Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays

Authors:
Hook L1, Vives J, Fulton N, Leveridge M, Lingard S, Bootman MD, Falk A, Pollard SM, Allsopp TE, Dalma-Weiszhausz D, Tsukamoto A, Uchida N, Gorba T.
In:
Source: Neurochemistry International
Publication Date: (2011)
Issue: 59 (2011): 432-444
Research Area:
Neurobiology
Culture Media:
Platform:
Nucleofector™ I/II/2b
Experiment

hNS can be efficiently transfected using nucleofection technology (Amaxa Nucleofector II, Lonza) using program G013 (chosen from various programs tested, namely A-033, C-013, G-013, O-003 and O-005; being G-013 the one that gave the brightest cells with normal morphology and higher transfection efficiency, 78% at 24 h post-nucleofection) with cells resuspended in ‘‘Basic Primary Neurons’’ solution (Lonza) and following the manufacturer’s instructions. 5  105–2.5  106 cells were transfected per reaction with Lonza’s pmaxGFP or linearized pCAGGFPIP reporter plasmid and pCAGbgeo-hCD2 fusion construct, or with anti-Nestin and anti-GFP siRNA.

Abstract

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.