Abstract
Angiogenesis and neurogenesis are functionally interconnected during brain development. However, the study of the vasculature has trailed other brain cell types because they are delicate and of low abundance. Here we describe a protocol extension to purify prenatal human brain endothelial and mural cells with FACS and utilize them in downstream applications, including transcriptomics, culture and organoid transplantation. This approach is simple, efficient and generates high yields from small amounts of tissue. When the experiment is completed within a 24 h postmortem interval, these healthy cells produce high-quality data in single-cell transcriptomics experiments. These vascular cells can be cultured, passaged and expanded for many in vitro assays, including Matrigel vascular tube formation, microfluidic chambers and metabolic measurements. Under these culture conditions, primary vascular cells maintain expression of cell-type markers for at least 3 weeks. Finally, we describe how to use primary vascular cells for transplantation into cortical organoids, which captures key features of neurovascular interactions in prenatal human brain development. In terms of timing, tissue processing and staining requires ~3 h, followed by an additional 3 h of FACS. The transplant procedure of primary, FACS-purified vascular cells into cortical organoids requires an additional 2 h. The time required for different transcriptomic and epigenomic protocols can vary based on the specific application, and we offer strategies to mitigate batch effects and optimize data quality. In sum, this vasculo-centric approach offers an integrated platform to interrogate neurovascular interactions and human brain vascular development.
Key points
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This protocol extension describes the purification of prenatal human brain endothelial and mural cells with FACS and their utilization in downstream applications, including cell culture, organoid transplantation and single-cell transcriptomics.
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This simple, efficient protocol has relatively few steps compared with other methods and uses inexpensive reagents. Robust yields of healthy vascular and perivascular cells can be obtained in 6 h.
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Data availability
ScRNA-seq data have been deposited at GEO and are publicly available with code GEO: PRJNA803255. In addition, the data can be downloaded at https://cells.ucsc.edu/?ds=vascular-dev. No original code was generated using this data. The flow cytometry data can be accessed at Mendeley Data (https://doi.org/10.17632/9j6chfbvxm.1). Other data produced during and/or analyzed in this study are available from the corresponding authors upon request.
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Acknowledgements
The authors thank E. Winkler and A. Yang for their discussion on the strengths and challenges of different methods to purify human brain vascular cells (Table 1). This study has been supported by the Eli and Edythe Broad Regeneration Medicine and Stem Cell Fellowship, Pediatric Scientist Development Program (5K12HD000850-34), UCSF Physician Scientist Scholars Program (PSSP), American Heart Association Career Development Grant 857876, CIRM Alpha Stem Cell Clinic Fellowship and National Institutes of Health (NIH) grants K08 NS116161 to E.E.C., NIH K99/R00 NS111731 to A.B., NIH K99/R00 MH125329 to M.G.A., and California Institute of Regenerative Medicine (CIRM) Bridges Science Master’s Fellowship (SFSU EDUC2-12693) to E.J.V., P01 NS083513 and U01 MH105989 to E.J.H. The UCSF Flow Cytometry Core is supported by DRC Center Grant NIH P30 DK063720.
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E.E.C. and E.J.H. conceived the project and designed the experiments. E.E.C. performed FACS experiments, 2D culture experiments and organoid transplants. L.D. performed FACS experiments with passaged cells and organoid transplants. E.J.V. performed FACS experiments. J.O., K.W.-P., J.C., T.J. and A.B. performed scRNA-seq bioinformatics. L.D., A.B. and M.G.A. performed and designed organoid experiments. E.E.C. and E.J.H. wrote the manuscript with inputs from all authors.
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Crouch, E. E. et al. Cell 185, 3753–3769.e18 (2022): https://doi.org/10.1016/j.cell.2022.09.004
This protocol is an extension to: Nat. Protoc. 13, 738–751 (2018): https://doi.org/10.1038/nprot.2017.158
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Crouch, E.E., Diafos, L.N., Valenzuela, E.J. et al. Profiling human brain vascular cells using single-cell transcriptomics and organoids. Nat Protoc 19, 603–628 (2024). https://doi.org/10.1038/s41596-023-00929-1
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DOI: https://doi.org/10.1038/s41596-023-00929-1
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