Young-Woong Kim ^1,9 , Greta Zara¹, HyunJun Kang¹, Sergio Branciamore², Denis O’Meally ³, Yuxin Feng⁴, Chia-Yi Kuan⁵, Yingjun Luo ⁶, Michael S. Nelson ⁷, Alex B. Brummer^2,10, Russell Rockne ², Zhen Bouman Chen ^6,8, Yi Zheng⁴, Angelo A. Cardoso^3,8 & Nadia Carlesso ^1,8

¹Department of Stem Cell Biology and Regenerative Medicine, Gehr Family Center for Leukemia Research, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. 
²Department of Computational and Quantitative Medicine, Division of Mathematical Oncology, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. 
³Center for Gene Therapy, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. 
⁴Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 
⁵Department of Neuroscience, Center for Brain Immunology and Glia (BIG), University of Virginia School of Medicine, Charlottesville, VA 22908, USA. 
⁶Department of Diabetes Complications and Metabolism, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. 
⁷Light Microscopy Core, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA. 
⁸Irell and Manella Graduate School of Biological Sciences, Duarte, USA. 
⁹Present address: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu Daejeon 34126, Republic of Korea. 
¹⁰Present address: Department of Physics and Astronomy, College of Charleston, Charleston, SC 29424, USA

Corresponding authors: Correspondence to Young-Woong Kim or Nadia Carlesso.

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue’s homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs’ ECs to decode mechanistic information and explore therapeutics.