Induction of Expansion and Folding in Human Cerebral Organoids

Abstract

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex.

Keywords: AKT; Dengue; PTEN; Zika; cerebral organoids; cortical development; human embryonic stem cells; microcephaly; outer SVZ; outer radial glia.

Figure 1.. Induction of expansion and folding in PTEN mutant human cerebral organoids.

A-B) CRISPR/Cas9-mediated targeting of the human PTEN locus, sequences of mutant WIBR3 hESC clones, and immuno-blotting for PTEN protein. C) ATP assay on 2D adherent NP culture, showing enhanced proliferation in WIBR3 PTEN mutants in the presence of low bFGF concentration. D-E) Representative image of EdU-Ki67 co-staining in WIBR3 wild-type NPs cultured with low bFGF (D), and quantification of cell cycle exit ratio (EdU+Ki67-/all EdU+). Low bFGF, 1ng/ml; high bFGF, 10ng/ml. Scale bars, 50um (top panel) and 10um (lower panels). F-G) Representative bright field (F) and light sheet images (G) of control and PTEN mutant WIBR3 cerebral organoids. Lower panels in (G) show the different angle view of the main panels above. Scale bar, 1mm (F) and 500um (G). H-I) Reconstructed models of control and mutant WIBR3 organoids, and quantification of volume, surface area and sphericity. Scale bar, 1mm. J) Quantification of control and mutant WIBR3 organoids volume and surface area on histological sections using stereological method. K-L) Images and quantification of surface folds density in Hoechst-stained control and mutant WIBR3 organoids at 6 weeks. Middle panels show higher magnification view of the top panels. Lower panels show algorithmic tracing of surface folds via Canny Edge Detection. Scale bar, 500um (upper panels), 100um (middle and lower panels). Results are mean +/− SEM. *p<0.05, ***p<0.001. See also Figure S1, S2, S3, S4.

Figure 2.. Lack of folding in Pten mutant mouse cerebral organoids.

A) Immuno-blotting shows a complete ablation of wild-type Pten protein in V6.5 mutant mouse ESCs. B-C) Bright field and light sheet images of control and Pten mutant mouse organoids at 4 days, 4 weeks and 8 weeks. Scale bar, 200um (upper panels in B), 500um (lower panels in B, C). D-F) Quantification of volume, sphericity and fold density on control and Pten mutant mouse organoids at 4 weeks. Results are mean +/− SEM. *p<0.05. See also Figure S4.

Figure 3.. PTEN deletion induces expansion and folding of forebrain neuroepithelia in human cerebral organoids.

A-C) Immuno-staining for NPs using antibodies against Nestin (A), Pax6 (B), and Sox2 (C) in control and mutant WIBR3 organoids at 4 weeks. Scale bars, 50um (A-B) and 20um (C). D-G) Light sheet images and quantification of control and mutant WIBR3 organoids treated with dorsomorphin for 14 days, demonstrating expansion and folding (D), increased folds density (G), patterning towards the forebrain fate (E) at the preclusion of none forebrain lineages (F). Results are mean +/− SEM. **p<0.01, ***p<0.001. See also Figure S4.

Figure 4.. PTEN deletion enhances proliferation and expands the NP pool.

A-B) Representative images and quantification of Ki67 immuno-staining in control and PTEN mutant WIBR3 cerebral organoids. Scale bar, 20um. C-E) Quantitative RT-PCR analysis of radial glial marker Pax6 (C), intermediate progenitor marker Tbr2 (D), and outer radial glia marker Hopx (E) demonstrates their temporal-specific over-expression in PTEN mutant WIBR3 organoids compared to controls. F) Immuno-staining for Pax6 and Tbr2 shows increased number of positive cells in PTEN mutant WIBR3 organoids. G-I) Representative images of the expanded neuroepithelium in 12-week-old control and PTEN mutant WIBR3 organoids stained with Sox2 and outer radial glia marker Hopx. Quantification was conducted by measuring and dividing the entire span of the neuroepithelium into 5 equal portions (bins), showing significantly more Sox2+ (H) and Hopx+ cells (I) in PTEN mutant WIBR3 organoids in the expanded neuroepithelium. Scale bar, 100um. J-K) Representative images of Sox2, Hopx, Ki67 co-staining in a PTEN mutant WIBR3 organoid (J), and quantification showing increased proliferation of VZ/iSVZ as well as oSVZ NPs in PTEN mutants compared to controls. L) Total neuroepithelial thickness at 8 and 12 weeks in control and PTEN mutant WIBR3 organoids. M-N) Representative images and quantification of Ki67 immuno-staining in control and Pten mutant mouse cerebral organoids. Scale bar, 20um. O) Immuno-staining for NP marker Sox2, outer radial glia marker Hopx, and neuronal marker Ctip2 in 3-week-old control and Pten mutant mouse organoids, showing lack of Hopx immuno-positivity. Scale bar, 20um. Results are mean +/− SEM. *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.

Figure 5.. Transient delay in neuronal differentiation expands the NP pool.

