Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture

doi: 10.3389/fcell.2021.708389

Figure Lengend Snippet: Viability of hiPSCs encapsulated and differentiated in alginate of different compositions. (A) The induction of early neuroectoderm differentiation from hiPSCs was achieved by dual SMAD inhibition, followed by the activation of SHH, Wnt, and FGF8 signaling pathways for patterning the midbrain fate. The committed neural progenitor cells were terminally differentiated into DA neurons by withdrawal of key neurogenic factors (BAGTCD). SM, StemMACS medium; RI, Rho associated kinase inhibitor; BAGTCD, BDNF, L-Ascorbic Acid, GDNF, TGFβ3, dbcAMP, DAPT. (B) Representative 3D reconstructions of cell aggregates stained with Calcein-AM (green, live cells) and Ethidium Homodimer-1 (red, dead cells). Scale bar represents 100 μm. (C) Cell viability over time was calculated as a percentage of green/red ratio. (D) Average size of the cell aggregates formed by viable cells was measured in millions of cubic micrometers. Statistical differences were calculated by two-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons ** p ≤ 0.005.

Article Snippet: On day 12, maturation of DA neurons was initiated by adding recombinant Human BDNF (Peprotech), recombinant Human GDNF (Peprotech), ascorbic acid (AA, Sigma), recombinant Human TGF-beta 3 (β3, Peprotech), dibutyryl-cyclic-AMP (dbcAMP, EnzoLifescience) and DAPT (Tocris).

Techniques: Inhibition, Activation Assay, Protein-Protein interactions, Staining