Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation

Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfra+ preadipocyte- and fibroblast-like cells during pregnancy, and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. In order to determine the molecular signature of these de-differentiated adipocytes in the mammary gland, we compared these cells with classical adipocytes. Using the AdipoChaser-mT/mG system, we pre-labeled mature adipocytes with GFP expression to characterize the features of these de-differentiated adipocytes (Figure 4A), and then purified CD31-/CD45-/PDGFRa+/Tomato+ and CD31-/CD45-/PDGFRa+/GFP+ cells from the stromal vascular fraction (SVF) of lactating mammary gland at the peak of lactation through FACS. Gene expression analyses showed that the CD31-/CD45-/PDGFRa+/Tomato+ cells were indeed enriched with Tomato expression, while the CD31-/CD45-/PDGFRa+/GFP+ cells were enriched with GFP expression (Figure 4C). We then collected CD31-/CD45-/PDGFRa+/GFP+ cells as single cells for subsequent single cell RNA-sequencing analysis (Figure 4D-G, Supplemental. Figure S1A-G). After the flow sorting and single cell RNA amplification, 26 CD31-/CD45-/PDGFRa+/GFP+ cells passed the quality control, and these cells were used for single-cell RNA-sequencing analysis. Due to technical difficulties in sorting single mature white adipocyte through flow cytometry, adipocytes differentiated from the immortalized murine-derived brown pre-adipocyte cell line were used as mature adipocyte control (Pradhan et al., 2017). Additionally, we also included population RNA-seq experiments, i.e. three mature white adipocyte samples, two GFP+, and six GFP- ones.