The Complete Hedgehog, Vol. 2 LINK
Proliferation in response to mitogens and the ability to self-renew are defining properties of NPCs. All proliferation experiments were performed in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF). A bromodeoxyuridine (BrDU) assay was used to determine the effects of Fas deficency on NPC proliferation. Lpr NPCs showed significantly less BrDU incorporation over 16vhours in complete media compared to wt NPCs. Lpr NPCs had 18% 1.6 less proliferation compared to wt (Figure 2(a)).
The Complete Hedgehog, Vol. 2
Taken together, we propose the following paradigm for the role of Fas in NPCs. Fas targeting leads to two unique physiological processes: first, Fas ablation promotes NPC survival in deprived-environments and second, Fas ablation promotes increased neuronal and oligo differentiation. Coupled together, Fas manipulation may produce a viable means of exogenous stem cell transplantation by overcoming the challenge of cell survival under harsh transplantation conditions, while promoting complete integration of NPCs into functional neuronal circuits by stimulation of NPC differentiation. The Shh signaling pathway, or other molecules identified here, may be a useful target in promoting this response. This paradigm offers novel candidates deserving further investigation because of their potential to advance NPC transplantation therapies.
NPCs were isolated from whole brains dissected from 4-day postnatal wild-type C57/BL6 or lpr mutant mice (C57/BL6 background). Brains were processed using NeuroCult® Enzymatic Dissociation Kit for CNS Tissue (Stem Cell Technologies). Resulting single-cell suspension was plated in EGF and FGF (both 10ng/mL) containing complete media (Gibco®Neural Basal Media supplemented with B27 and 1:1:2). Neurospheres were chemically dissociated (Kit, Stem Cell Technologies) into a single-cell suspension and plated on poly-D Lysine and laminin (both Sigma) coated Nunc® flasks or plates for passaging and experimentation. Cells were plated at a density of 16,000 cells/mL media and grown to confluency (3 - 4 days) before experimentation or switching to minimal media. All experiments were performed on passage-matched wt or lpr NPC lines and only cells with passage numbers between 3 and 6 were used. Proliferation studies were performed in EGF and FGF containing media, cell death studies and early differentiation timepoints were performed in minimal media (Neural Basal Media with only B27 and 1:1:2 [penicillin/streptomycin: L-glutamine: glucose]), and extended differentiation experiments were performed in minimal media containing 0.2% FCS (to prevent extensive cell death in the absence of growth factors). All cells incubated in 37C/5% CO2 conditions.
Death & Apoptosis Assay (UV/Annexin stain): UV Live/Dead Dye® and Annexin Alexa Fluor-647 both used according to manufacturer instructions (Invitrogen). UV dye labels dead cells because of disruptions in the cell membrane. Viable cells to not stain positive for UV dye because of an intact cell membrane. Annexin binds strongly to phosphatidylserine residues that have flipped to the exterior surface of the cell, thus labeling early apoptotic cells. Briefly, 200 µL of diluted UV dye (2 µL per mL 1 PBS) was added to cell suspension containing 10,000 cells/µL. Cells were incubated on ice, in the dark, for 30 min. After cells with 1 PBS and aspirating the supernatant, 100 uL of diluted Annexin (1 µL per 400 µL HEPES buffer) was added to cells. Cells grown in complete media (growth factors present) were used as a negative control because >90% of cells in these cultures are live/healthy. Cells briefly treated with 3% paraformaldehyde were used as positive controls because >90% of cells are dead/dying.
Single-cell suspensions were plated in uncoated 24-well plates (seeding density of 100,000 cells/mL) using NPC media containing EGF and FGF. After 7 days, 2-3 pictures of random frames from each well (3 wells per cell type) were taken at 20X using a phase-contrast microscope. After taking pictures, neurospheres were dissociated using Neurocult® Chemical Dissociation Kit and then filtered through a 40 µm nylon cell strainer (BD FalconTM). Cells were then counted, plated as described above, and grown for 7 days before the next passage time point was recorded (this same process completed four total times over four weeks). Exact cell circumferences were measured using Neurolucida software.
Performed as previously described . Briefly, RNA was extracted from cells using RNeasy® Mini Kit (Qiagen) according to manufacturer protocol. For RT-qPCR, cDNA was prepared using Superscript® III First-Strand Synthesis Supermix for qRT-PCR. RT-qPCR completed using Taqman® Master Mix and applied Biosystems 7500 Fast Software. Primers were purchased from Applied Biosystems (Taqman Assay on Demand). 041b061a72