Abstract: TH-PO788
Characterizing Podocyte Cell Cultures and Genetic Markers of Podocyte Maturity
Session Information
- Cellular Crosstalk in Glomerular Diseases - I
October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Soare, Thomas, Goldfinch Bio, Cambridge, Massachusetts, United States
- Zhang, Wei, Goldfinch Bio, Cambridge, Massachusetts, United States
- Westerling-Bui, Amy Duyen, Goldfinch Bio, Cambridge, Massachusetts, United States
- Hoang, Hien G., Goldfinch Bio, Cambridge, Massachusetts, United States
- Tebbe, Adam, Goldfinch Bio, Cambridge, Massachusetts, United States
- Walsh, Liron, Goldfinch Bio, Cambridge, Massachusetts, United States
- Mundel, Peter H., Goldfinch Bio, Cambridge, Massachusetts, United States
- Tibbitts, Thomas T., Goldfinch Bio, Cambridge, Massachusetts, United States
Background
Podocytes are terminally differentiated cells that are vital to kidney function. Injury and loss of these cells leads to the presence of protein in the urine, scarring of the kidney, and ultimately renal failure. Conditionally-immortalized differentiated podocytes provide an in vitro model to test hypotheses regarding mechanisms of disease and responses to treatment. However, podocytes in culture lack slit diaphragms and may lack expression of certain podocyte-specific proteins, leading to doubts about maturation of cultured podocytes. Objective: To assess cultured podocytes for maturity, we characterized transcriptomic changes of podocyte differentiation at the single-cell resolution.
Methods
Undifferentiated and differentiated mouse podocytes were disassociated, prepped with a methanol fixation protocol (10X Genomics), and sequenced on an Illumina HiSeq machine (26x40). To assess transcriptomic changes associated with differentiation and passage number, cells were clustered with principal component analysis (PCA) and visualized with t-Distributed Stochastic Neighbor Embedding (tSNE). Cell clusters were labeled by visual inspection of expression of published cell-type specific markers. We leveraged publicly-available scRNAseq data of mouse kidney to identify differentially expressed genes associated with podocyte maturation and to infer the trajectory of differentiation along the podocyte lineage.
Results
We analyzed 1.8e9 reads in 17467 cells, identifying marker genes expressed in differentiated mouse podocytes. Differentiated podocytes in culture expressed markers of S-shaped bodies/early podocytes (Col4a1, Col4a2, Col4a5, Pax2) as well as markers of mature podocytes (Synpo, Podxl, Nupr1, Plce1, Foxd1). Other markers of mature podocytes were not expressed in cultured podocytes (Wt1, Nphs1, Nphs2, Col4a3, Col4a4).
Conclusion
Though differentiated podocytes in culture share transcriptomic signatures with mature podocytes in vivo, important differences remain. Future work will continue to differentiate mature mouse podocytes in culture as well as mature podocytes in human kidney organoids. Podocytes are critical to healthy kidney function. A robust in vitro model of mature podocytes is vital to assessing mechanisms of kidney disease and responses to treatment.
Funding
- Commercial Support – Goldfinch Bio