A 3D human neural cell culture system for modeling Alzheimers disease

A 3D human neural cell culture system for modeling Alzheimers diseaseAuthors: Young Hye Kim, Se Hoon Choi, Carla DAvanzo, Matthias Hebisch, Christopher Sliwinski, Enjana Bylykbashi, Kevin J Washicosky, Justin B Klee, Oliver Brstle, Rudolph E Tanzi & Doon Yeon KimIntroductionAlzheimers disease (AD)An age-related neurological disorderMemory loss and cognitive impairmentEarly-onset or familial forms of AD (FAD)Caused by mutations in genes encoding:Amyloid precursor protein (APP)Presenilin 1 (PESN1)Presenilin 2 (PESN2)Introduction (cont.)Sporadic AD result of environmental and genetic factorsSlight differences in types of AD, but it has two key points:1. presence of extracellular amyloid plaques (made up of A peptides, which are derived from APP)2. presence of neurofibrillary tangles (made up of aggregation of hyperphosphorylated tau (p-tau) proteins

3Introduction (cont.)Existing animal models could not show all aspects of AD pathologyInduced pluripotent stem cells (iPSCs) are more accurate models.Genetically engineered human neural stem cells that overexpressed FAD-related genes combined with 3D culture condition led to AD pathogenesis, including 2 of the previously mentioned hallmarks.A3Dcellcultureis an artificially-created environment in which biological cells are permitted to grow or interact with its surroundings in all three dimensions. This is an improvement over the previous method of growing cells in 2D (on a petri dish) because the3Dmodel more accurately models the in vivo cells.4ObjectivesTo describe a procedure for the production and analysis of 3D human neural culture models of Alzheimers diseaseTo prove that the 3D models are more accurate than 2D models because secreted As aggregation is disrupted by diffusion throughout the mediumDiffusion is less likely in 3D model due to Matrigel culture matrixWe specifically look at using 3D human neural cell cultures to generate genetically modified human neural progenitor cells (hNPCs) with FAD mutations; then we analyze the AD pathogenesis.Development of 3D human neural cell culture models of ADUsed immortalized hNPC cell line ReNcell VM (or ReN) because they can be maintained easily and for ~45 passages.ReN cells were transfected with vectors containing FAD-related genes encoding human amyloid precursor protein (APP) with the following, as well as GFP or mCherry as reporter for viral infection:Both the Swedish and London mutations (APPSL)PSEN1 with the E9 mutation (PSEN1(E9))APPSL/PSEN1

VectorsThe green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.

CMV = promoter63D culture Protocol

Cells then sorted by Fluorescence-activated cell sorting to enrich cells with highest expression levels.3D culture will help promote extracellular deposition of A.3D culture formats and data analysis(a) A 24-well culture plate generates thick-layer culture.Used for molecular, biochemical, and ELISA analyses.(b) 96-well plate, (c) 35-mm dish, and (d) 8- or 16-chambered cover-glass compartments generate thin cultures.Used for high-resolution immunofluorescent staining

ELISA: enzyme-linked immunosorbent assay8ResultsArrowheads indicate A aggregates.Immuno-histochemical (IHC) analysis of A deposition, detected by anti-A 40 BA27 antibodies, in control cells (a-c) and FAD ReN cells (d-f).(g-i) shows an enlargement of the images (d-f).

Immunohistochemistryor IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

9Results (cont.)(a-e)Confocal microscopy image showing the levels of phosphorylated Tau detected by the paired helical filament (PHF)-1 antibody in the ReN control cells.(a) ReN control cells(b-e) ReN cells after 3D differentiation in culture.

Enriched and control cells were 3D-differentiated for 8 weeks.

Conclusions3D Matrigel is an effective 3D seeding structure for A aggregation once the aggregation is initiated; however, it is still permeable to A species.Not as much as with 2D culture.Increase in p-tau levels and A42 levels compared to control cells were seen with iPSC-derived human neurons with either APP or PSEN1 FAD mutations.Limitations of Protocol3D culture model lacks human microgliaThey are essential to the processes involving clearance of A, brain inflammation, and synaptic and neural damage.The model is not specific for any particular area of the brain, including the ones most affected by AD (e.g. hippocampus).The model is more representative of the late stages of AD because it incorporates both A accumulation and hyperphosphorylation of tau.Early stages of AD show A accumulation without higher levels of p-tauP-tau = phosphorylated tau12ReferencesKim YH, Choi SH, D'Avanzo C, Hebisch M, Sliwinski C, Bylykbashi E, Washicosky KJ, Klee JB, Brstle O, Tanzi RE, Kim DY. A 3D human neural cell culture system for modeling Alzheimer's disease. Nat Protoc. 2015 Jul;10(7):985-1006. doi:10.1038/nprot.2015.065. Epub 2015 Jun 11. PubMed PMID: 26068894.