3D cell culture technologies aim to provide improved predictive cellular models for research, drug discovery or regenerative medicine applications. Historically, In vitro human cell models of Alzheimer’s disease (AD) have been challenging due to high levels of soluble and insoluble toxic amyloid β (Aβ) species that do not recapitulate the true AD pathology. Recently, Kim et. al created a three-dimensional (3D) human neural stem cell model of Alzheimer’s Disease using β-amyloid precursor protein and presenilin-1 overexpressing ReNcell™ VM human neural stem cell lines. This 3D cell model was able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, and high levels of phosphorylated tau in the soma and neurites, as well as filamentous tau. This model is a valuable tool to study age-related AD dementia.1,2,3
ReNcell lines are highly published human neural stem cell lines derived from developing human brains. ReNcell® VM and CX cells are generated from the ventral mesencephalon and cortical regions of the brain, respectively, and transduced with the myc transcription factor. Both cell lines offer phenotype and genotype stability, in addition to the multipotential neuronal and glial differentiation capacity over long-term culture.
Figure 1. Fluorescence images of ReNcell VM infected with APPSL-GFP (A) or PSEN1-RFP (B) Lentivirus, MOI =20. By day 3 post viral transduction >80% ReNcell VM express the Alzheimer’s associated transgenes.
Figure 2. Alzheimer’s In a Dish™ clonal FAD ReNcell® VM human neural stem cell lines.Alzheimer’s in a Dish™ is a proprietary collection of immortalized single cell derived ReNcell® VM human neural progenitor cells that express stable levels of fluorescently tagged AD genes with multiple FAD mutations. The clonal FAD neural progenitor cells secrete different amounts of total Aβ and differ in the Aβ42/40 ratios. All clonal FAD cell lines express neural stem cell markers Nestin (B) and Sox-2 (C). For example: the ReN-mGAP10 Clone D4 neural progenitor cells (SCC008FAD2) were double infected with polycistronic lentiviruses expressing APP (Swe/Lon)-GFP, (D) PS1 (deltaE9)-mCherry (E), merged images (F).
Figure 3. A) Anti-β-Amyloid antibody staining (MAB348) staining on Alzheimer’s diseased cells. Immunoreactivity is seen as staining on plaque deposits (dark brown). B) Anti-Phospho Tau staining (AB9668) of Alzheimer’s diseased cells. Immunoreactivity is clearly not nuclear and it follows the length of the neuron’s axon.
Figure 4. Analysis of amyloid aggregates in 3D culture by Congo red staining. Clonal FAD ReNcell NSCs (SCC008FAD2, SCC008FAD3, SCC008FAD5) were 3D differentiated for 7 weeks and extracted with mild detergent. The pellet (insoluble) fractions were resuspended in PBS, loaded and fixed in glass slides and stained with 1% Congo red solution (HT60).