Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Bob A. Hersbach; Tatiana Simon; Giacomo Masserdotti

doi:10.3791/64175

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Method Article

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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DOI:

10.3791/64175

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July 7th, 2022

Bob A. Hersbach¹^,²^,³ , Tatiana Simon² , Giacomo Masserdotti¹^,²

¹Institute of Stem Cell Research, Helmholtz Zentrum München, German Research Center for Environmental Health, ²Department of Physiological Genomics, Biomedical Center Munich, Ludwig-Maximilians University, ³Graduate School of Systemic Neurosciences, BioCenter, Ludwig-Maximilians University

Summary

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Abstract

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Introduction

The mammalian central nervous system (CNS) is highly complex, consisting of hundreds of different cell types, including a vast number of different neuronal subtypes¹^,²^,³^,⁴^,⁵^,⁶. Unlike other organs or tissues⁷^,⁸^,⁹, the mammalian CNS has a very limited regenerative capacity; neuronal loss following traumatic brain injury or neurodegeneration is irreversible and often results in moto....