. 2022 Sep 29;185(20):3753-3769.e18.

doi: 10.1016/j.cell.2022.09.004.

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

Elizabeth E Crouch¹, Aparna Bhaduri², Madeline G Andrews³, Arantxa Cebrian-Silla⁴, Loukas N Diafos⁵, Janeth Ochoa Birrueta⁵, Kaylee Wedderburn-Pugh⁶, Edward J Valenzuela⁵, Neal K Bennett⁷, Ugomma C Eze⁸, Carmen Sandoval-Espinosa⁹, Jiapei Chen¹⁰, Cristina Mora¹¹, Jayden M Ross³, Clare E Howard¹², Susana Gonzalez-Granero¹³, Jaime Ferrer Lozano¹⁴, Maximo Vento¹⁵, Maximilian Haeussler¹⁶, Mercedes F Paredes¹⁷, Ken Nakamura¹⁸, Jose Manuel Garcia-Verdugo¹³, Arturo Alvarez-Buylla¹⁹, Arnold R Kriegstein¹⁷, Eric J Huang²⁰

Affiliations

¹ Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA; The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: elizabeth.crouch@ucsf.edu.
² Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA; The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA 94158, USA.
⁸ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA.
¹³ Laboratorio de Neurobiología Comparada, Instituto Cavanilles de Biodiversidad y 21 Biología Evolutiva, Universitat de València - Centro de Investigación Biomédica en Red 22 sobre Enfermedades Neurodegenerativas, Valencia, Spain.
¹⁴ Department of Pathology, Hospital Universitari i Politecnic La Fe, Valencia, Spain.
¹⁵ Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain; Division of Neonatology, University and Polytechnic Hospital La Fe, Valencia, Spain.
¹⁶ Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA 95060, USA.
¹⁷ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁸ Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁹ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
²⁰ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA; Pathology Service 113B, San Francisco Veterans Affairs Healthcare System, San Francisco, CA 94121, USA. Electronic address: eric.huang2@ucsf.edu.

PMID: 36179668
PMCID: PMC9550196
DOI: 10.1016/j.cell.2022.09.004

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

Elizabeth E Crouch et al. Cell. 2022.

. 2022 Sep 29;185(20):3753-3769.e18.

doi: 10.1016/j.cell.2022.09.004.

Authors

Affiliations

¹ Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA; The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: elizabeth.crouch@ucsf.edu.
² Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA; The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA 94158, USA.
⁸ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA.
¹³ Laboratorio de Neurobiología Comparada, Instituto Cavanilles de Biodiversidad y 21 Biología Evolutiva, Universitat de València - Centro de Investigación Biomédica en Red 22 sobre Enfermedades Neurodegenerativas, Valencia, Spain.
¹⁴ Department of Pathology, Hospital Universitari i Politecnic La Fe, Valencia, Spain.
¹⁵ Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain; Division of Neonatology, University and Polytechnic Hospital La Fe, Valencia, Spain.
¹⁶ Genomics Institute, University of California, Santa Cruz, Santa Cruz, CA 95060, USA.
¹⁷ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁸ Department of Neurology, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, CA 94158, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
¹⁹ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA.
²⁰ The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Science Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Developmental & Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA; Pathology Service 113B, San Francisco Veterans Affairs Healthcare System, San Francisco, CA 94121, USA. Electronic address: eric.huang2@ucsf.edu.

PMID: 36179668
PMCID: PMC9550196
DOI: 10.1016/j.cell.2022.09.004

Abstract

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Keywords: angiogenesis; arterial endothelial cells; blood brain barrier; cortical organoids; endothelial cells; human prenatal brain development; mural cells; pericytes; smooth muscle cells; tip cells; venous and capillary endothelial cells; ventricular zone.

Published by Elsevier Inc.

PubMed Disclaimer

Conflict of interest statement

Author contributions E.E.C. and E.J.H. conceived the project and designed the experiments. E.E.C. performed immunohistochemistry and quantification of blood vessel density, FACS experiments, 2D culture experiments, and organoid transplants. A.C.-S., S.G.-G., J.M.G.-V., and A.A.-B. performed and analyzed TEM data. A.B., L.N.D., J.O.B., C.E.H., and C.S.-E. performed bioinformatics. A.B. and U.C.E. performed RNA velocity analysis. M.G.A. and J.M.R. performed organoid experiments and L.N.D. and K.W-P. analyzed the results with supervision from E.E.C., J.C., and L.N.D. performed FACS experiments and E.J.V. performed mitochondrial quantifications. N.K.B. and K.N. performed and analyzed the Seahorse experiments. C.M., J.F.L., and M.V. contributed to the collection and preparation of human tissues. M.H. designed the web browser. M.F.P. and A.R.K. contributed reagents and expertise. E.E.C. and E.J.H. wrote the manuscript with inputs from all authors. Declaration of interests A.R.K. and A.A-B. are founding members of Neurona Therapeutics.