A) Immuno-staining for DCX and Nestin at 4 weeks in control and PTEN mutant WIBR3 cerebral organoids. Scale bar, 200um. B-C) Representative images of EdU-Ki67 co-staining in 4-week-old control and mutant WIBR3 organoids (B), and quantification of cell cycle exit ratio (EdU+Ki67-/all EdU+, C) in 4 and 6 weeks old organoids. Scale bars, 20um. D) Immuno-staining for DCX and EdU in 4-week-old control and mutant WIBR3 organoids. Scale bars, 20um. E-F) Quantitative cell fate analysis of EdU+ cells in 4-week-old organoids, showing PTEN mutants have increased retention as Sox2+ NP (E) and decreased propensity for differentiation into DCX+ immature neurons (F). G-H) Immuno-staining for DCX and Nestin at 8 weeks. Scale bar, 200um. I-K) Differential gene expression analyses by quantitative RT-PCR on control and mutant WIBR3 organoids at 4, 10 and 16 weeks. Genes analyzed are representative of NPs (PAX6, TBR2, HOPX), pan-neuronal markers (TUBB3, DCX, MAP2, RBFOX3), early-born neurons (CTIP2, TBR1) and late-born neurons (BRN2, SATB2, CUX1, CUX2). Majority of neuronal markers were transiently down-regulated in PTEN mutants at 4 weeks, but greatly normalized at 16 weeks. Gene expressions are normalized to control-1 at each time point. P value reflects controls vs. mutants. L-N) Immuno-staining for markers of outer radial glia (Hopx), early-born neurons (Tbr1 and Ctip2) and late-born neurons (Satb2 and Brn2) in control and mutant WIBR3 organoids at 12 weeks. Scale bars, 50um. O) PTEN mutant WIBR3 organoids had folded cortical plate at 16 weeks. Images show immuno-staining for markers of neurons (Ctip2) and astrocytes (GFAP). White dash lines outline the cortical surface. Scale bars, 50um. Results are mean +/− SEM. *p<0.05, **p<0.01, ***p<0.001. See also Figure S6.

Figure 6.. PTEN-AKT signaling controls expansion and folding in human cerebral organoids.

A) Schematic overview depicting the strategy to rescue PTEN mutant WIBR3 hESCs via lentivirus re-expression of PTEN. B) Immuno-blotting for PTEN shows the presence of PTEN-GFP fusion protein in rescued PTEN mutant WIBR3 hESCs. C) Light sheet images of Hoechst-stained WIBR3 organoids at 6 weeks generated from control, PTEN mutant, and PTEN-rescued hESCs. Scale bar, 500um. D) Immuno-staining images of pAKT and Nestin, in control and mutant WIBR3 organoids. Increased pAKT immuno-staining is present in mutants at 1.5 weeks, prior to the onset of expansion and folding (upper panels). Increased pAKT signal concentrates in the Nestin+ NPs, and is highest at the apical surface. Scale bars, 100um (top panels), 20um (middle and lower panels). E) Light sheet images of control and mutant WIBR3 organoids at 6 weeks, treated with AKT inhibitors GDC-0068 (1uM) or MK-2206 (100nM). Scale bar, 500um. F) Differential gene expression analyses by quantitative RT-PCR on control and mutant WIBR3 organoids, treated with AKT inhibitors GDC-0068 (1uM) or MK-2206 (100nM). Gene expressions are normalized to control-1. P value reflects controls vs. AKT inhibitor treated mutants. G) Light sheet images of 6-week-old human organoids generated from PTEN mutant WIBR3 alone, or transduced with lentivirus encoding DN-AKT or CA-AKT. Scale bars, 500um. H) Quantitative analysis of the density of surface folds in 6-week-old organoids from PTEN mutant alone, or PTEN mutant transduced with DN-AKT or CA-AKT. I-J) Representative images and quantification of Ki67 immuno-staining in 6-week-old organoids from PTEN mutant alone, or PTEN mutant transduced with DN-AKT or CAAKT. Scale bar, 20um. Results are mean +/− SEM. *p<0.05, ***p<0.001. See also Figure S7.

Figure 7.. ZIKV infection impairs expansion and folding in human cerebral organoids.

A-B) Schematic diagram and light sheet images of PTEN mutant WIBR3 cerebral organoids at day 23 (19+4 days) showing widespread caspase activity induced by ZIKV but not Dengue virus. Scale bar, 500um. C) Light sheet images and immuno-staining of mutant WIBR3 organoids at day 29 (19+10 days) shows reduced organoid size and increased apoptosis caused by ZIKV but not Dengue virus. C-caspase 3, cleaved-caspase 3 as detected by immuno-staining. Scale bars, 500um (upper) and 50um (lower). D-E) Quantitative analysis of organoid at day 29 (19+10 days) shows reduced size (D) and loss of surface folds density (E) upon ZIKV exposure. F-G) Schematic diagram, and representative images of immuno-staining for ZIKV infected control and mutant WIBR3 organoids at day 34 (30+4 days). Scale bar, 200um. H-I) Representative images of immuno-staining in mutant WIBR3 organoids show ZIKV infection coincides with elevated apoptosis (cleaved-caspase 3) and reduced proliferation (phosphorylated-H3). Scale bars, 50um. J) Light sheet images show mutant WIBR3 organoids treated with ZIKV at day 30 displayed widespread apoptosis, as revealed by whole-mount caspase activity staining. Scale bar, 500um. Results are mean +/− SEM. ***p<0.001.