The neurosphere assay: an effective in vitro technique to study neural stem cells

Neural stem cells (NSCs) are known to be present in the adult mammalian brain where they constitutively differentiate into neuronal, astroglial, and oligodendroglial lineages, defined processes termed neurogenesis, astrogliogenesis oligodendrogenesis, respectively (reviewed Braun Jessberger, 2014). During development, NSCs throughout brain, becoming progressively restricted regions. In mainly areas classically as neurogenic niches, i.e. subventricular zone (SVZ), along lateral walls of ventricles, subgranular zone, located dentate gyrus (DG) hippocampus. These particularly enriched with NSCs, which not only multipotent but also proliferative ability self-renew, thus maintaining their own pool cells. fact, oligodendrogenesis highly intricate comprising several steps, including proliferation, differentiation, migration, functional integration newly formed existing circuitry, regulated by a plethora factors. differentiated adult-born have capacity continuously modulate function plasticity, constantly reacting external or internal stimuli 1960s, Altman Das (1965) described for first time presence dividing were later demonstrated neural cells-derived neurons. Ever since its description, neurogenesis has been extensively studied physiological pathological contexts, being considered promising therapeutic avenue both neurological neuropsychiatric disorders (Braun Physiologically, SVZ contributes olfactory system whereas injury conditions (e.g. stroke), newborn neurons may repopulate from striatum cortex (Sawada et al., Beyond can give rise oligodendrocyte precursor contexts multiple sclerosis), these able migrate damaged sites, helping restore myelin sheaths (Xing hippocampus, plays critical role learning, memory cognition, well mood regulation response stress, affected neurodegenerative Alzheimer’s disease) depression) disorders. particular, DG support hippocampus-dependent involved reorganization circuits Apart originate astrocytes, play fundamental roles synaptic activity plasticity (Steiner 2004). Undoubtedly, assessing behavior vast number repair strategies tested myriad and/or disorders, specifically replenishing either astroglial loss disfunction. Importantly, vivo vitro techniques developed further explore NSC biology. Recently, our group potential an technique established more than two decades ago, neurosphere assay (NSA) obtain high yields main niches study inherent properties NSCs. The NSA is simple, yet effective, method used renewable source undifferentiated that easily expanded culture (Soares 2020). Firstly Reynolds Weiss (1992), constitutes very useful biology dynamics, bypassing some deterring issues use animal models, namely large necessary animals, exposure animals toxic substances, limited amount obtained tissue. powerful allows studying controlled conditions. Cells cultured chemically serum-free medium specific growth factors, epidermal factor basic fibroblast factor- factor-responsive and, when exposed mitogens, enter period active while do survive conditions, grow spherical aggregates called neurospheres passaged expand Indeed, we primary composed positive markers, SOX2 Nestin. expansion precise selection using this obtaining higher yield clones tissue, reducing cells, grown characteristic sphere-shaped structures suspension, seeded withdrawn mitogens medium, form pseudo-monolayer heterogeneous out neurosphere, three lineages 2020; Figure 1).Figure 1: Schematic representation assay, applications limitations.Isolation SVZ- DG-derived 450 μm coronal sections containing ventricles respectively. factors suspension. Cell passage performed times, step dissociation neurospheres. self-renewal cell-pair assays access For beyond marker, it another Nestin marker doublecortin immature neurons). Primary plated coated plates without allowing differentiation neurons, oligodendrocytes astrocytes. differentiative cell survival, proliferation performed. Some limitations displayed on bottom figure. DG: Dentate gyrus; SVZ: zone.The phylogenetically distinct groups proved exist, such mammals mouse, rat human), fish, fruit fly birds Chapouton 2007; Fernández-Hernández 2013) viability neuronal glial contexts. capability evaluate postnatal investigate neurodevelopment at cellular molecular levels. Moreover, genetically modified models disease understand mechanisms regulate those processed analysis RNA protein expression, immunolabelling expression electrophysiological studies single-cell calcium-imaging heterogeneity makes potent tool mimic microenvironment niche. there methods associated different NSC, particular stemness (self-renewal assay), (proliferation (differentiation survival (cell assay) 1). investigating actions intrinsic extrinsic (Figure All well-established advantages simplicity technique, compared other protocols monolayer system), reason why methodology widely many researchers areas. Recent suggested exists regions hypothalamus, amygdala, septum, cortex, spinal cord substantia nigra Jurkowski 2020) dorsal vagus complex, optic nerve auditory (Charrier 2006; Völker 2019; Bernstein still under debate, evidence supporting idea endogenous pools non-canonical others migration classic novel sites. Notably, screened together evaluation markers (SOX2 Nestin), (BrdU, thymidine analogue) multipotency (TUJ1, O4 GFAP, respectively) (Völker characterize sub-regions rodent hypothalamus terms potential. Additionally, progenitor isolated human A recent mice precursors basolateral amygdala proliferate neurosphere-like shape mature Interestingly, rodents identified vagal pathway lamina region revealing through Although generated corroborate hypothesis existence areas, needs explored vivo. This versatile adapted according experimental protocol. variations applied fields cancer biology, cardiology enteric nervous generate tumorspheres, cardiospheres enterospheres, was firstly described, sphere-formation published types Deleyrolle 2008). relevant derived identify region-specific processes, Firstly, does take account quiescent fact self-renewal. Therefore, counting accurate measure determine properties. However, limitation overcome simultaneously measuring diameter, given progenitors, due reduced stem-like smaller On hand, direct correlation between difficult aggregate. possibility aggregation proportional density. solve problem, single passaging, advantageous increase yield, decreases passages concentrations shown deregulate spatial identity (Hack although completely bi-dimensional, might represent limitation, improved three-dimensional systems similar Finally, despite simplicity, requires experienced researcher fine tune all steps protocol, resulting frequency dependent dissection procedure, process, components interfere neurosphere-forming capability. SVZ-derived rate (Seaberg van der Kooy, 2002). similarly what happens vivo, differences DG. example, generating much (Palmer 1997). Consequently, preserves lineage, showing potency. Furthermore, substrate essential guarantee good neurospheres, compromising influence type predominantly during promotion versus differentiation) shows opportunity composition neurosphere-derived migration. Overall, contributed evolution new area research, regenerative neurobiology ultimately, challenged helped “no neurogenesis” dogma Ramon y Cajal. broadened understanding underlying opening door designing innovative therapeutics. More 25 years passed then, subjected intense scrutiny validated viable technique. It now proven model demonstrate behind dynamics work supported IF/01227/2015 UID/BIM/50005/2019, projeto financiado pela Fundação para Ciência e Tecnologia (FCT)/Ministério da Ciência, Ensino Superior (MCTES) através de Fundos Orçamento Estado. RS (SFRH/BD/128280/2017), FFR (IMM/CT/35-2018), DML (PD/BD/141784/2018), RSR (SFRH/BD/129710/2017) received fellowship FCT.