Figures

**Figure 1.. Angiogenesis in the periventricular region of the germinal matrix in the prenatal human brain.**
A. Gross photos of 19 GW prenatal human brain. Top: Lateral view showing the coronal planes for the bottom panels. B. Confocal images of the periventricular area of human brain at 14 and 22 GW. C. Immunostaining of CD31, NG2 and PDGFR-β in representative sections of the human medial ganglionic eminence (MGE) at 14 and 22 GW. **D-E.** Quantification of CD31⁺ and NG2⁺ surface area in MGE. F. Confocal image of 17 GW MGE immunostained for CD31, NG2 and PDGFR-β. 3D Imaris rendering shows CD31 (gray) and NG2 immunostaining with a gradient. Red indicates the highest NG2 expression to the lowest level in purple. The areas in the white box highlight filopodia (arrows). **G-H.** Quantification of the number of filopodia (G) or branch points (H) in MGE. I. Confocal image of the 17 GW MGE immunostained for CD31, Ki-67 and PDGFR-β. 3D images show Ki-67⁺ endothelial cell in i and Ki-67⁺ mural cell in ii. **J-K.** Quantification of Ki-67⁺ endothelial (J) or mural (K) cells in MGE. In D, E, G, H, J, and K, each data point represents the average of 5 sections from one case. Statistics used ANOVAs with post-hoc Student’s t-tests to assess differences within groups. Data are mean ± S.E.M. Non-significant comparisons not shown.

**Figure 2.. Ultrastructural features of developing human MGE and cortical vasculature.**
A. Composite images of ultrathin sections from 17 GW MGE. The magenta outlines the vasculature, the dense magenta spots highlight the lumen of each blood vessel, and blue shows radial glial fibers. **B-E.** Blood vessel in the VZ composed of intermediate (blue) and light (purple) endothelial cells and a mural cell (magenta). C. High magnification of cells in B showing the mural cell cytoplasm with abundant RER (arrowheads) and adherens junctions (arrow). Intermediate and light endothelial cells show tight junctions (arrows). A dotted black line indicates the blood vessel lumen. D. Tight junctions (arrows) between endothelial cells and discontinuous basal lamina (yellow). E. High magnification from B highlighting the caveolae (black arrowheads) and clathrin coated vesicles (white arrowhead). **F-G.** Blood vessel in the oSVZ showing light (EL), intermediate (Ei) and dark (ED) endothelial cells and mural cell expansions (M, magenta). Note the long tight junctions (arrows) and discontinuous basal lamina (yellow). **H-I.** A blood vessel composed of intermediate (blue) and dark (green) endothelial cells and a mural cell (magenta). I. High magnification of endothelial cells showing intraluminal filopodia (arrowheads) and Weibel-Palade bodies (arrows). J. High magnification of an intermediate endothelial cell showing microtubules (arrowheads) and mural cell with its characteristic dilated RER (arrows) and abundant mitochondria (asterisk). K. Mural cell (magenta) interdigitated with endothelial cells. L. Composite images of ultrathin sections from the 17 GW cortex showing vascular distribution (magenta). **M-O** Cortical blood vessels with dark (E_D, green), intermediate (E_i, blue), and light (E_L, purple) endothelial cells and a mural cell (magenta). N. Higher magnification from M showing the junctional complexes between different types of endothelial cells (arrows) and the near obliterated vascular lumen (magenta line). O. High magnification from N showing clathrin-coated vesicles (white arrowhead) and caveolae (black arrowhead) at the thin luminal surface. **P-Q.** Blood vessels from the VZ at 17 GW (P) and 21 GW (Q) highlighting all 3 subtypes of endothelial cells and their mitochondria (arrows). **R-T.** Higher magnification images to show the mitochondria and RER in each endothelial subtype (Light, R; Intermediate, S; Dark, T) at 17GW. U. Quantification of mitochondrial surface area of each endothelial cell subtype at 17 and 21GW. Scale bars: A, L (5 μm); M, N: (2 μm); P, Q (1 μm); R-T (200 nm). In U, each data point represents a blood vessel cell of the dark, intermediate, or light subtype from the 17 or 21 GW case. Statistics used ANOVA with post-hoc Student’s t-tests to assess differences within groups. Data are mean ± S.E.M.. Non-significant comparisons not shown.

