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Engineering of human brain organoids with a functional vascular-like system


Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.

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Fig. 1: Characterization of vasculature-like structures in vhCOs.
Fig. 2: Single-cell analysis of vhCOs.
Fig. 3: vhCOs demonstrate BBB characteristics.
Fig. 4: vhCOs possess a functional vascular system.

Data availability

Single-cell transcriptome data are available in the Gene Expression Omnibus under accession code GSE134049.

Code availability

The DCMRSoft code that support the findings of this study are available on request from the corresponding author.


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I.-H.P. was partly supported by the NIH (grant nos. GM111667-01, R01AA025080-01, R01CA203011-2), CSCRF (grant nos. 14-SCC-YALE-01 and 16-RMB-YALE-04), Kavli Foundation, Simons Foundation and the KRIBB/KRCF research initiative program (grant no. NAP-09-3). This work was supported by the College of Medicine, University of Arkansas for Medical Sciences to Sang-Hun Lee, the Core Facilities of the Center for Translational Neuroscience, an award (no. P30 GM110702) from the IDeA program at NIGMS. Y.-S.Y was partly supported by the NIH (grant nos. R01HL127759 and DP3DK108245) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, Republic of Korea (grant nos. HI15C2782 and HI16C2211). L.E.N was partly supported by NIH (1R21 EB024889, 1R01 HL148819), M.P. was partly supported by Fonds de recherche du Québec – Santé in Canada, and F.H. was partly supported by NIH (EB023366-02, MH067528-02). Computation time was provided by Yale University Biomedical High Performance Computing Center.

Author information




B.C. and I.-H.P. conceived the study. B.C., Y.X., B.P., Y.-J.K., Y.T., K.-Y.K., P.S., Sang-Ho Lee and Y.-S.Y. performed the experiments. M.H.K., M.S.B.R. and L.E.N. performed perfusion setup and analysis. Y.Y. performed ECIS analysis. M.P. and F.H. performed MRI analysis. C.-S.H. performed surgeries. K.C., J.D. and P.P. performed TEER analysis. Y.-J.K. and S.-H.L. performed electrophysiological recordings. B.C., Y.X. and I.-H.P. wrote the manuscript.

Corresponding author

Correspondence to In-Hyun Park.

Ethics declarations

Competing interests

I.-H.P. serves on the scientific advisory boards of ELGEN Therapeutics and INSTEMCARE with financial interest. However, the work presented in this manuscript is not related to ELGEN Therapeutics or INSTEMCARE.

Additional information

Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Characterization of ETV2-induced cells in hCOs.

(a) Left, immunostaining for SOX2 and TBR1 in control hCOs and vhCOs (30-day old). Right, immunostaining for CTIP2 and SATB2 in 120-day old control and vhCOs. Data are representative images of 5 organoids from three independent experiments. (b) Expression of pluripotency and neural genes from control hCOs and vhCOs (30- and 70-day old). Gene expression was measured relative to HES3 hESCs and normalized to β-Actin. Data represent the mean ± SEM (n=5, from three independent batches). (c) Quantification of vascularization in vhCOs and control hCOs at different time points. CD31 labeled row shows the projections of z-stacks of CD31 stained red whole mount images. Angiotool analysis of the CD31 projections, thick red lines show the vascular paths, blue dots represent vascular junction points and the thin white line demonstrate the entire area over which vascular structures formed within organoid. Data represent the mean ± SEM (n=3, from three independent batches). (d-e) Co-immunostaining for CD31 and mCherry (d), CDH5 and mCherry (e) in 70-day old hCOs. Data are representative images of three independent experiments. (f) Representative electron microscopy images of vhCO at day 30 and 180. The E represents the endothelial cells, black arrowhead points at tight junction and white arrowhead indicates the astrocytic end feet. (n=2, from two independent batches). The scale bar represents 50 μm in a, c and d, and 100 μm in e, and 2 and 1 μm at day 30 and 180, respectively in f.

Supplementary Figure 2 Characterization of ETV2-induction in hCOs.

