Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells

Abstract

Although new culture conditions enable homogeneous and long-term propagation of radial glia-like neural stem (NS) cells in monolayer and serum-free conditions, the efficiency of the conversion of NS cells into terminally differentiated, functionally mature neurons is relatively limited and poorly characterized. We demonstrate that NS cells derived from adult mouse subventricular zone robustly develop properties of mature neurons when exposed to an optimized neuronal differentiation protocol. A high degree of cell viability was preserved. At 22 days in vitro, most cells (65%) were microtubule-associated protein 2 þ and coexpressed c-aminobutyric acid (GABA), GAD67, calbindin and parvalbumin. Nearly all neurons exhibited sodium, potassium and calcium currents, and 70% of them fired action potentials. These neurons expressed functional GABA A receptors, whereas activable kainate, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartic acid receptors were present in approximately 80, 30 and 2% of cells, respectively. Antigenic and functional properties were efficiently and reliably reproduced across experiments and cell passages (up to 68). This is the first report showing a consistent and reproducible generation of large amounts of neurons from long-term passaged adult neural stem cells. Remarkably, the neuronal progeny carries a defined set of antigenic, biochemical and functional characteristics that make this system suitable for studies of NS cell biology as well as for genetic and chemical screenings.

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments

“…The adult SVZ-derived NS cell line aNS-1 was derived from the SVZ of a 2-month-old wild-type (CD1 strain) mouse, as described by Goffredo et al [11]. For routine passaging, all NS cells were dissociated with Accutase (Sigma) and split 1:4 or 1:5 every 2-3 days.…”

“…Seals between the electrode and the cell were established in a bath solution containing 155 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 3 mM KCl and 10 mM HEPES/NaOH (pH 7.4). Patch pipettes were filled with 128 mM KCl, 10 mM NaCl, 11 Whole-cell currents and potentials were recorded using an Axopatch 200B amplifier (Axon Instruments, Burlingame, CA), digitized at sampling intervals of 10-50 ls using a DigiData 1322A AD/DA converter (Axon Instruments). Stimulation, acquisition, and data analysis were carried out using pCLAMP (Axon Instruments) and ORIGIN software (Microcal Software, Northampton, MA).…”

“…Fetal NS cells were compared to NS cells derived from the SVZ of adult brain (aNS-1) [9,11] and to ESC-derived NS 46C CAG and NS R1 cell lines [8,22] (Fig. 1a).…”

“…To test the neural differentiation ability of fetal NS cells, we set up a new one-step protocol (Fig. 4a) because of the limited differentiation and reduced survival using the protocol developed for the ESC-derived NS cells [22] or aNS-1 [11] (not shown). The protocol involved EGF withdrawal and a gradual decrease in FGF-2, with a simultaneous increase in BDNF in the differentiation medium up to a final concentration of 30 ng/ml (Fig.…”

“…They can be derived from embryonic stem cells (ESCs), as well as from fetal and adult mouse brain, and remain highly neurogenic even after long-term expansion in vitro ([100 passages). Interestingly, NS cells derived from the adult subventricular zone (SVZ), exposed to an optimized differentiation protocol, consistently and reproducibly differentiate into mature GABAergic neurons, most of which can fire action potentials and express functionally active neurotransmitter receptors [11]. Furthermore, ESC-derived NS cells, subjected to a newly developed neuronal differentiation protocol, exhibit an 85% yield of molecularly and electrophysiologically mature neurons in the presence of limited cell death [22].…”