Cellular Analysis of Adult Neural Stem Cells for Investigating Prion Biology

Abstract

Traditional primary and secondary cell cultures have been used for the investigation of prion biology and disease for many years. While both types of cultures produce highly valid and immensely valuable results, they also have their limitations; traditional cell lines are often derived from cancers, therefore subject to numerous DNA changes, and primary cultures are labor-intensive and expensive to produce requiring sacrifice of many animals. Neural stem cell (NSC) cultures are a relatively new technology to be used for the study of prion biology and disease. While NSCs are subject to their own limitations-they are generally cultured ex vivo in environments that artificially force their growth-they also have their own unique advantages. NSCs retain the ability for self-renewal and can therefore be propagated in culture similarly to secondary cultures without genetic manipulation. In addition, NSCs are multipotent; they can be induced to differentiate into mature cells of central nervous system (CNS) linage. The combination of self-renewal and multipotency allows NSCs to be used as a primary cell line over multiple generations saving time, costs, and animal harvests, thus providing a valuable addition to the existing cell culture repertoire used for investigation of prion biology and disease. Furthermore, NSC cultures can be generated from mice of any genotype, either by embryonic harvest or harvest from adult brain, allowing gene expression to be studied without further genetic manipulation. This chapter describes a standard method of culturing adult NSCs and assays for monitoring NSC growth, migration, and differentiation and revisits basic reactive oxygen species detection in the context of NSC cultures.

Prion protein N1 cleavage peptides stimulate microglial interaction with surrounding cells

“…NSC cultures were used to differentiate mature cultures containing neurons, astrocytes and oligodendrocytes. Differentiation of NSCs results in mature cells appearing in 4-6 days 35,36 with progenitors negligible (<1%) by 5 days 36 and markers of mature cells are strongly detected by 7 days within these cultures 31 . The mature cultures do not differentiate equal populations but contain ~80% astrocytes, <20% neurons and <5% oligodendrocytes [37][38][39] .…”

“…Dichlorofluorescein diacetate (DCFDA) assay. The DCFDA assay has been described in detail previously 31 . Briefly, cells were loaded with CM-H 2 -DCFDA by incubating in a 5 µM in PBS working solution for 20 minutes at 30 °C in the dark.…”

A Purpose-Built System for Culturing Cells as In Vivo Mimetic 3D Structures

“…Stem cells, including induced pluripotent stem cells appear to need a wide variety of times -some need only a few days while others never fully recover in vivo functionality [27,28]. As a general rule primary cells seem to retain (most of) their functionality but use some of their mortal time to re-establish partial tissue organisation [29,30,31].…”