PU.1 drives specification of pluripotent stem cell-derived endothelial cells to LSEC-like cells

Abstract

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Gene Signatures Detect Damaged Liver Sinusoidal Endothelial Cells in Chronic Liver Diseases

“…The expression of some of these markers ( STAB2 and CLEC4G ) has been shown to decrease during chronic liver disease ( 47 ). Other genes in this signature are less known but have been mentioned mainly in gene profiling studies ( OIT3, NPL ) ( 24 , 56 , 57 ). Genes that showed a higher expression after acute liver injury in mice were not included in the LSEC signature, such as LYVE1 and STAB1 .…”

Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery

“…This resulted in the creation of endothelial cells that expressed a number of typical LSEC scavenging receptors, such as FCGR2B and MRC1. More importantly, these scavenging receptors were functional as they supported uptake of FSA-FITC as well as labelled IgG, and this to the same degree as primary murine LSECs [109]. However, ETV2-SPI1 LSEC-like cells did not display fenestrations, and a full comparison with primary human LSECs was not conducted.…”

“…Despite the important role of LSECs in liver homeostasis and disease, very few protocols have aimed to create cells with LSEC features from PSCs [103,109,110] (Table 2, Figure 2D), even if multiple methods have been described to create PSC-derived endothelial cells [111,112]. Using mouse PSC, Arai et al already demonstrated in 2011 that cells with LSEC features could be generated from embryoid bodies by the modulation of andrenomedullin-RAMP2 signaling.…”

Organ-Specific Endothelial Cell Differentiation and Impact of Microenvironmental Cues on Endothelial Heterogeneity

“…The identified functions can then be tested. As for LSECs, (specific) functions that can be tested include (Figure 3C): (i) the binding, uptake and lysosomal degradation of fluorescently labeled macromolecules (reflecting the presence of scavenger receptors such as mannose receptor or MRC1, the Fc gamma receptor IIb or Stabilins) [59,107,[109][110][111][112], (ii) the binding of viral proteins (reflecting the expression of receptors interacting with these viruses such as L-SIGN) [107]; and (iii) the production of coagulation Factor VIII [110,113].…”