High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord.

Marie-Josée Beaudet,Sébastien Cadau,Mathieu Blais,François Gros-Louis,Sabrina Bellenfant,Qiurui Yang,François Berthod

doi:10.1038/srep16763

Abstract

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases.

Highlights

In order to extract at the same time motor neurons (MNs), astrocytes and microglia from mouse embryos, we have used in the past a discontinuous density gradients system[5]
An optimized and simplified approach using this purification method allowed us to obtain higher recovery rates of purified embryonic MNs extracted from one single embryo
This number was increased to 2 million MNs and 891,000 glial cells by collecting E14.5 mouse embryonic spinal cords (48 embryonic mouse spinal cords were isolated) while 2.7 million MNs and 855,000 glial cells in average were collected from Hxlb9-GFP transgenic E14.5 embryos (57 embryonic mouse spinal cords were isolated) (Table 1)

Summary

Introduction

Attempts have been previously described for time-consuming MNs extraction protocols from single embryos using immune-affinity purification These protocols typically use a p75 (NTR)-antibody-based cell-sorting panning technique, p75 being an extracellular protein exclusively expressed in the spinal cord by MNs8–11. This method yielded up to 80% pure spinal MNs, it was not possible to isolate more than 15 000 cells from individual embryo[10]. The possibility to extract, purify and culture adult astrocytes and microglia from mice at different specific disease stages would be essential to better understand neurodegenerative mechanisms. The possibility to extract, purify and culture adult astrocytes from old mice at different specific disease stages such as in ALS would be extremely valuable to better understand neurodegenerative mechanisms. Most protocols for adult microglia extraction have been designed for the brain, instead of the spinal cord[26,31]

Objectives

Results

Conclusion