Droplet-based Single-cell RNA Sequencing Research Articles

Abstract 472: Large-Scale Single-Cell RNA-Seq Identifies Heterogeneous Populations of Human Primary and Induced Pluripotent Stem Cell-Derived Endothelial Cells

Rationale: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation and of primary endothelial cells isolated from human coronary arteries. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs sorted by CD144 antibody-conjugated magnetic sorting exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively. These findings were then cross-analyzed with the primary EC data. Conclusions: Massively parallel scRNA-seq allowed analysis of thousands of human iPSCs subjected to EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity. Cross-analysis with scRNA-seq data of human coronary artery ECs reveals transcriptional similarities and dissimilarities of iPSC-ECs to the primary ECs that will be addressed in subsequent studies.

Single-Cell RNA Sequencing of Lymph Node Stromal Cells Reveals Niche-Associated Heterogeneity

Stromal cells (SCs) establish the compartmentalization of lymphoid tissues critical to the immune response. However, the full diversity of lymph node (LN) SCs remains undefined. Using droplet-based single-cell RNA sequencing, we identified nine peripheral LN non-endothelial SC clusters. Included are the established subsets, Ccl19hi T-zone reticular cells (TRCs), marginal reticular cells, follicular dendritic cells (FDCs), and perivascular cells. We also identified Ccl19lo TRCs, likely including cholesterol-25-hydroxylase+ cells located at the T-zone perimeter, Cxcl9+ TRCs in the T-zone and interfollicular region, CD34+ SCs in the capsule and medullary vessel adventitia, indolethylamine N-methyltransferase+ SCs in the medullary cords, and Nr4a1+ SCs in several niches. These data help define how transcriptionally distinct LN SCs support niche-restricted immune functions and provide evidence that many SCs are in an activated state.

Single-cell RNA-seq identifies the neuropeptide NMU as a novel regulator of ILC2 function

Abstract Type 2 innate lymphoid cells (ILC2s) are important for both mucosal homeostasis as well as for initiation and progression of allergy and asthma. Alarmin cytokines produced by damaged or stressed epithelial cells activate ILC2s, however, the role of other pathways regulating the homeostatic versus inflammatory functions of ILC2s remain poorly defined. We generated transcriptional profiles of thousands of lung-resident ILCs isolated from mice challenged with either PBS or the alarmin cytokines IL-25 or IL-33 using massively parallel droplet-based single-cell RNA sequencing (scRNA-seq). We found significant transcriptional heterogeneity within activated ILCs and identified differentially expressed genes as potential novel regulators of ILC function. One of the genes, neuromedin U receptor 1 (NMUR1), is a receptor for the neuropeptide neuromedin U (NMU) and is specifically expressed by ILC2s and not by other immune cells. Nmur1 is particularly expressed by ILC2s at steady-state and after IL-25 treatment. In vivo NMU synergized with IL-25 to induce more severe airway inflammation than was seen with either IL-25 or NMU alone and this effect was dependent on ILCs. Mechanistically, IL-25 + NMU expanded highly proliferative and inflammatory ILCs with distinct transcriptional profiles and increased expression of IL-5 and IL-13. We observed reduced ILC2 frequency and function in NMU- or NMUR1-deficient mice after allergen challenge and found that ILCs from these mice had a lower score for an inflammatory ILC2 gene signature. Our data therefore establish a novel neuro-immune pathway that amplifies ILC2-driven airway inflammation.