Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M-/-CIITA-/- CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their Fc, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.
© 2023. The Author(s).

Decreased left ventricle (LV) function caused by genetic mutations or injury often leads to debilitating and fatal cardiovascular disease. LV cardiomyocytes are, therefore, a potentially valuable therapeutical target. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are neither homogeneous nor functionally mature, which reduces their utility. Here, we exploit cardiac development knowledge to instruct differentiation of hPSCs specifically toward LV cardiomyocytes. Correct mesoderm patterning and retinoic acid pathway blocking are essential to generate near-homogenous LV-specific hPSC-CMs (hPSC-LV-CMs). These cells transit via first heart field progenitors and display typical ventricular action potentials. Importantly, hPSC-LV-CMs exhibit increased metabolism, reduced proliferation, and improved cytoarchitecture and functional maturity compared with age-matched cardiomyocytes generated using the standard WNT-ON/WNT-OFF protocol. Similarly, engineered heart tissues made from hPSC-LV-CMs are better organized, produce higher force, and beat more slowly but can be paced to physiological levels. Together, we show that functionally matured hPSC-LV-CMs can be obtained rapidly without exposure to current maturation regimes.
Crown Copyright © 2023.

Flow cytometry is a powerful method, widely used to identify cell types present in tissues, to describe their phenotypes, and to purify cells for functional analyses. As a single cell technique, flow cytometry relies on identifying and excluding cell doublets and aggregates present in samples in the initial gating steps. This identification is based on detection of events generating electrical pulses falling outside of linear variations of pulse height, width, and area in a singlet population with increasing cell sizes. In heterogeneous cell mixtures, however, with cell types varying extensively in size and granularity, exclusion of doublets has the risk of removing single cells that co-localize with doublets of another cell type. This is particularly the case when doublets of a smaller cell type overlap with large cells of a distinct, larger cell type. Here, we describe a gating method to reduce this risk. In this protocol, initial gating steps aim to segregate cells according to physical characteristics (such as size and granularity) and gene expression properties in order to obtain more homogeneous cell clusters. Doublet exclusion is then performed separately in each cluster, minimizing the risk of confusion between single cells and doublets. To illustrate this protocol, human blood monocytes are separated and analyzed. By implementing this protocol, we were able to reveal the existence of a population of large monocytes previously unrecognized using conventional gating strategies. In subsequent functional assays, we have shown that this novel population exhibits unique inflammatory responses, highlighting the need and pertinence of this approach to identify and characterize infrequent-yet functionally relevant-cell populations present in complex cell mixtures. © 2021 Wiley Periodicals LLC. Basic Protocol: Distinguishing single cells from doublets in heterogeneous cell mixtures by flow cytometry.
© 2021 Wiley Periodicals LLC.

The efficacy of immune checkpoint inhibitors (ICIs) remains unexpected and in some patients the resistance to anti-programmed death-1 (anti-PD-1) and anti-programmed death ligand 1 (anti-PD-L1) agents is observed. One of possible explanation may be PD-L2 activity. PD-1 ligands: PD-L1 and PD-L2 are present on cancer cells but also, not without significance, on alveolar macrophages (AMs) contributing to immune-suppression in the tumor microenvironment. The aim of this study was to analyse PD-L2, PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) in relation to PD-1 positive T lymphocytes.
Seventeen patients with lung cancer were investigated. BALF cells from the lung with cancer (clBALF) and from the opposite "healthy" lung (hlBALF) and peripheral blood (PB) lymphocytes were investigated. Flow cytometry method was used.
We found that 100% of CD68+ AMs from the clBALF were PD-L1 and PD-L2-positive. Unexpectedly, fluorescence minus one (FMO) PD-L1 and PD-L2 stained controls and isotype controls also showed strong autofluorescence. The hlBALF AMs exhibited a similar PD-L1 and PD-L2 autofluorescence. The median proportion of PD-1+ T lymphocytes was higher in the clBALF than the hlBALF and PB (28.9 vs. 23.4% vs. 15.6%, P=0.0281).
We discussed the opportunities of exploring the PD-1-PD-L1/PD-L2 pathway in the lung cancer environment, which may help to find new potential biomarkers for immunotherapy. We concluded that precise identification by flow cytometry of macrophages in the BALF is possible, but our study showed that the autofluorescence of macrophages did not allow to assess a real expression of PD-L2 as well as PD-L1 on AMs.
2021 Translational Lung Cancer Research. All rights reserved.