Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells

Brito, Catarina; Simão, Daniel; Costa, Inês; Malpique, Rita; Pereira, Cristina I.; Fernandes, Paulo; Serra, Margarida; Schwarz, Sigrid C.; Schwarz, Johannes; Kremer, Eric J.; Alves, Paula M.

doi:10.1016/j.ymeth.2012.03.005

Cited by 43 publications

(42 citation statements)

References 30 publications

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“…The model system here characterized has several applications ranging from drug screening to disease modeling (Brito et al, 2012). In vivo gene transfer using viral vectors is still the primary strategy for delivering novel genes to the CNS (Bjorklund and Kordower, 2010), and previous work performed by our group has proven the differentiated human neural aggregates' amenability to transduction by canine adenovirus derived vectors (CAV) (Brito et al, 2012). Our setup enables the visualization of the 3D network of the transduced cells inside the neural aggregates (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

“…In Figure 2C an aggregate was imaged with a 60× objective and the recorded image has been saturated to allow the visualization of neurites. Deep inside the sample, fluorescent spots are still detected, corresponding to neuronal somas, increasing the penetration depth up to 1.5-fold when compared to spinning-disk confocal microscopes (Brito et al, 2012). …”

Section: Resultsmentioning

confidence: 99%

“…All Human midbrain-derived neural progenitor cells (hmNPC) were differentiated as neural aggregates using stirred culture systems with orbital shaking, in the presence of morphogens and under low oxygen conditions, as previously described (Brito et al, 2012). …”

Section: Methodsmentioning

confidence: 99%

“…Transduction of differentiated neural aggregates with CAVGFP vectors (an E1-deleted CAV-2 vector expressing GFP) (Bru et al, 2010) was performed as previously described (Brito et al, 2012). Briefly, CAVGFP vectors were added to the culture, with 50% reduction of the working volume, according to the intended Multiplicity of Infection (MOI).…”

Section: Methodsmentioning

confidence: 99%

“…Here we describe the implementation of a SPIM/DSLM system and exploit its potentialities through a detailed characterization of differentiated human neural aggregates, derived from stem cells. A 3D strategy based on stirred systems was explored by our group for its potential to support CNS differentiation, resulting in a 3D CNS cell model composed of cells from the three neural lineages (neurons, astrocytes, and oligodendrocytes) (Brito et al, 2012). This has important applications ranging from drug screening to disease modeling and its coupling with the LSFM microscope developed represents one step forward in conceding the neural model system developed its full potential.…”

Section: Introductionmentioning

confidence: 99%

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Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Gualda

Simão

Pinto

et al. 2014

Front. Cell. Neurosci.

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The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Gualda

Simão

Pinto

et al. 2014

Front. Cell. Neurosci.

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The Mechanical Properties of Neurospheres

Okwara

Ghaemi

et al. 2021

Adv Eng Mater

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Stem cell‐derived 3D tissues such as spheroids are excellent models for investigating mechanisms of tissue formation and responses to physiological and mechanical cues. Neuronal spheroids, also known as neurospheres, have attracted particular interest. A lot is now known about the differentiation and maturation of neurospheres, as well as their responses to biochemical cues. However, understanding about their mechanical properties pales in comparison, which is all the more galling in light of newfound insights about how mechanical stimuli trigger the onset of neurodegenerative conditions. Herein, formative steps are taken to fill this knowledge gap. Neurospheres are generated from murine neural stem cells and are subjected to compressive forces. It is observed that neurospheres exhibit stress relaxation under static compression and viscoelastic behavior at low strains. The suitability of the Tatara model for characterizing the mechanical properties of neurospheres is also evaluated. The study is the first study of its kind to investigate the mechanical properties of in vitro 3D tissues. Moreover, the methodologies developed in the study can also be used to improve the quality and safety of cell and tissue biomanufacturing processes.

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Materials for Neural Differentiation, Trans‐Differentiation, and Modeling of Neurological Disease

et al. 2018

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Neuron regeneration from pluripotent stem cells (PSCs) differentiation or somatic cells trans-differentiation is a promising approach for cell replacement in neurodegenerative diseases and provides a powerful tool for investigating neural development, modeling neurological diseases, and uncovering the mechanisms that underlie diseases. Advancing the materials that are applied in neural differentiation and trans-differentiation promotes the safety, efficiency, and efficacy of neuron regeneration. In the neural differentiation process, matrix materials, either natural or synthetic, not only provide a structural and biochemical support for the monolayer or three-dimensional (3D) cultured cells but also assist in cell adhesion and cell-to-cell communication. They play important roles in directing the differentiation of PSCs into neural cells and modeling neurological diseases. For the trans-differentiation of neural cells, several materials have been used to make the conversion feasible for future therapy. Here, the most current applications of materials for neural differentiation for PSCs, neuronal trans-differentiation, and neurological disease modeling is summarized and discussed.

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Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells

Cited by 43 publications

References 30 publications

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

The Mechanical Properties of Neurospheres

Materials for Neural Differentiation, Trans‐Differentiation, and Modeling of Neurological Disease

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