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Dorsal-ventral Patterned Neural Cyst From Human Pluripotent Stem Cells in a Neurogenic Niche

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Dorsal-ventral Patterned Neural Cyst From Human Pluripotent Stem Cells in a Neurogenic Niche

Y Zheng et al. Sci Adv.

Abstract

Despite its importance in central nervous system development, development of the human neural tube (NT) remains poorly understood, given the challenges of studying human embryos, and the developmental divergence between humans and animal models. We report a human NT development model, in which NT-like tissues, neuroepithelial (NE) cysts, are generated in a bioengineered neurogenic environment through self-organization of human pluripotent stem cells (hPSCs). NE cysts correspond to the neural plate in the dorsal ectoderm and have a default dorsal identity. Dorsal-ventral (DV) patterning of NE cysts is achieved using retinoic acid and/or sonic hedgehog and features sequential emergence of the ventral floor plate, P3, and pMN domains in discrete, adjacent regions and a dorsal territory progressively restricted to the opposite dorsal pole. This hPSC-based, DV patterned NE cyst system will be useful for understanding the self-organizing principles that guide NT patterning and for investigations of neural development and neural disease.

Figures

Fig. 1
Fig. 1. hESCs form NE cysts in a bioengineered 3D neurogenic niche.
(A and B) Schematic of neural induction in a 3D in vitro culture system comprising a gel bed and an ECM overlay (Gel-3D). For comparison, culture systems were generated without gel bed but with ECM overlay (Glass-3D), with gel bed but without ECM overlay (Gel-2D), or without either gel bed or ECM overlay (Glass-2D). (C) Representative confocal micrographs showing multicellular structures at day 8 under different culture conditions as indicated stained for PAX6 and N-CAD. DAPI-counterstained nuclei. (D) Representative cystic tissues in Gel-3D at day 8 stained for PAX6, NESTIN, SOX2, N-CAD, SOX17, BRACHYURY, CDX2, and EOMES as indicated. DAPI counterstained nuclei. (E) Representative cystic tissues in Gel-3D at day 13 stained for ZO-1 and SOX1 as indicated. DAPI counterstained nuclei. (F) Representative cystic tissues in Gel-3D at day 8 stained for EdU and pH3 as indicated. DAPI counterstained nuclei. n = 3 independent experiments. Scale bars, 50 μm (C to F).
Fig. 2
Fig. 2. Dorsalization and ventralization of NE cysts in Gel-3D.
(A) NE cysts obtained at days 9 and 18 under neural induction condition as indicated. Bright-field images show representative cyst morphologies. (B) Representative confocal micrographs showing NE cysts obtained at days 9 and 18 stained for PAX3, PAX6, MSX1, FOXA2, OLIG2, and NKX2.2 as indicated. DAPI counterstained nuclei. (C) Dorsalization with CHIR and ventralization with SAG, or RA and SAG, or RA, SAG, and SHH from day 4 to day 9. Bright-field images show representative NE cyst morphologies at day 9. (D) Representative confocal micrographs showing cysts at day 9 stained for PAX3, PAX6, MSX1, FOXA2, OLIG2, and NKX2.2 as indicated. DAPI counterstained nuclei. n = 3 independent experiments. Scale bars, 200 μm (A and C) and 50 μm (B and D).
Fig. 3
Fig. 3. Self-organized, emergent DV patterning of NE cysts in Gel-3D.
(A) Schematic of patterning of NE cysts with RA and SAG or RA and SHH from day 4 to day 9. (B and C) Representative confocal micrographs showing RA/SAG-treated NE cysts at day 18 stained for dorsal and ventral markers as indicated (B). (C) plots percentages of different patterned cysts. (D to F) Representative confocal micrographs showing RA/SHH-treated NE cysts at day 18 stained for dorsal and ventral markers as indicated [(D) SHH, 10 nM; (E) SHH, 100 nM]. (F) plots percentages of different patterned cysts under indicated conditions. Data in (C) and (F) represent means ± SEM. A total of 150 cysts were pooled from n = 3 independent experiments under both RA/SAG and RA/SHH conditions. Scale bars, 50 μm (B, D, and E).
