Endothelial contribution to COVID-19: an update on mechanisms and therapeutic implications.

The global propagation of SARS-CoV-2 leads to an unprecedented public health emergency. Despite that the lungs are the primary organ targeted by COVID-19, systemic endothelial inflammation and dysfunction is observed particularly in patients with severe COVID-19, manifested by elevated endothelial injury markers, endotheliitis, and coagulopathy. Here, we review the clinical characteristics of COVID-19 associated endothelial dysfunction; and the likely pathological mechanisms underlying the disease including direct cell entry or indirect immune overreactions after SARS-CoV-2 infection. In addition, we discuss potential biomarkers that might indicate the disease severity, particularly related to the abnormal development of thrombosis that is a fatal vascular complication of severe COVID-19. Furthermore, we summarize clinical trials targeting the direct and indirect pathological pathways after SARS-CoV-2 infection to prevent or inhibit the virus induced endothelial disorders.

Single-Cell RNA-Seq Reveals that CD9 Is a Negative Marker of Glucose-Responsive Pancreatic ß-like Cells Derived from Human Pluripotent Stem Cells.

To date, it remains unclear if there are specific cell-surface markers for purifying glucose-responsive pancreatic ß-like cells derived from human pluripotent stem cells (hPSCs). In searching for this, we generated an efficient protocol for differentiating ß-like cells from human embryonic stem cells. We performed single-cell RNA sequencing and found that CD9 is a negative cell-surface marker of ß-like cells, as most INS+ cells are CD9-. We purified ß-like cells for spontaneous formation of islet-like organoids against CD9, and found significantly more NKX6.1+MAFA+C-PEPTIDE+ ß-like cells in the CD9- than in the CD9+ population. CD9- cells also demonstrate better glucose responsiveness than CD9+ cells. In humans, we observe more CD9+C-PEPTIDE+ ß cells in the fetal than in the adult cadaveric islets and more Ki67+ proliferating cells among CD9+ fetal ß cells. Taken together, our experiments show that CD9 is a cell-surface marker for negative enrichment of glucose-responsive ß-like cells differentiated from hPSCs.

Single-cell transcriptomics uncover distinct innate and adaptive cell subsets during tissue homeostasis and regeneration.

Recently, immune cell-mediated tissue repair and regeneration has been an emerging paradigm of regenerative medicine. Immune cells form an essential part of the wound as induction of inflammation is a necessary step to elicit tissue healing. Rapid progress in transcriptomic analyses by high-throughput next-generation sequencing has been developed to study gene regulatory network and establish molecular signatures of immune cells that could potentially predict their functional roles in tissue repair and regeneration. However, the identification of cellular heterogeneity especially on the rare cell subsets has been limited in transcriptomic analyses of bulk cell populations. Therefore, genome-wide, single-cell RNA sequencing (scRNA-Seq) has offered an unprecedented approach to unravel cellular diversity and to study novel immune cell populations involved in tissue repair and regeneration through unsupervised sampling of individual cells without the need to rely on prior knowledge about cell-specific markers. The analysis of gene expression patterns at a single-cell resolution also holds promises to uncover the mechanisms and therefore the development of therapeutic strategy promoting immunoregenerative medicine. In this review, we will discuss how scRNA-Seq facilitates the characterization of immune cells, including macrophages, innate lymphoid cells and T and B lymphocytes, discovery of immune cell heterogeneity, identification of novel subsets, and tracking of developmental trajectories of distinct immune cells during tissue homeostasis, repair, and regeneration.

Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner.

The neonatal mouse heart is capable of transiently regenerating after injury from postnatal day (P) 0-7 and macrophages are found important in this process. However, whether macrophages alone are sufficient to orchestrate this regeneration; what regulates cardiomyocyte proliferation; why cardiomyocytes do not proliferate after P7; and whether adaptive immune cells such as regulatory T-cells (Treg) influence neonatal heart regeneration have less studied. Methods: We employed both loss- and gain-of-function transgenic mouse models to study the role of Treg in neonatal heart regeneration. In loss-of-function studies, we treated mice with the lytic anti-CD25 antibody that specifically depletes Treg; or we treated FOXP3DTR with diphtheria toxin that specifically ablates Treg. In gain-of-function studies, we adoptively transferred hCD2+ Treg from NOD.Foxp3hCD2 to NOD/SCID that contain Treg as the only T-cell population. Furthermore, we performed single-cell RNA-sequencing of Treg to uncover paracrine factors essential for cardiomyocyte proliferation. Results: Unlike their wild type counterparts, NOD/SCID mice that are deficient in T-cells but harbor macrophages fail to regenerate their injured myocardium at as early as P3. During the first week of injury, Treg are recruited to the injured cardiac muscle but their depletion contributes to more severe cardiac fibrosis. On the other hand, adoptive transfer of Treg results in mitigated fibrosis and enhanced proliferation and function of the injured cardiac muscle. Mechanistically, single-cell transcriptomic profiling reveals that Treg could be a source of regenerative factors. Treg directly promote proliferation of both mouse and human cardiomyocytes in a paracrine manner; and their secreted factors such as CCL24, GAS6 or AREG potentiate neonatal cardiomyocyte proliferation. By comparing the regenerating P3 and non-regenerating P8 heart, there is a significant increase in the absolute number of intracardiac Treg but the whole transcriptomes of these Treg do not differ regardless of whether the neonatal heart regenerates. Furthermore, even adult Treg, given sufficient quantity, possess the same regenerative capability. Conclusion: Our results demonstrate a regenerative role of Treg in neonatal heart regeneration. Treg can directly facilitate cardiomyocyte proliferation in a paracrine manner.

