Single-cell dissections of Rosette-to-Lumen stage embryoids - and the Glucose-6-phosphate isomerase-induced arthritis model

Abstract

Single-cell omics (SCO), particularly single-cell RNA-sequencing (scRNA-seq), are widely used to describe dynamic biological and pathophysiological processes. This thesis explores the utility of SCO in characterizing dynamic processes in the in vitro generation of embryo-like structures and a pathophysiological condition in tissue usually sparse in cells, the joint. More specifically, scRNA-seq was used to analyze the transcriptional signatures of cells obtained from a new stem-cell-based embryo model, the Rosette-to-Lumen stage embryoids (RtL-embryoids) and an established glucose-6-phosphate isomerase (G6PI) regulatory T cells (Tregs) depletion model for the induction of acute and chronic arthritis. <br /> In 3 dimensional (3D) cell culture environments, blastocyst-derived stem cell lines are self-organized into embryolike structures. Using a 3D cell co-culture system, embryo-like structures can be generated based on transcription factor-mediated reprogramming of embryonic stem cells. In these cultures, embryonic stem cells self-organize into compartmentalized, elongated structures that mimic the inner regions of the early postimplantation embryos. The Smart-Seq 2 scRNA-seq protocol was used to identify transcriptomic profiles similar to epiblasts, primitive-/visceral endoderm, and extraembryonic ectoderms of early murine embryos around E4.5 to E5.5. SCOs revealed how stem-cell-based embryos progressed from rosette formation to lumenogenesis, followed by epiblast-like cells development from naïve- to primed pluripotency. In addition, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells were observed respectively in epiblast- or visceral endoderm-like compartments. Applying SCO to RtL-embryoids led to new findings in early embryogenesis. <br /> Murine models of induced arthritis are valuable tools for studying the pathogenic process of arthritic inflammation (ArInf). For example, G6PI-induced arthritis is a spontaneously remitting experimental arthritis model in which the depletion of Tregs can further modulate to induce non-remitting, chronic, and destructive arthritis. The Seq-Well scRNA-seq platform was applied to describe mesenchymal and immune cells in ArInf. Sampling over time allowed us to define dynamic processes of transcription in individual cells during ArInf. Here, an increased gradient of Notch signaling was identified between sublining fibroblasts (SLFs) and lining layer fibroblasts (LLFs). Furthermore, in ArInf, a counteractive <em>Prg4</em> gradient was observed in fibroblasts of the synovium, which was tightly restricted to the lining layer of fibroblasts in the healthy synovium. This counteractive <em>Prg4</em> gradient was connected to the loss of synovial barrier integrity formed by a distinct population of Cx3cr1⁺ tissue-resident macrophages, which get lost in ArInf. Arthritis remission and expansion of this specialized macrophage subcluster resulted in a reduced expression of <em>Prg4</em> in the SLFs. ScRNA-seq provided a cellular framework for understanding the pathomechanisms of ArInf, which is a prerequisite for developing new therapies for this disease. <br /> In conclusion, scRNA-seq provided considerable insight into the development of RtL-embryoids but is equally suited to identifying rare cell populations in complex tissue under pathophysiological conditions.