Fig. 4
Fig. 4. Dynamics of DV patterning of NE cysts in Gel-3D.
(A) Representative confocal micrographs showing cysts stained for PAX3 and OLIG2 at different days as indicated. (B) Pie charts showing percentages of different types of cysts at different days as indicated. Cysts were grouped into five categories as indicated (PAX3+OLIG2, PAX3OLIG2, PAX3+OLIG2+ patterned, PAX3+OLIG2+ unpatterned, and PAX3+OLIG2+). (C) Representative confocal micrographs showing cysts stained for NKX2.2 and FOXA2 at different days as indicated. (D) Pie charts showing percentages of different types of cysts at different days as indicated. Cysts were grouped into five categories as indicated (NKX2.2FOXA2+, NKX2.2FOXA2, NKX2.2+FOXA2+ patterned, NKX2.2+ FOXA2+ unpatterned, and NKX2.2+ FOXA2). Data in (B) and (D) represent the mean. A total of 150 cysts were counted from n = 3 independent experiments at each time point. Scale bars, 50 μm (A and C).
Fig. 5
Fig. 5. Independent effects of RA and SHH on patterning of NE cysts in Gel-3D.
(A) Effect of RA stimulation (1 or 0.1 μM) alone from day 4 to day 9. Representative confocal micrographs show cysts at day 18 stained for dorsal and ventral markers as indicated. (B) Percentages of different patterned cysts as a function of RA dose. Data represent means ± SEM. Fifty cysts were counted from each independent experiment. n = 3 independent experiments at each RA dose. (C) Effect of RA stimulation (1 or 0.1 μM) alone from day 4 to day 18. Representative confocal micrographs show cysts at day 18 stained for dorsal and ventral markers as indicated. (D) Percentages of different patterned cysts as a function of RA dose. Data represent means ± SEM. Fifty cysts were counted from each independent experiment. n = 3 independent experiments at each RA dose. (E) Effect of SHH stimulation alone from day 4 to day 9. Representative confocal micrographs show cysts at day 18 stained for dorsal and ventral markers as indicated. (F) Effect of inhibition of SHH signaling with cyclopamine from day 4 to day 9. Representative confocal micrographs show cysts at day 18 stained for dorsal and ventral markers as indicated. (G) qRT-PCR analysis of PAX3, OLIG2, NKX2.2, FOXA2, and SHH expression for cysts at day 18 with or without cyclopamine treatment. Data are normalized against GAPDH and represent means ± SEM. n = 3 independent experiments. P values were calculated using unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars, 50 μm (A, C, D, and E).
Fig. 6
Fig. 6. Induction of spinal MNs from NE cysts in Gel-3D.
(A and B) Induction of spinal MNs with RA and SAG supplemented from day 4 to day 9. Representative confocal micrographs in (B) show cysts at days 9 and 18 stained for βIII-TUBULIN, MAP2, ISLET1/2, and HB9 as indicated. DAPI counterstained nuclei. (C and D) Induction of spinal MNs with RA and SAG supplemented from day 4 to day 9 and neurotrophic factors BDNF, GDNF, CNTF, IGF-1, cAMP, and AA supplemented from day 9 to day 18. Representative confocal micrographs in (D) show cysts at day 18 stained for ISLET1/2, MAP2, HB9, and βIII-TUBULIN as indicated. DAPI counterstained nuclei. The zoomed-in image shows a magnified view of the area highlighted by the white square. (E and F) Induction of spinal MNs with RA and SHH supplemented from day 4 to day 9 and neurotrophic factors from day 12 to day 25. Representative confocal micrographs in (F) show cysts at day 25 stained for FOXA2, OLIG2, HB8, and ISLET1/2. (G) Percentage of different patterned cysts. Data represent means ± SEM. ncyst = 126 and 134 for HB9 staining and ISLET1/2 staining, respectively. n = 3 independent experiments. Scale bars, 50 μm (B, D, and F).

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