (refereegranskat)abstract
- Recent progress in stem cell biology and epigenetic reprogramming has opened up previously unimaginable possibilities to study and develop regenerative approaches for neurological disorders. Human neurons and glial cells can be generated by differentiation of embryonic and neural stem cells and from somatic cells through reprogramming to pluripotency (followed by differentiation) as well as by direct conversion. All of these cells have the potential to be used for studying and treating neurological disorders. However, before considering using human neural cells derived from these sources for modelling or regenerative purposes, they need to be verified in terms of functionality and similarity to endogenous cells in the central nervous system (CNS).In this chapter, we describe how to assess functionality of neurons and astrocytes derived from stem cells and through direct reprogramming, using calcium imaging and electrophysiology.
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| 4. |
- Quist, Ella, et al.
(författare)
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Transcription Factor Programming of Human Pluripotent Stem Cells to Functionally Mature Astrocytes for Monocultures and Cocultures with Neurons
- 2021
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Ingår i: Neural Reprogramming: Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616017 - 9781071616000 ; 2352, s. 133-148
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Bokkapitel (refereegranskat)abstract
- Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.
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