Int J Stem Cells.  2022 Feb;15(1):85-94. 10.15283/ijsc21157.

Generation of Cortical Brain Organoid with Vascularization by Assembling with Vascular Spheroid

• Kook MG
• Lee SE
• Shin N
• Kong D
• Kim DH
• Kim MS
• Kang HK
• Choi SW
• Kang KS

• KMID: 2526740
• DOI: http://doi.org/10.15283/ijsc21157

Abstract

Background and Objectives
Brain organoids have the potential to improve our understanding of brain development and neurological disease. Despite the importance of brain organoids, the effect of vascularization on brain organoids is largely unknown. The objective of this study is to develop vascularized organoids by assembling vascular spheroids with cerebral organoids.
Methods and Results
In this study, vascularized spheroids were generated from non-adherent microwell culture system of human umbilical vein endothelial cells, human dermal fibroblasts and human umbilical cord blood derived mesenchymal stem cells. These vascular spheroids were used for fusion with iPSCs induced cerebral organoids. Immunostaining studies of vascularized organoids demonstrated well organized vascular structures and reduced apoptosis. We showed that the vascularization in cerebral organoids up-regulated the Wnt/β-catenin signaling.
Conclusions
We developed vascularized cerebral organoids through assembly of brain organoids with vascular spheroids. This method could not only provide a model to study human cortical development but also represent an opportunity to explore neurological disease.

Keyword

Organoid; Brain; Cerebral organoid; iPSCs; Vascularization

Figure

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  Fig. 1 Phenotypical characterization of vascular spheroids. (A) Representative images of vascular spheroids. HUVECs, hDFs and hUCB-MSCs were cultured directly for 10 days. Angiogenesis is shown using RFP fluorescence in the vascular spheroid (Scale bar: 500 μm). (B) Representative H&E and picrosirius staining images of vascular spheroids. Collagen deposition is shown in the vascular spheroid (Scale bar: 100 μm). (C) Representative images from day 10 spheroids immunostained with CD31, VE-cadherin, vWF and collagen IV. Nuclei are stained with DAPI (Scale bar: 100 μm).

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  Fig. 2 Characterization of vascular structure in the assembloid of vascular spheroids and cortical organoids. (A) Morphology and size of organoids on days 20, 30 and 43. Quantification of diameter and area from each group at different stages (Scale bar: 500 μm). (B) Representative CD31 immunostaining images of organoids on day 43 (Scale bar: 100 μm). (C) Immunostaining for CD31 and endothelial markers vWF and CDH5 in sectioned organoids at different time points (days 43 and 60). Expression of CD31 is shown in the vascular spheroid and cerebral organoid. The endothelial markers CDH5 and vWF are expressed in the vascular spheroid. White dash line indicates the location of the vascular spheroid (Scale bar: 100 μm). One-way ANOVA analysis. (D) Immunostaining images of whole-mount organoids at different time points (days 43 and 60) showing vascular-like structures in the organoids. Total vessel length was quantified by angiogenesis analysis (Scale bar: 1 mm). **p<0.01.

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  Fig. 3 Neuronal characterization of cerebral organoids. (A) Representative images of each organoid on day 43 stained with NeuN, CTIP2, TUJ1 and MAP2. Nuclei are stained with DAPI (Scale bar: 100 μm). (B) Immunostaining images of α-ZO1 in each group on day 60 (Scale bar: 100 μm). Student’s t-test, n=3, independent organoids from 2 different batches. *p<0.05; n.s. not significant.

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  Fig. 4 Vascularization of hCOs with vascular spheroids reduces apoptosis. (A) Representative images of the TUNEL assay for detecting apoptotic cells. Quantification showing the percentage of TUNEL＋ cells that were positive for DAPI in each group (Scale bar: 100 μm). (B) Protein levels of rH2AX and cleaved caspase-3. (C) Immunostaining images and quantification of rH2AX and SOX2 in each group on day 60 (Scale bar: 100 μm). (D) Immunostaining images and quantification of cleaved caspase-3 and PAX6 in each group on day 60 (Scale bar: 100 μm). Student’s t-test, n=4, independent organoids from 2 different batches. *p<0.05, ***p<0.001; n.s. not significant.

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  Fig. 5 Wnt/β-catenin signaling are upregulated in ahCOs. (A) Western blot of Wnt7a, β-catenin, GSK3β, AXIN2 and GFAP in each group of organoids. Western blot analysis revealed that decrease tendency of GSK3β and up-regulation of axin2 along with Wnt7a activation and that β-catenin was increased in the ahCO group compared to the hCO group. (B) Immunostaining images and quantification of SOX2 and NeuN in each group on day 60 (Scale bar: 100 μm). (C) Immunostaining images and quantification of MAP2 and GFAP in each group on day 60 (Scale bar: 100 μm). Student’s t-test, n=4, independent organoids from 2 different batches. **p<0.01; n.s. not significant.

Reference

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