A comparison of proliferative capacity and passaging potential between neural stem and progenitor cells in adherent and neurosphere cultures

Abstract

Neural stem and progenitor cells (NSPCs) can be isolated from the fetal or adult brain and expanded in culture for potential use in basic research, drug discovery and cell therapy. In the present study, two culture systems have been commonly used to maintain and expand NSPCs isolated from mammalian CNS: neurosphere and adhesive substrate-bound monolayer culture. NSPCs were isolated from the neuroepithelium of E14 embryonic rat cerebral cortex and maintained and expanded on fibronectin substrates or within neurospheres in serum-free medium. Ultrastructural study under transmission electron microscope revealed similar characteristics of immature morphology of NSPCs in adherent and neurosphere cultures. NSPCs cultured on adherent substrates and within neurospheres shared the properties of self-renewal and multipotency, but little is known about proliferation capacity and passaging potential of adherent NSPCs compared to neurosphere culture. We found that the self-renewal capacity of NSPCs in adherent culture was higher than that in neurosphere culture in the P1 and P3 passages, and reduced after the P5 passage. At the same time, comparative analysis using BrdU incorporation and immunostaining for nestin indicated that NSPCs grew significantly faster in primary cultures on adherent substrates than within neurospheres. Whereas, NSPCs in adherent culture could not maintain such robust growth for more than 6 passages. The growth of NSPCs within neurospheres was slower than that in adherent culture, but increased steadily and could be maintained for more than 10 passages. These data provide useful information for large scale in vitro expansion of NSPCs required by potential drug screening and cell therapy.

Later Passages of Neural Progenitor Cells from Neonatal Brain Are More Permissive for Human Cytomegalovirus Infection

“…Ex vivo expression of NPC-specific markers is maintained for longer than 20 weeks (50,51). However, the capacity of NPCs to proliferate decreases with increasing culture time/number of passages, suggesting that extended passage in vitro may induce differentiation.…”

IGF-1 Acts as Controlling Switch for Long-term Proliferation and Maintenance of EGF/FGF-responsive Striatal Neural Stem Cells

“…NSCs are represented by expression of Nestin; a most prominent signature associated with neural precursors of central nervous system 32. Immunocytochemical staining of NSCs was performed to characterise the isolated cell population.…”

“…NSCs are characterised as undifferentiated cells with the capacity to self-renew (while maintaining an undifferentiated state) that can give rise to all neural lineages: including neurons, astrocytes and oligodendrocytes 3, 8, 32, 36, 41. The major pitfall that has been encountered till now in growing NSCs is their long-term maintenance in vitro, due to unavailability of optimal guidance cues for their microenvironmental niche.…”

Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse

“…However, spontaneous differentiation has been seen in mouse NSCs maintained as monolayer cells (based on our own observation). Thus although both neurosphere and adherent culture systems have pros and cons 7 , neurospheres, which provide a three-dimensional environment, may be better for maintenance of NSCs 8 . Therefore, neurosphere culture remains the most popular and reliable method for isolating and maintaining adult NSCs.…”