Fig. 1
From: Modeling PTEN overexpression-induced microcephaly in human brain organoids
PTEN-OE brain organoids model human microcephaly. A Immuno-staining of control and PTEN-OE hPSCs for markers of pluripotency (OCT4). Presence of GFP indicates overexpression of PTEN-GFP fusion protein. B Quantitative RT-PCR shows increased expression of PTEN in PTEN-OE hPSCs. Each data point represents one independent hPSC line (n = 3 for each group). C Immuno-blotting analysis shows increased total PTEN protein level (higher molecular weight band indicates PTEN-GFP fusion protein) in PTEN-OE hPSCs. D Quantification of total PTEN protein levels (endogenous PTEN and PTEN-GFP) in control and PTEN-OE hPSCs, normalized to Actin. Each data point represents one independent hPSC line (n = 3 for each group). E Representative images of control and PTEN-OE organoids at 6 weeks. F Quantification of control and PTEN-OE organoid size at around 1 week (day 7-10, 3 weeks (day 21-24), and 5-6 weeks (day 35-42). Each data point represents the area of a single organoid. N = 18 for each group at each time point from 3 hPSC lines and 2 independent differentiation experiments. ANOVA revealed significant effects of age (F2,102=669.4, p < 0.0001), genotype (F1,102=34.65, p < 0.0001), and the interaction between the two (F2,102=12.20, p < 0.0001). G and H Representative images (G) and quantification (H) of KI67 immuno-staining in 3-week-old control and PTEN-OE brain organoids. White dashed lines indicate the apical edge of the ventricular zone. Each data point represents one hPSC line in an independent differentiation, from the average of measurement from 3 organoids. N = 6 for each group from 3 hPSC lines and 2 independent differentiation experiments. I and J Quantitative RT-PCR analysis of neural precursor markers SOX2, TBR2 (I) and neuronal markers DCX, CTIP2 (J) in 3-week-old control and PTEN-OE brain organoids. Each data point represents one independent hPSC line (n = 3 for each group). K Representative images of immuno-staining for DCX and NESTIN (upper panels), CTIP2 and SOX2 (lower panels) in 3-week-old control and PTEN-OE organoids. L Quantification of CTIP2 immuno-staining in 3-week-old control and PTEN-OE brain organoids. Each data point represents one hPSC line in an independent differentiation, from the average of measurement from 3 organoids. N = 6 for each group from 3 hPSC lines and 2 independent differentiation experiments. M Immuno-blotting analysis shows reduced phospho-AKT protein level in 3-week-old PTEN-OE brain organoids generated from hPSC line Control-1, Control-2, PTEN-OE-1, and PTEN-OE-2. N Quantification of immuno-blotting results (Figure  1M and Additional file 1: Figure S1D) shows reduced phospho-AKT to total AKT ratio in 3-week-old PTEN-OE brain organoids. Each data point represents one hPSC line in an independent differentiation. N = 4 for each group from 3 hPSC lines and 2 independent experiments. O Representative images of 6-week-old brain organoids treated with vehicle or AKT inhibitor MK-2206 (100 nM). P Size quantification of vehicle and MK-2206 treated brain organoids at 5-6 weeks. Each data point represents the area of a single organoid. N = 18 for each group from 3 hPSC lines and 2 independent differentiation experiments. Q and R Quantitative RT-PCR analysis of neural precursor markers SOX2, TBR2 (Q) and neuronal markers DCX, CTIP2 (R) in vehicle and MK-2206 treated 6-week-old brain organoids. Each data point represents one independent hPSC line (n = 3 for each group).Results are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001