Profiling human brain vascular cells using single-cell transcriptomics and organoids

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.

Figure 1:. Capture, culture, and profiling of developing human brain vascular cells.

FACS strategy to simultaneously purify endothelial, and mural cells with ANPEP and CD31 (Steps 1-55) and downstream applications: 2D and 3D culture (Steps 56-96), organoid transplants (Steps 96-114), and transcriptomics (Steps 115-132).

Figure 2:. Detailed steps for dissociation, digestion, and isolation of prenatal human brain vascular cells.

Schema with detailed steps for dissociation, digestion, and isolation of prenatal human brain vascular cells. Once selected, pieces of 1 cm³ brain tissue should be finely minced with a scalpel (Step 6), then distributed into 3 conical tubes with collagenase/dispase in solution (Step 8). At the completion of the digestion, triturate the solution (Step 13). Layer the digested cells on top of 22% Percoll solution, distributing each digestion reaction into two Percoll tubes (Steps 14-16). After the Percoll centrifugation, pool the cells (Steps 19-20). Perform immunostaining (Steps 22-35) and Fluorescence Activated Cell Sorting (Steps 46-55).

Figure 3:. Prenatal and adult human brain vascular cells can be purified with FACS.

a. FACS plots showing the initial purification of prenatal human brain vascular cells. b. FACS plots showing CD31 expression in adult human brain endothelial cells and absence of ANPEP. c. Violin plots of PECAM1/CD31, PDGFRB, CD34, TIE1, and RGS5 in scRNA-seq data of endothelial and mural cells, microglia and radial glia. Experiments were performed with n>5 samples and data are represented as mean ± S.E.M.

Figure 4:. Prenatal human brain vascular cells can be maintained in culture.

a. Growth of passaged endothelial and mural cells after 3 passages in vitro. P1, passage 1; P2, passage 2; and P3, passage 3. b. Mural cells in culture. Left: Microscopic image of mural cells in culture, passage 3. Right: Flow cytometry and quantification of PDGFR-β expression in mural cells up to passage 3. c. Endothelial cells in culture. Left: Microscopic image of endothelial cells in culture, passage 3. Right: Flow cytometry and quantification of CD31 expression in endothelial cells up to passage 3. Data are mean ± SEM, n=3.

Figure 5. Organoid transplantation, embedding, and immunostaining.

a. Schema with detailed steps for prenatal human brain vascular cell viral infection, organoid transplantation, and freezing. Once desired cells are sorted and acquired via FACS (Steps 46-55), immediately infect them using AAV-CMV-GFP and transplant them onto the organoids (Steps 96-104). After culturing the organoids for two weeks, organoids should be transferred on to a 24-well plate, fixed, washed, and soaked in 30% sucrose solution. Organoids are frozen using O.C.T. within a disposable embedding mold (Steps 105-114). b. Fixing and Embedding Organoids. 1. Images of organoids in culture (circled in red, step 104) 2. Transfer organoids using wide-opening pipette tips (Step 105). 3. Fix the organoids in a 24-well plate (Step 105). 4. Submerge for 30 minutes in PFA (Step 106). 5. Wash with 1X PBS (Step 107). 6. Immerse the organoids in 30% sucrose for 6 hours or overnight (Step 108). 7. On Day 2 remove all the sucrose and add blue OCT/sucrose solution (Step 109). 8. Immerse the organoids in blue OCT/sucrose solution overnight (Step 109). 9. Transfer blue stained organoid to a base mold with a wide-opening pipette tips (Step 112). 10. Image demonstrating how the organoid sits in the base mold (Step 112). 11. Freeze over dry ice (Step 113). c. Confocal images for GFP and CD34 (endothelial cells) or PDGFR-β (mural cells) in the transplanted organoids.

Figure 6:. Alternative digestion and purification methods.

a. Example FACS plot with mechanical dissociation. Left plot shows that approximately 85% of FACS events are debris with this approach. Middle FACS plot shows the absence of vascular cells. b. Example FACS plot with liberase. c. Example FACS plot with papain. d. Example FACS plot with magnetic bead purification with CD31 conjugated antibody (left, middle) and sequential purification with ANPEP-APC and anti-APC magnetic antibody (right). Left and middle panels show the same data as a pseudocolor plot and histogram for clarify. Note how endothelial cells are enriched but not purified with CD31 magnetic antibody and ANPEP+ cells are completely lost. Data are mean ± SEM and n=4 replicates. e. Example FACS plot with >24 hour PMI. Mural cells survive the dissociation and digestion but endothelial cells do not. f. Feature plots for mural (PDGFRB, RGS5) and endothelial (PECAM1, CD34, TIE1) genes in a sample with >24 hour PMI.