Wnt-Notch Signaling Interactions During Neural and Astroglial Patterning of Human Stem Cells

Abstract

The human brain formation involves complicated processing, which is regulated by a gene regulatory network influenced by different signaling pathways. The cross-regulatory interactions between elements of different pathways affect the process of cell fate assignment during neural and astroglial tissue patterning. In this study, the interactions between Wnt and Notch pathways, the two major pathways that influence neural and astroglial differentiation of human induced pluripotent stem cells (hiPSCs) individually, were investigated. In particular, the synergistic effects of Wnt-Notch pathway on the neural patterning processes along the anterior-posterior or dorsal-ventral axis of hiPSC-derived cortical spheroids were explored. The human cortical spheroids derived from hiPSCs were treated with Wnt activator CHIR99021 (CHIR), Wnt inhibitor IWP4, and Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester [DAPT]) individually, or in combinations (CHIR + DAPT, IWP4 + DAPT). The results suggest that CHIR + DAPT can promote Notch signaling, similar or higher than CHIR alone, whereas IWP4 + DAPT reduces Notch activity compared to IWP4 alone. Also, CHIR + DAPT promoted hindbrain marker HOXB4 expression more consistently than CHIR alone, while IWP4 + DAPT promoted Olig2 expression, indicating the synergistic effects distinctly different from that of the individual small molecule. In addition, IWP4 simultaneously promoted dorsal and ventral identity. The patterned neural spheroids can be switched for astroglial differentiation using bone morphogenetic protein 4. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from hiPSCs for disease modeling, drug screening, as well as for hiPSC-based therapies. Impact Statement Wnt signaling plays a central role in neural patterning of human pluripotent stem cells. It can interact with Notch signaling in defining dorsal-ventral and rostral-caudal (or anterior-posterior) axis of brain organoids. This study investigates novel Wnt and Notch interactions (i.e., Wntch) in neural patterning of dorsal forebrain spheroids or organoids derived from human induced pluripotent stem cells. The synergistic effects of Wnt activator or inhibitor with Notch inhibitor were observed. This study should advance the derivations of neurons, astroglial cells, and brain region-specific organoids from human stem cells for disease modeling and drug screening, as well as for stem cell-based therapies. The results can be used to establish better in vitro culture methods for efficiently mimicking in vivo structure of central nervous system.

Keywords: Notch; Wnt; astroglial; neural patterning; pluripotent stem cells; spheroids.

FIG. 1.

Effects of Wnt and Notch modulators on the expression of β-catenin and Notch ligand Jagged 1 (Wnt-Notch interactions). The neural spheroids were treated with different Wnt and Notch modulators on day 16 and were analyzed for expression of β-catenin and Jagged 1. (A) Fluorescent images of β-catenin and Jagged 1. Scale bar: 100 μm. (B) (i) Representative Western blots of β-catenin; (ii) β-catenin Western blot quantification (normalized expression) using averages from different experimental repeats (n = 3). (C) Western blots of Jagged 1 expression. *, **, and # indicate p < 0.05. Color images are available online.

FIG. 2.

Effects of Wnt and Notch modulators on the expression of Notch receptor Notch-1 and YAP localization (Notch-YAP interactions). The neural spheroids treated with different Wnt and Notch modulators were analyzed for Notch-1 expression and YAP localization. (A) Fluorescent images of Notch-1 and YAP. Scale bar: 100 μm. (B) RT-PCR analysis of Notch-1 mRNA expression (n = 3). (C) (i) Western blots of the Notch-1 transmembrane segment and Notch-targeted Hes 1 expression. Western blot quantification (normalized expression) for (ii) Notch-1 and (iii) Hes 1 proteins (n = 3). *, **. #, and $ indicate p < 0.05. RT-PCR, reverse transcription–polymerase chain reaction analysis. Color images are available online.

FIG. 3.

Impacts of Wnt and Notch modulation on neural tissue patterning of hiPSCs. The neural spheroids treated with Wnt and Notch modulators on day 16 were analyzed for different brain regional markers. Fluorescent images of different markers (at day 20): dorsal forebrain-FOXG1; dorsal cortical layer IV-TBR1; hippocampal marker-PROX1; ventral marker-Nkx2.1; midbrain/hindbrain marker-OTX2, hindbrain marker-HOXB4. Scale bar: 100 μm. The six conditions were labeled as follows: Control, CHIR (C), IWP4 (I), DAPT (D), C + D, and I + D. hiPSC, human induced pluripotent stem cell; DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Color images are available online.

FIG. 4.

Quantification of brain regional marker expression of neural spheroids derived from hiPSCs. The neural spheroids treated with Wnt and Notch modulators on day 16 were analyzed using flow cytometry (at day 21). (A) Flow cytometry analysis of dorsal forebrain marker TBR1, ventral marker Nkx2.1, and hindbrain marker HOXB4 (n = 3). (B) Representative flow cytometry plots of TBR1, HOXB4, and Nkx2.1. (i) HOXB4; (ii) TBR1; (iii) Nkx2.1. Black line: negative control; Red line: marker of interest. *, **, and # indicate p < 0.05. Color images are available online.

FIG. 5.

Additional brain regional marker expression of neural spheroids derived from hiPSCs. (A) Flow cytometry plots of FOXG1 and additional hindbrain/midbrain marker (Olig2) on day 21. Black line: negative control; Red line: marker of interest. (B) (i) Western blots of the brain regional markers HOXB4, OTX2 (hindbrain/midbrain), Olig2, and TBR1; (ii) Quantification (normalized expression) of Western blot band density (n = 3). The six conditions were labeled as follows: Control, CHIR (C), IWP4 (I), DAPT (D), C + D, and I + D. *, **, #, and $ indicate p < 0.05. Color images are available online.

FIG. 6.

Quantification of brain regional marker mRNA expression using RT-PCR analysis. The neural spheroids treated with Wnt and Notch modulators on day 16 were analyzed using RT-PCR (at day 21). RT-PCR analysis of hindbrain genes (HOXB4 and Olig2), forebrain gene (dorsal-TBR1 and ventral-Nkx2.1), and cerebellar genes (MATH1 and NEPH3) (n = 3). The six conditions were labeled as follows: Control, CHIR (C), IWP4 (I), DAPT (D), C + D, and I + D. *p < 0.05.

FIG. 7.

Impacts of Wnt and Notch modulation on astroglial tissue patterning of cortical spheroids when treated with BMP4. (A) Western blots of the astrocyte markers GFAP, S100B, V/vimentin, and Aldolase C; (B) Quantification (normalized expression) of Western blot band density (n = 3). The six conditions were labeled as follows: Control, CHIR (C), IWP4 (I), DAPT (D), C + D, and I + D. *, **, and # indicate p < 0.05. BMP, bone morphogenetic protein.