RNAseq analysis of bone marrow endothelial cells

Although endothelial cells (ECs) have been shown to contribute to HSC maintenance in bone marrow (BM), differential contributions of EC subtypes remain unknown, owing to the lack of methods to separate with high purity arterial (AEC) from sinusoidal (SEC) endothelial cells. We show that combination of podoplanin (PDPN) and Sca-1 expression distinguishes AEC from SEC where Sca1brightPDPN—CD45—Ter119— cells exhibit an arterial gene signature and PDPN+Sca1dimCD45—Ter119— marks sinusoids. PDPN can be substituted for antibodies against the adhesion molecules ICAM1 or E-selectin that also mark SEC. We performed functional analysis of these different types of endothelial cells and found that SCF secreated from AECs regulate HSCs. We sorted live, singlet, CD45-Ter119-CD31+PDPN-Sca-1brightNestin-GFPdim artery endothelial cells (AECs), and live, singlet, CD45-Ter119-CD31+PDPN-Sca-1bright Nestin-GFPbright AECs and live, singlet, CD45-Ter119-CD31+PDPN+Sca-1dim sinusoidal endotehlial cells(SECs) from digested bone marrow cells from Nestin-GFP. RNA was extracted, amplification and sequenced on Hiseq 2500 in Single Cell Genomics and Epigenomics core facility at Albert Einstein College of Medicine.