Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

In This Article

Abstract

Source: Hersbach, B. A. et al., Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes. J. Vis. Exp. (2022)

This video showcases the direct reprogramming of astrocytes expressing transforming factors into neurons. Transformation factors trigger a cascade of molecular events facilitating astrocyte-to-neuron conversion. The process is further supported by supplying specific agents and supplements through a neuronal differentiation medium.

Protocol

1. Seeding of astrocytes for reprogramming

NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).

Prepare 24-well plates with poly-D-lysine coated glass coverslips in the same way culture flasks were prepared previously. To determine the number of plates needed, consider that astrocytes are plated at a density of 5-5.5 x 10⁴ cells per well in a 24-well plate. Usually, isolating six spinal cords from postnatal day 2 (P2) mice yields around 1 x 10⁶ cells.
Aspirate media from the T25 culture flasks containing the cultured astrocy....

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Results

Cell differentiation and maturation process diagram; images of stained cells in multiple stages.
Figure 1: Overview of astrocyte culture and reprogramming. (A)Timeline of astrocyte-to-neuron direct conversion. Each black line represents an important step in the protocol. (B) Representative brightfield images of cultured spinal cord-derived astrocytes a.......