**Figure 3.. Single-cell transcriptomics of endothelial and mural cells from prenatal human brain in the second trimester.**
A schematic diagram for isolating endothelial and mural cells from the prenatal human brain. Left: Coronal sections of 21 GW prenatal human brain and higher power views depicting the cortex and GE and brief dissociation details. Right: Scattered dot plots showing the exclusion of CD45⁺ myeloid cells and selection of endothelial and mural cells. B. Immunofluorescent images of CD31⁺ endothelial and PDGFR-β⁺ mural cells in 2D cultures in EGM-2 media. **C-D.** Images of 3D Matrigel cultures of endothelial and mural cells showing their ability to form tubes (C). Quantification of the number of branch points established by endothelial and mural cells in 3D cultures (D). Each data point represents one well of cells from one case. Statistics used ANOVAs with post-hoc Student’s t-tests to assess differences within groups. Data are mean ± S.E.M. Non-significant comparisons not shown. E. UMAP plots showing the clustering of endothelial and mural cells based on their subtypes, brain regions, and gestational ages. F. Feature plots on the UMAP space showing the expression of pan-endothelial cell markers (*PECAM1*, *CD34*, and *TIE1*) and mural cell markers (*ANPEP*, *RGS5*, and *PDGFRB*) in each cluster.

**Figure 4.. Development of endothelial cells in the nascent vasculature of the second trimester human brain.**
A. UMAP plots highlighting 15–23 GW endothelial cells according to subtypes and brain regions. B. Heatmap showing mitotic, artery, tip cell, and capillary/venous gene expression in distinct groups. C. Feature plots for representative genes for the mitotic, capillary/venous, tip cell, and arterial endothelial cells on the UMAP space. D. RNA velocity analysis based on the scRNA-seq data of 15 and 23 GW endothelial cells indicating the stage-dependent lineage trajectories. **E-F.** RNAscope for *ADGRG6* (capillary/venous endothelial cell marker) in 15 and 23 GW human brain sections. Arrows indicate areas of the RNAscope signals. G. Immunostaining for *MFSD2A* (capillary/venous endothelial cell marker) in 17 GW human brain sections. Arrow indicates vascular areas positive for the antibody staining. **H-I.** RNAscope for *ADM* and *ANGPT2* (tip cell markers) in 15 and 23 GW human brain sections. Arrows indicate vascular areas of the RNAscope signals. Note the absence of ADM and ANGPT2 in the cortical plate (Box 1), distant from the ventricular zone. J. Immunostaining for ADM (tip cell marker, arrow) in 17 GW human brain sections. **K-L.** RNAscope for *GJA4* and *FBLN5* (arterial markers) in 15 and 23 GW week human brain sections. Arrows indicate vascular areas of the RNAscope signals. M. Immunostaining for ELN (arterial marker) in 17 GW human brain sections. Arrow indicates vascular areas positive for the antibody staining, and arrowhead indicates an ELN-negative area. N. Volcano plot of differentially expressed genes in young (left side) versus old (right side) endothelial cells. O. Gene ontology terms for the genes enriched in young (left) and old (right) endothelial cells. P. FACS plots showing the selection of endothelial cells (left panel) and a histogram of Mitotracker Red expression in 16–19 GW, 21–23 GW, and adult cases (right panel). Q. Quantification of low (10³–10⁴) and high (10⁴–10⁵) Mitotracker Red expression in endothelial cells from different gestational ages. **R-S.** Quantification of OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) in primary human brain endothelial cells. Values are normalized to starting point. T. Quantification of the basal levels of OCR and ECAR in 16 and 24 GW human brain endothelial cells. In Q, each data point represents a separate case. In **R-S**, points represent technical replicates of ≥ 4 wells of cells from the given gestational age. Statistics used ANOVA with post-hoc Student’s t-tests to assess differences within groups (Q) or Student’s t-tests (R-T). Data are mean ± S.E.M.. Non-significant comparisons not shown.

**Figure 5.. Development of mural cells in the nascent vasculature of the second trimester human brain.**
A. UMAP plots indicating 15–23 GW mural cells according to subtypes, brain regions, and prenatal ages. B. Heatmap demonstrating mitotic, classic pericyte, smooth muscle cell, and fibroblast gene expression in distinct groups. C. Feature plots showing representative genes for the mitotic, smooth muscle cell, classic pericyte, and fibroblast subtypes of mural cells on the UMAP space. D. RNA velocity analysis based on scRNA-seq data of 15 and 23 GW mural cells indicating the stage-dependent lineage trajectories. **E-F.** RNAscope for *ATP1A2* and *KCNJ8* (classic pericyte markers) in 15 and 23 GW human brain sections. Arrows indicate vascular areas where the RNAscope probe was localized. G. Immunostaining for CD248 (classic pericyte marker) in 17 GW human brain sections. Note the gradient of expression highest at the ventricular surface. **H-I.** RNAscope for *LUM* and *SERPING1* (fibroblast markers) in 15 and 23 GW human brain sections. Arrows indicate vascular areas where the RNAscope probe was localized. J. Immunostaining for LUM in 17 GW human brain sections. **K-L.** RNAscope for *MYL9* and *TAGLN* (smooth muscle cell markers) in 15 and 23 GW human brain sections. Arrows indicate vascular areas where the RNAscope probe was localized. M. Immunostaining for MYL9 in 17 GW human brain sections.

