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Adult neural precursor cells are an integral part of the brain and have been a focus of intense research for half a century. Even though adult neural stem/progenitor cells in the human brain have been identified over 20 years ago, progress in this area has been slow, and the existence of lifelong neurogenesis in humans is still debated. Remarkable species differences exist between humans and rodents in astrocyte development and diversity, suggesting similar differences may exist in neural stem/progenitor cells and underscoring the need for further research in human tissue.This chapter provides a guideline for dissociation of adult human brain tissue from biopsy or autopsy specimens. While protocols for subsequent culturing of neural precursors are included, the main focus is the preparation of a suspension of viable single cells that may also be useful in other experimental paradigms, such as genomic profiling., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Adult neural precursor cells are an essential part of the brain, and a focus of two decades of intense research (Ming and Song, Neuron 70:687-702, 2011). Even though adult human stem/progenitor cells have been identified early on (Kirschenbaum et al., Cereb Cortex 4:576-589, 1994; Eriksson et al., Nat Med 4:1313-1317, 1998), progress in the field of adult human neurogenesis has been slow. The reasons for this may be more advanced neighboring fields of pluripotent stem cell research, and lacking study material as well as well-established and standardized protocols. Furthermore, adult precursor cells in humans seem to have greater potential than in rodents (Walton et al., Development 133:3671-3681, 2006). This may be attributed to species differences in astrocyte development and diversity (Oberheim et al., Neurosci 29:3276-3287, 2009). In this chapter, we provide a guideline for adult human brain tissue dissociation, be it from biopsy or autopsy specimens. This is by no means the only way of culturing adult neural precursor cells, but it may help in streamlining research on this fascinating topic, as well as help introducing others into this field. We describe our methodology for establishing and maintaining long-term cultures from white and grey matter, as well as a simple protocol for differentiating these cells.

It has been thought for a long time that the adult brain is incapable of generating new neurons, or that neurons cannot be added to its complex circuitry. However, recent technology has resulted in an explosion of research demonstrating that neurogenesis, or the birth of new neurons from adult stem cells constitutively occurs in two specific regions of the mammalian brain; namely the subventricular zone and hippocampal dentate gyrus. Adult CNS stem cells exhibit three main characteristics: (1) they are "self-renewing," i.e., they possess a theoretically unlimited ability to produce progeny indistinguishable from themselves, (2) they are proliferative (undergoing mitosis) and (3) they are multipotent for the different neuroectodermal lineages of the CNS, including the different neuronal, and glial subtypes. CNS stem cells and all progenitor cell types are broadly termed "precursors." In this chapter, we describe methods to identify, isolate and experimentally manipulate stem cells of the adult brain. We outline how to prepare a precursor cell culture from naive brain tissue and how to test the "stemness" potential of different cell types present in that culture, which is achieved in a three-step paradigm. Following their isolation, stem/progenitor cells are expanded in neurosphere culture. Single cells obtained from these neurospheres are sorted for the expression of surface markers by flow cytometry. Finally, putative stem cells from cell sorting will be subjected to the so-called neural colony-forming cell assay, which allows discrimination between stem and progenitor cells. At the end of this chapter we will also describe how to identify neural stem cells in vivo.