Abstract
Lineage reprogramming of astroglial cells isolated from different brain regions leads to the generation of different neuronal subtypes. This protocol describes the isolation and culture of neocortical and cerebellar astrocytes from postnatal mice. We also present a comprehensive description of the main steps towards successful gene delivery in these cells using nucleofection. Neocortex and cerebellum astrocyte cultures obtained with these methods are suitable for the study of molecular and cellular mechanisms involved in direct cell lineage reprogramming into induced neurons (iNs).
Keywords: Primary cell culture, Murine astrocytes, Nucleofection, Cell lineage reprogramming
Background
Astrocyte culture has been extensively described in the literature (Saura, 2007; Schildge et al., 2013). Several protocols, mostly differing in the number of steps for cell isolation, yield astrocyte cultures with sufficient purity (up to 98%). However, most protocols described to date focus on the isolation of astrocytes from the neocortex and use chemical dissociation (Schildge et al., 2013). Here, we provide an alternative method to generate highly enriched astrocyte monolayers without the necessity of chemical tissue dissociation, what makes the protocol rather faster as compared to previous methods. Additionally, we also describe the isolation and culture of neocortical and cerebellum astrocytes, highlighting some important differences between these two cell populations. Finally, we describe an alternative, cost-effective approach for gene delivery in astrocytes using nucleofection (Chouchane et al., 2017). This technique consists of the direct delivery of DNA molecules following electroporation of the cells using a specific voltage and reagent. Nucleofection of proneural genes into astrocytes is a cheap, fast and relatively efficient method that leads to similar results as compared to retroviral-mediated transfection (Heins et al., 2002; Berninger et al., 2007; Heinrich et al., 2012; Chouchane et al., 2017). It also presents a great advantage as quiescent astrocytes are easily targeted, hence bypassing the precondition of having dividing cells.
Materials and Reagents
Equipment
Software
Procedure
Note: The following protocol should be held in a sterile environment. Familiarity with basic cell culture is expected.
Data analysis
Quantification of neuronal reprogramming and iNs phenotype in vitro must be performed in at least three independent batches of cell culture. However, the exact sampling size must be determined for each experiment considering the effect size, variation and statistical power. Statistical tests can be performed using GraphPad Prism version 5.00 for Windows or other statistic programs. The choice of the statistical test must be determined according to the experimental design. For examples of possible analyses using the astrocyte culture described herein, please refer to Chouchane and coworkers (2017) (https://www.ncbi.nlm.nih.gov/labs/articles/28602612/).
Recipes
Acknowledgments
The National council for scientific and technological development (CNPq) supported this work. The current protocol was adapted from Heins et al., 2002 and Berninger et al., 2007. The authors declare no conflicts of interest or competing interests.
References
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