Single cell approaches to address adipose tissue stromal cell heterogeneity

Abstract

A central function of adipose tissue is in the management of systemic energy homeostasis that is achieved through the co-ordinated regulation of energy storage and mobilization, adipokine release, and immune functions. With the dramatic increase in the prevalence of obesity and obesity-related metabolic disease over the past 30 years, there has been extensive interest in targeting adipose tissue for therapeutic benefit. However, in order for this goal to be achieved it is essential to establish a comprehensive atlas of adipose tissue cellular composition and define mechanisms of intercellular communication that mediate pathologic and therapeutic responses. While traditional methods, such as fluorescence-activated cell sorting (FACS) and genetic lineage tracing, have greatly advanced the field, these approaches are inherently limited by the choice of markers and the ability to comprehensively identify and characterize dynamic interactions among stromal cells within the tissue microenvironment. Single cell RNA sequencing (scRNAseq) has emerged as a powerful tool for deconvolving cellular heterogeneity and holds promise for understanding the development and plasticity of adipose tissue under normal and pathological conditions. scRNAseq has recently been used to characterize adipose stem cell (ASC) populations and has provided new insights into subpopulations of macrophages that arise during anabolic and catabolic remodeling in white adipose tissue. The current review summarizes recent findings that use this technology to explore adipose tissue heterogeneity and plasticity.

Keywords: plasticity; single cell RNA-seq; stromal cells; white adipose tissue.

Figure 1.. Schematic Diagram of Basic Steps in Droplet-Based Microfluidic scRNAseq.

Cells and DNA-barcoded beads are partitioned into a microfluidic device and mixed with oil to generate nanoliter-sized single cell droplets. Each bead contains primers, cellular barcodes, and a unique molecular identifier (UMI). Cells are lysed within droplets, and reverse transcription either takes place within the droplets or following primer annealing and demulsification. Downstream steps differ slightly between platforms (not shown) to generate libraries for sequencing. Following sequencing, reads are demultiplexed, aligned, and then UMI counts per cell are quantified. Modified from (78).

Figure 2.. Integrated scRNAseq of Immune-Depleted Stromal Cells Isolated from eWAT of Adult Mice.

Publicly available scRNAseq datasets (64, 104) from immune-depleted stromal cells isolated from eWAT were integrated using Seurat (105). (A) tSNE plots displaying the overlay of integrated datasets (left) and cell cluster identification (right). (B) tSNE plots displaying the log2 expression levels for select genes commonly used to purify adipose stem cells by FACS and/or used in lineage tracing experiments. (C) Violin plots displaying relative log2 expression levels of select marker genes in individual clusters. Abbr: Ly6a (stem cell antigen-1 (Sca-1)), Itgb1 (CD29).

Figure 3.. Integrated scRNAseq of Immune-Depleted Stromal Cells Isolated from iWAT of Adult Mice.

Publicly available scRNAseq datasets (64, 102, 103) from immune-depleted stromal cells isolated from iWAT were integrated using Seurat (105). (A) tSNE plots displaying the overlay of integrated datasets (left) and cell cluster identification (right). (B) tSNE plots displaying the log2 expression levels for select genes commonly used to purify adipose stem cells by FACS and/or used in lineage tracing experiments. (C) Violin plots displaying relative log2 expression levels of select marker genes in individual clusters. Abbr: Ly6a (stem cell antigen-1 (Sca-1)), Itgb1 (CD29).

Figure 4.. Immunohistochemical Staining of Total ASC (Pdgfrα) and ASC2 (DPP4, Pi16) in eWAT and iWAT of Adult Male Mice.

(A, B) Immunofluorescence for Pdgfrα (green) and either DPP4 (red, top panel) or Pi16 (red, bottom panel) was evaluated in formaldehyde-fixed tissue sections (10 μM) from (A) eWAT and (B) iWAT. (A) ASC2 cells (Pdgfrα+DPP4+ or PI16+) cells are localized in areas underlying the mesothelium in eWAT. Note the strong immunoreactivity of DPP4 surrounding eWAT, which is also expressed in mesothelial cells from scRNAseq (Fig. 2C). (B) ASC2 progenitors are localized primarily within the fascia that encapsulates the inguinal adipose depot and is also present in a thin layer of connective tissue lining individual fat lobules. ASC1 cells (Pdgfrα+DPP4-) are localized within the parenchyma body in association with mature adipocytes and surrounding vessels. DAPI was used for nuclear staining as indicated in blue. Orange arrows denote some areas immunoreactive for both Pdgfrα and either DPP4 or Pi16. Abbr: Mesothelium (Mesoth.).