Neural Stem Cells

This study was carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health. All procedures used were approved by the University of Texas
Southwestern Medical Center Institutional Animal Care and Use Committee APN
2007-0065. All efforts were made to minimize suffering.
Ascl1^CreERT2 knock-in mice were generated by
replacing the Ascl1 coding region with
CreER^T2[8] (link) and
Frt-Neo-Frt cassettes. The targeting strategy was the same used to generate
Ascl1^GFP knock-in mice [9] (link). The endogenous ATG was
replaced by a short sequence containing a PacI site and a consensus Kozak site.
The correct targeting event was identified by Southern analysis of EcoRI
digested DNA using 5′ and 3′ probes. After obtaining germ line
transmission in the Ascl1^{CreERT2-Frt-Neo-Frt} mice,
they were crossed with FLPe mice [10] (link) to remove the neomycin
cassette resulting in Ascl1^CreERT2 mice.
For PCR genotyping, the following primers were used: 5′-AAC TTT CCT CCG GGG CTC GTT
TC-3′ (Sense Ascl1 5′UTR) and 5′-CGC CTG GCG ATC CCT GAA CAT
G-3′ (Anti sense Cre) giving a PCR product of 247 bp.
Tamoxifen (TAM) induction of Cre recombinase was accomplished by intraperitoneal
injection of
Ascl1^CreERT2/+;R26R^YFP/YFPpostnatal day 50 (P50) mice with 180 mg/kg/day TAM (Sigma, T55648) in sunflower
oil on five consecutive days. Brains were harvested at the times specified after
TAM and processed as described [7] (link), [11] (link). R26R^YFP and
Nestin::GFP mice have been previously described [12] (link), [13] (link).
For immunofluorescence staining, free floating sections or sections mounted on
slides were incubated in the appropriate dilution of primary antibody in
PBS/3% donkey (or goat) serum/0.2% NP-40 (or 0.2% Triton
X-100), followed by appropriate secondary antibody conjugated with AlexaFluor
488, 568, or 594 (Molecular Probes). Mouse monoclonal antibodies used were:
Ascl1 (1∶750, RDI Fitzgerald, 10R-M106B), NeuN (1∶1000, Chemicon,
MAB377), GFAP (1∶400, Sigma, G3893). Rabbit polyclonal antibodies used
were: GFP (1∶500, Molecular Probes, A6455), GFAP (1∶500, DAKO,
Z0334), Ki67 (1∶500, Neomarker), Sox2 (1∶2000, Millipore). Goat
polyclonal antibodies used were: DCX (1∶200, Santa Cruz) and NeuroD1
(1∶200, Santa Cruz). Chick GFP (1∶500, Aves Lab) was also used.
Confocal imaging was carried out on a Zeiss LSM510 confocal microscope.
Ascl1⁺ fluorescence intensity levels were classified as high
or low using ImageJ and setting a threshold of pixel intensity for
Ascl1^Low (314–599 units) and Ascl1^High (>600
units). For cell number counts, three Nestin::GFP mice were
analyzed to place Ascl1⁺ progenitors in the adult neural stem
cell lineage. For in vivo genetic tracing experiments using the
Ascl1^CreERT2 knock-in line, at least two
Ascl1^CreERT2/+;R26R^YFP/YFPmice per each harvest time point (7, 30, or 180 days post-TAM) were used. For
co-localization data with each stage-specific marker, 150–500
YFP⁺ cells per animal were counted.

An existing protocol, based on the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, was adapted to generate neural progenitor cells (NPCs), which were then further induced to differentiate into neurons (not enriched in DAn) (Chambers et al, 2009 (link)). Briefly, the iPSCs were maintained in mTeSR™ medium until confluence. EBs were generated and maintained in suspension for 48 h. Subsequently, the medium was changed to a proneural medium (PN) and maintained for 5 days. PN medium consists of DMEM/F12 medium (GIBCO), neurobasal medium (GIBCO), 0.5× B27 minus vitamin A supplement (GIBCO), 0.5× N2 supplement (GIBCO), 2 mM Ultraglutamine (Lonza), β‐mercaptoethanol (Life Technologies), and penicillin–streptomycin (Lonza). To obtain neural rosettes, EBs were seeded on poly‐l‐ornithine/laminin (POLAM)‐coated plates with PN medium supplemented with Noggin (200 ng/ml; Peprotech) and SB431542 (10 μM; Tocris). After 8–12 days, the neural rosettes were picked and expanded in new POLAM‐coated dishes. Neural rosettes were enzymatically dissociated and seeded in new POLAM‐coated plates with PN medium supplemented with FGF2 (10 ng/ml; Peprotech) and EGF (10 ng/ml; Peprotech) to promote the generation and proliferation of neural progenitor cells (NPCs). After forming a homogenous cell population, the NPCs were further differentiated into GABA and glutamatergic neurons on POLAM‐coated plates with PN medium during 3–5 weeks.