Single-cell transcriptomics reveal that PD-1 mediates immune tolerance by regulating proliferation of regulatory T cells.

BACKGROUND: We have previously reported an antigen-specific protocol to induce transplant tolerance and linked suppression to human embryonic stem cell (hESC)-derived tissues in immunocompetent mice through coreceptor and costimulation blockade. However, the exact mechanisms of acquired immune tolerance in this model have remained unclear. METHODS: We utilize the NOD.Foxp3hCD2 reporter mouse line and an ablative anti-hCD2 antibody to ask if CD4+FOXP3+ regulatory T cells (Treg) are required for coreceptor and costimulation blockade-induced immune tolerance. We also perform genome-wide single-cell RNA-sequencing to interrogate Treg during immune rejection and tolerance and to indicate possible mechanisms involved in sustaining Treg function. RESULTS: We show that Treg are indispensable for tolerance induced by coreceptor and costimulation blockade as depletion of which with an anti-hCD2 antibody resulted in rejection of hESC-derived pancreatic islets in NOD.Foxp3hCD2 mice. Single-cell transcriptomic profiling of 12,964 intragraft CD4+ T cells derived from rejecting and tolerated grafts reveals that Treg are heterogeneous and functionally distinct in the two outcomes of transplant rejection and tolerance. Treg appear to mainly promote chemotactic and ubiquitin-dependent protein catabolism during transplant rejection while seeming to harness proliferative and immunosuppressive function during tolerance. We also demonstrate that this form of acquired transplant tolerance is associated with increased proliferation and PD-1 expression by Treg. Blocking PD-1 signaling with a neutralizing anti-PD-1 antibody leads to reduced Treg proliferation and graft rejection. CONCLUSIONS: Our results suggest that short-term coreceptor and costimulation blockade mediates immune tolerance to hESC-derived pancreatic islets by promoting Treg proliferation through engagement of PD-1. Our findings could give new insights into clinical development of hESC-derived pancreatic tissues, combined with immunotherapies that expand intragraft Treg, as a potentially sustainable alternative treatment for T1D.

Genome-Wide Transcriptome Profiling of the Neoplastic Giant Cell Tumor of Bone Stromal Cells by RNA Sequencing.

Giant cell tumor of bone (GCTB) is the most common non-malignant primary bone tumor reported in Hong Kong. Failure of treatment in advanced GCTB with aggressive local recurrence remains a clinical challenge. In order to reveal the molecular mechanism underlying the pathogenesis of this tumor, we aimed to examine the transcriptome profiling of the neoplastic stromal cells of GCTB in this study. RNA-sequencing was performed on three GCTB stromal cell samples and one bone marrow-derived MSC sample and 174 differentially expressed genes (DEGs) were identified between these two cell types. The top five up-regulated genes are SPP1, F3, TSPAN12, MMP13, and LGALS3BP and further validated by qPCR and Western Blotting. Knockdown of SPP1 was found to induce RUNX2 and OPG expression in GCTB stromal cells but not the MSCs. Ingenuity pathway analysis (IPA) of the 174 DEGs revealed significant alternations in 23 pathways; variant calling analysis revealed 1915 somatic variants of 384 genes with high or moderate impacts. Interestingly, four canonical pathways were found overlapping in both analyses; from which VEGFA, CSF1, PLAUR, and F3 genes with somatic mutation were found up-regulated in GCTB stromal cells. The STRING diagram showed two main clusters of the DEGs; one cluster of histone genes that are down-regulated in GCTB samples and another related to osteoblast differentiation, angiogenesis, cell cycle progression, and tumor growth. The DEGs and somatic mutations found in our study warrant further investigation and validation, nevertheless, our study add new insights in the search for new therapeutic targets in treating GCTB. J. Cell. Biochem. 118: 1349-1360, 2017. © 2016 Wiley Periodicals, Inc.