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Abstract: PO0615

The ATP-Binding Cassette Protein ABCG2 Marks Kidney Resident Endothelial Colony-Forming Cell Activity in Multiple Endothelial Clusters Identified by Single-Cell RNA Sequencing

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Myslinski, Jered, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Go, Ellen, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Collett, Jason Andrieu, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Yoder, Mervin C., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Hato, Takashi, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Basile, David P., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

Side-population cells (SP) were originally identified in hematopoietic stem cells based on their ability to efflux the DNA binding dye Hoechst 33342, an activity thought to be mediated by members of the ATP-binding cassette protein family such as ABCG2. We hypothesized that ABCG2-expressing endothelial cells (EC) are enriched in colony forming cell (ECFC) activity, and may contribute to vascular homeostasis in kidney.

Methods

The fate of ABCG2 expressing EC was investigated using adult Abcg2-CreERT X Td-tomato Rosafl/fl reporter mice (ABCG2-TT). Transgene with tamoxifen (TMX; 50 µg/g, 1X) followed by FACS analysis for TdTomato in EC. For single-cell RNA sequencing, mouse kidney ECs were isolated following digestion with collagenase, and CD45 depleted/CD31+(positive) magnetic selection. Isolated single cells were sequenced using the 10X platform. Data were analyzed with Seurat.

Results

24 hours following TMX, 2.9% of kidney EC (CD31+/CD45-) expressed TdTomato. The percentage of Td-Tomato+ EC progressively increased to 5.3% (p=0.4) by 1 week and 15.4% (p<0.0001) by 6 weeks post injection. To determine the EC subtypes expressing ABCG2-associated progenitor activity, scRNAseq was conducted on isolated kidney endothelial cells of ABCG2-TT mice 24 hours following TMX injection. A total of 10 endothelial clusters were identified. Analysis of top expressing genes suggested these clusters correspond to different kidney EC populations such as peritubular capillaries, venules, arteries, arterioles, AVR, DVR and lymphatics. The expression of the reporter was based on identification of WPRE response element expressed Rosa mice following Cre activation. Interestingly, no single discrete cluster of ECs expressing WPRE were identified. Rather, a variable percentage (4.3 to 38.7%) of WPRE expressing cells were identified in each cluster.

Conclusion

Taken together these data suggest that ABCG2+ expressing cells contribute to vascular maintenance in adult kidney and that such cells are found in most kidney EC populations. In addition, reporter expressing EC cells do not represent a transcriptionally distinct subset of EC but are transcriptionally similar to the surrounding tissue endothelial cell subsets.

Funding

  • NIDDK Support