Dataset Information

A single cell transcriptome atlas of murine endothelial cells

ABSTRACT: The heterogeneity of endothelial cells (ECs), lining blood vessels, across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomic data from 11 tissues of the model organism Mus musculus. We propose a new classification of EC phenotypes based on transcriptome signatures and inferred putative biological features. We identified top-ranking markers for ECs from each tissue. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) resembled each other across tissues, but only arterial, venous and lymphatic (not capillary) ECs shared markers, illustrating a greater heterogeneity of capillary ECs. We identified high-endothelial-venule and lacteal-like ECs in the intestines, and angiogenic ECs in healthy tissues. Metabolic transcriptomes of ECs differed amongst spleen, lung, liver, brain and testis, while being similar for kidney, heart, muscle and intestines. Within tissues, metabolic gene expression was heterogeneous amongst ECs from different vascular beds, altogether highlighting large EC heterogeneity.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Shawez Khan

PROVIDER: E-MTAB-8077 | ArrayExpress | 2020-02-13

SECONDARY ACCESSION(S): ERP115884

REPOSITORIES: ArrayExpress, ENA

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Publications

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

Kalucka Joanna J de Rooij Laura P M H LPMH Goveia Jermaine J Rohlenova Katerina K Dumas Sébastien J SJ Meta Elda E Conchinha Nadine V NV Taverna Federico F Teuwen Laure-Anne LA Veys Koen K García-Caballero Melissa M Khan Shawez S Geldhof Vincent V Sokol Liliana L Chen Rongyuan R Treps Lucas L Borri Mila M de Zeeuw Pauline P Dubois Charlotte C Karakach Tobias K TK Falkenberg Kim D KD Parys Magdalena M Yin Xiangke X Vinckier Stefan S Du Yuxiang Y Fenton Robert A RA Schoonjans Luc L Dewerchin Mieke M Eelen Guy G Thienpont Bernard B Lin Lin L Bolund Lars L Li Xuri X Luo Yonglun Y Carmeliet Peter P

Cell 20200213 4

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues ...[more]

PMID: 32059779

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Project description:BACKGROUND:Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically. METHODS:We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function. RESULTS:We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors. CONCLUSION:ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.