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eLifeDevelopmental BiologyPeer-reviewed journal article

Generation of Vascularized Brain Organoids to Study Neurovascular Interactions

Xin-Yao Sun, Xiang-Chun JuiD, Yang LiiD, Peng-Ming ZengiD, Jian Wu, Yingying ZhouiD, Libing SheniD, Jian DongiD, Yuejun CheniD, Zhen‐Ge LuoiD

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Abstract

The recently developed brain organoids have been used to recapitulate the processes of brain development and related diseases. However, the lack of vasculatures, which regulate neurogenesis, brain disorders, and aging process, limits the utility of brain organoids. In this study, we induced vessel and brain organoids respectively, and then fused two types of organoids together to obtain vascularized brain organoids. The fused brain organoids were engrafted with robust vascular network-like structures, and exhibited increased number of neural progenitors, in line with the possibility that vessels regulate neural development. Fusion organoids also contained functional blood-brain-barrier (BBB)-like structures, as well as microglial cells, a specific population of immune cells in the brain. The incorporated microglia responded actively to immune stimuli to the fused brain organoids. Thus, the fusion organoids established in this study allow modeling interactions between the neuronal and non-neuronal components in vitro, in particular the vasculature and microglia niche.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (4 Jan 2022) · CC BY 4.0

Fusing stem cell-derived vessel organoids with cerebral organoids yields brain organoids containing vascular networks, BBB-like features and responsive microglia, with more neural progenitors than unfused controls.

Brain organoids lack vasculature, which the authors say regulates neurogenesis, and they describe earlier routes (grafting, transcription-factor-driven endothelial cells, co-culture) as not giving an integrated in vitro vascular network or microglia. Here a separately induced vessel organoid, which also contains microglia, is fused to a cerebral organoid.

Key results

  1. ShownSingle-cell profiling of day-40 vessel organoids shows a brain-vessel-like cell repertoire (endothelial, pericyte, smooth muscle, fibroblast, microglia). Pseudotime places pericytes earlier than smooth muscle cells.

    About 7000 cells after QC, 9 main clusters; two independent batches highly correlated. Immunostaining for αSMA, PDGFRβ, CD31, TREM2 and TMEM119 agrees; DLL4 and EPHB4 mark separate EC populations. · Cell composition of brain-specific VOrs…; Single-cell RNA-Seq data analysis

  2. SuggestedVessel-organoid cell types resemble in vivo counterparts, and the endothelial cells cluster with endothelial rather than epithelial-derived brain endothelial-like cells.

    GO analysis of the 149 up-regulated DEGs from the VOr-vs-mouse and human-vs-mouse EC comparison (Fig. 3H, J); a large fraction of up-regulated genes overlapped between the two comparisons. · Cell composition of brain-specific VOrs…; Single-cell RNA-Seq data analysis; Figure legends

  3. SuggestedFused organoids show BBB-like features: tight-junction markers, neurovascular unit-like structures and selective uptake of a BBB-penetrating peptide that brain organoids alone take up much less.

    CLDN5, ZO-1 and p-glycoprotein staining; TEM (D80) shows pericytes and astrocyte end-feet around the basement membrane. Angiopep-2 gave strong signal, scrambled peptide none; BOrs alone weaker (n = 3 organoids per sample, paired t-test, p < 0.05). · Generation of fusion vascularized brain…; Figure legends

  4. SuggestedMicroglia-like cells from vessel organoids migrate into the neural part of fusion organoids and are depleted by a CSF1R inhibitor; LPS-induced TNFα and IL-6 expression is attenuated when they are depleted, suggesting they take part in the response.

    IBA1+GFP+ cells in fVBOrs, none in BOrs; IBA1+ cells rose from D25 to D40 (n = 16, p < 0.001). PLX5622 (2 μM, 7 days) nearly eliminated IBA1 cells; LPS raised TNFα and IL-6 qPCR, attenuated with PLX5622 (n = 3 experiments, one-way ANOVA). · Microglia cells in fVBOrs are responsive to…; Figure legends

  5. ShownFusion organoids have more neural progenitors and less central apoptosis than brain organoids, with no change in neuronal differentiation markers.

    At D25 rosette thickness, PAX6+ and p-VIM+ density higher in fVBOrs (p < 0.001; e.g. PAX6 n = 25 rosettes from 4 organoids). DCX p = 0.308 and TBR1 p = 0.8340 not different. Cleaved caspase-3 lower in fVBOrs: p = 0.0303 (D40), p = 0.0286 (D70). · Increased neural progenitors in the fusion…; Figure legends

Methods, data and reproducibility

Human H9 embryonic stem cells were differentiated separately into vessel organoids (mesoderm induction, endothelial maturation, neurotrophic supplements) and cerebral organoids, then combined in one Matrigel droplet. Characterised by scRNA-seq with comparison to public datasets, immunostaining, qPCR, TEM and a peptide permeability assay.

  • scRNA-seq of day-40 vessel organoids (two independent batches), 10x Genomics Chromium · human (H9 hESC-derived) · n = about 7000 cells after QC · SRP338043 (SRR15992286, SRR15992285)
  • Public human neocortical scRNA-seq (eight adult and four embryonic cortexes) used for comparison · human · phs001836
  • Public mouse brain vascular scRNA-seq used for comparison · mouse · GSE98816
  • Fusion (fVBOr), vessel (VOr) and brain (BOr) organoids for imaging, qPCR, TEM and permeability assays · human (H9 hESC-derived) · n = e.g. 55 rosettes from 6 fVBOrs and 60 rosettes from 7 BOrs (thickness)
Methods & tools
Cell Ranger (hg38), Seurat v3.0, Monocle v2.10.1, limma v3.38.3, clusterProfiler, AngioTool, FlowJo, Imaris, 10x Genomics Chromium, PLX5622, Angiopep-2 peptide
Reagents & environment
Cell Ranger with hg38 (v1.2.0); Seurat v3.0 under R v3.5.2; Monocle v2.10.1; limma v3.38.3.
Reproducibility gaps
No analysis code repository is listed; the paper says only that all software is open and freely available. Only vessel-organoid scRNA-seq is deposited.
Limitations (4)
  • The fusion organoids still lack active blood flow; the authors suggest microfluidic organ-on-a-chip combinations as a possible remedy. · stated by the authors
  • The factors that define microglial identity in vessel organoids are unclear, and astrocyte roles in BBB maturation and immune surveillance remain to be investigated. · stated by the authors
  • scRNA-seq was done only on vessel organoids; the fused organoids were not profiled. Everything derives from one hESC line (H9). · noted in this summary
  • BBB function rests on one peptide with scrambled control and n = 3 organoids; permeability was not tested with a range of molecules. · noted in this summary

Read this if you want a vessel-plus-brain organoid fusion protocol with microglia and BBB-like readouts, and a vessel-organoid scRNA-seq resource compared against mouse and human data.

Citations
385
FWCI
28.59
Field percentile
top 1%

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