**Figure 6.. Molecular pathways for cell-cell communication between endothelial and mural cells in the prenatal human brain.**
A. Rank order of significant ligand-receptor pairs utilized by the endothelial and mural cells in the cell-cell communication in the second trimester human brain. B. (Top panels) Chord plots summarizing expression of collagen genes and their receptors by endothelial and mural cells at 15 and 23 GW. (Bottom panels) Violin plots of *COL4A1* and *ITGA1* in vascular subtypes. C. Confocal images showing immunostains for COL4A1, ITGA1, and CD31 in the ventricular zone of 23 GW prenatal human brain. D. (Top panels) Chord plots summarizing expression of midkine and its receptors by endothelial and mural cells at 15 and 23 GW. (Bottom panels) Violin plots of *MDK*, *SDC2*, and *ITGB1* in vascular subtypes. E. Confocal images showing immunostains for MDK, ITGB1, and CD31 in the ventricular zone of 23 GW prenatal human brain. F. Confocal images showing immunostains for MDK, SDC2, and CD31 in the ventricular zone of 23 GW prenatal human brain. G. 3D Matrigel cultures of endothelial and mural cells in the presence of MDK or iMDK. Images shown at 3 and 6 hours. **H-I**. Quantifcation of number of branches in Matrigel cultures at 3, 6, and 12 hours in endothelial or mural cells with MDK or iMDK. Each data point represents the average of 3 wells from a separate biological replicate. Statistics used ANOVA with post-hoc Student’s t-tests to assess differences within groups. Upper asterisk compares between control and MDK; lower asterisk between control and iMDK. Data are mean ± S.E.M.. Non-significant comparisons not shown.

**Figure 7.. Human endothelial and mural cells promote neurogenesis and reduce cell stress in cortical organoids.**
A. Schematic diagram for transplanting AAV-GFP labeled endothelial or mural cells into cortical organoids. B. Microscopic images of transplanted endothelial and mural cell organoids after 7 days in culture. C. Confocal images for immunostains for GFP and Ki-67 in the transplanted organoids. D. Quantification of Ki-67⁺ cells in transplanted organoids. E. Confocal images showing *LUM*, *SERPING1*, *KCNJ8*, *ATP1A2*, *MYL9* and *TAGLN* RNA in transplanted mural cells. F. Graph showing the subtype of transplanted mural cells in the cortical organoids as determined by RNAscope probes. G. Confocal images of *PRDX3*, *ADGRG6*, *GJA4*, *FBLN5*, *ANGPT2*, and *ADM* RNA in transplanted endothelial cells. H. Graph showing the subtype of transplanted endothelial cells in the cortical organoids as determined by RNAscope probes. I. Confocal images for GFP, BCL11B (CTIP2), and RBFOX3 (NeuN) in the transplanted organoids. **J-K.** Quantification of RBFOX3⁺ or BCL11B⁺ cells in organoids in GFP⁺ or control regions. L. Confocal images for GFP, PGK1, and CD34 (endothelial cells) or PDGFR-β (mural cells) in organoids. M. Quantification of PGK1⁺ cells in the organoids in GFP⁺ or control regions. In D, J, K, and M, each data point represents the average of 5 sections from one transplant. Statistics used Student’s t-tests. Data are mean ± S.E.M.. Non-significant comparisons not shown.

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Cerebrovasculature pumps up progenitors.
Fame RM. Fame RM. Cell. 2022 Sep 29;185(20):3645-3647. doi: 10.1016/j.cell.2022.09.014. Cell. 2022. PMID: 36179664

References

1. Ando K, Tong L, Peng D, Vázquez-Liébanas E, Chiyoda H, He L, Liu J, Kawakami K, Mochizuki N, Fukuhara S, et al. (2022). KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling. Dev. Cell - PubMed
1. Armulik A, Genové G, Mäe M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom P, Strittmatter K, et al. (2010). Pericytes regulate the blood-brain barrier. Nature 468, 557–561. - PubMed
1. Armulik A, Genové G, and Betsholtz C (2011). Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215. - PubMed
1. Attwell D, Mishra A, Hall CN, O’Farrell FM, and Dalkara T (2016). What is a pericyte? J. Cereb. Blood Flow Metab 36, 451–455. - PMC - PubMed
1. Ayloo S, Lazo CG, Sun S, Zhang W, Cui B, and Gu C (2022). Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier. Neuron 110, 1641–1655.e6. - PMC - PubMed

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Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

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Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

Authors

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Abstract

Conflict of interest statement

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