Fig 1.
Mini-Brain maturation through neural induction and differentiation.
(A) Neural induction matures the neuroectodermic layers at the outer surface of the EB, the edge of which appears optically translucent (arrow). (B) Neural differentiation in Matrigel droplets expands neuroepithelial buds (arrowheads) and increases their size (primitive organoid). (C) Organoids develop in size and morphology for 2 months, reaching their maximal size of 3–4 mm in diameter at the end of this period. (D) Hematoxylin and eosin staining shows the presence of discrete brain regions such as ventricle like-cavities (upper panel, 1-month old MBs) and choroid plexus-like structures (lower panel, 4-month old MBs). Scale bars 250 μm (A, B, C) and 100 μm (D).
Fig 2.
Chemical induction of APP cleavage in vitro upregulates both Aβ42 and Aβ42/Aβ40 ratio without altering Aβ40 levels.
(A) Schematic representation of treatments with Aftin-5; 1- and 2-month-old MBs (upper and lower representations respectively) were treated once with Aftin-5 for 4 days before collecting conditioned media. (B) Aβ40 (left) and Aβ42 (right) mean concentration levels in conditioned media measured by ELISA (MSD); concentrations of soluble Aβ40 and Aβ42 peptides from supernatant of vehicle-treated with DMSO (Ctrl) were compared with concentrations obtained from MBs treated with Aftin-5 (concentration of 150μM (A150)). (C) Aβ42/Aβ40 ratios corresponding to 1 month- and 2-month old organoids measured by ELISA (MSD) in conditioned media of MBs treated with control vehicle or with Aftin-5. Statistical analysis: unpaired nonparametric Mann-Whitney test. On charts *: p = 0.02; ***: p = 0.009; ns: not significant.
Fig 3.
APP and PrPC expression in MBs during culture.
(A) Real-time PCR analysis shows APP and PRNP gene expression at different times during culture. Expression during MB generation and differentiation was normalized with iPSCs expression, which was used as the baseline. (1) iPSCs; (2) embryoid bodies, (EBs); (3) embryoid bodies after neural induction (NI); (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m). (B) Western Blot analysis of APP and PrPC using specific antibodies: PrPC antibody SAF32 is specific for the octapeptide repeat region at the N-terminal site which contains the octarepeat region; full-length diglycosylated PrPC appears at ∼ 35 kDa (arrow) whereas unglycosylated and truncated forms appear at a lower molecular weight. 22C11 antibody recognizes amino acid sequence 66–81 and allows identification of a full-length APP695 isoform at ∼ 100 kDa (arrow) as well as an N-truncated form of APP protein. The Bradford method was used to measure the protein load (5 μg of total protein per well); α-tubulin (∼ 55 kDa) was used as loading control. (4) MBs after 19 days of culture (d19); (5) 2-month old MBs (2m); (6) 4-month old MBs (4m); (>6) 7-month old MBs (7m). (C) Tissue sections of 1 month- (left panel) and 3-month old MBs (right panel), immunostained with specific antibodies recognizing PrPC (SAF32 antibody, specific for an N-terminal epitope of protein, upper panel) and Aβ peptides (BAM10 antibody, lower panel).