(a) Immunostaining for endothelial markers, CD31 and CDH5, and tight junction marker, α-ZO1, in 30-day old H1-derived hCOs. Data are representative images of 5 organoids from two independent experiments. (b) Image of the FITC-dextran perfusion system for organoids via connected tubes. (c) Left, Image of FITC-dextran and CDH5 in perfused hCOs with confocal microscopy. Right, the percentage of lumens containing FITC-dextran in vhCOs and control hCOs. Data represent the mean ± SEM (n=5, from three independent batches); (T=9.761 DF=4 and *p=0.0001). (d) Left, HIF-1α staining of organoids after 30-,70- and 120-day culture. Right, quantification of HIF-1α +/DAPI+ cells in control hCOs and vhCOs at days 30, 70 and 120. Data represent the mean ± SEM (n=3, from three independent batches). (D70: T=7.91 DF=4 and *p=0.00138, D120: T=16.07 DF=4 and **p=0.000087). (e) Left, expression of the synaptic marker SYN1 in control hCOs and vhCOs. Right, quantification of SYNP +/DAPI+ cells in control hCOs and vhCOs at days 30, 70. Data represent the mean ± SEM (n=3, from three independent batches). (D70: T=11.18 DF=4 and *p=0.00036, D120: T=7.372 DF=4 and **p=0.0018). The unpaired two-tail t-test was used for all comparisons. The scale bar represents 100 μm in d, e, and 50 μm in a, and c.

Supplementary Figure 3 Classification, annotation and evaluation of cell clusters from vhCOs.

(a) tSNE plot of single cells colored by functional clusters. Data are representative of 20026 cells. (b) Schematic representation of cluster annotation. (c) Expression patterns of genes related with neuronal growth cone, early neurogenesis, proteoglycans and EMT. Data are representative of 20026 cells. (d) Comparison of total UMI counts, mitochondria-derived reads, GO enrichment and unique markers for lineage commitment and cellular event among clusters. (e) Exclusive expression pattern of TBR1 and GFAP genes in single cells. Cells co-expressing both genes (colored by blue) were considered doublets. (f) Histogram of gene expression related to vasculogenesis. Two-sided t-test p-value=1.91e-02 (VTN) and 2.2e-16 (HAND1). Data are representative of 2257 cells.

Supplementary Figure 4 Functional characterization of vhCOs.

(a) Immunostaining for α-ZO1 in hCOs and vhCOs at day 70. (b-d) Immunostaining for β-Catenin (b), OCLN and KDR (c), and GFAP and S100β (d) in 30-day old hCOs and vhCOs. Arrows show co-localization of OCLN and KDR in vhCOs. Data are representative images of 5 organoids from three independent experiments in a, b, c, and d. (e) Co-immunostaining for GFAP and PDGFRβ in control and vhCOs (day 70). Arrows indicate regions where pericytes and astrocytes are in close juxtaposition. (n=2, from two independent batches) (f) Expression of tight junctions and transporters with and without Aβ1-42-oligo treatment of vhCOs. Gene expressions were measured relative to control hCOs and normalized to β-Actin. Data represent the mean ± SEM (n=3, from three independent batches). The scale bar represents 50 μm in a, b, c, d and e.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Notes 1–3 and Protocol.

Reporting Summary

Supplementary Video 1

Vascular networks in vhCOs at day 30.

Supplementary Video 2

Vascular networks in hCOs at day 30.

Supplementary Video 3

FITC-dextran perfusion of a vhCO at day 30.

Supplementary Video 4

FITC-dextran perfusion of vhCO explant stained for human-specific CD31 and hNUC.

Supplementary Video 5

FITC-dextran perfusion of vhCO explant.

Supplementary Video 6

FITC-dextran perfusion of control hCO explant.

Supplementary Video 7

Mouse brain explant with FITC-dextran perfusion.

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Cakir, B., Xiang, Y., Tanaka, Y. et al. Engineering of human brain organoids with a functional vascular-like system. Nat Methods 16, 1169–1175 (2019).

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