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A 3D human neural cell culture system for modeling Alzheimer's disease.

Nature protocols (2015-06-13)
Young Hye Kim, Se Hoon Choi, Carla D'Avanzo, Matthias Hebisch, Christopher Sliwinski, Enjana Bylykbashi, Kevin J Washicosky, Justin B Klee, Oliver Brüstle, Rudolph E Tanzi, Doo Yeon Kim
ABSTRACT

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1-2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10-14 weeks.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-MAP2 Antibody, Chemicon®, from chicken
Sigma-Aldrich
ReNcell VM Human Neural Progenitor Cell LIne, ReNcell VM is an immortalized human neural progenitor cell line with the ability to readily differentiate into neurons & glial cells.
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Alzheimer′s In A Dish APPSL-GFP Lentivirus
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Ethidium bromide solution, BioReagent, for molecular biology, 10 mg/mL in H2O
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hEGF, EGF, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥99.0% (T)
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Anti-Microtubule-Associated Protein 2 (MAP2) Antibody, Chemicon®, from rabbit
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N-Lauroylsarcosine, purum p.a., ≥98.0% (GC)
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Congo Red, BioXtra, certified by the Biological Stain Commission
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Gelatin from bovine skin, Type B, powder, BioReagent, suitable for cell culture
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Donkey serum
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Alzheimer′s In A Dish PSEN1-RFP Lentivirus
Sigma-Aldrich
Sodium fluoride, ACS reagent, ≥99%
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Formic acid, reagent grade, ≥95%