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Abstract: TH-PO642

Identification of Nephron Progenitor Cell (NPC) Markers Derived from Human Embryonic Stem Cells (hESCs) by Single-Cell RNA Sequencing

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Nguyen, Mien, Duke-NUS Medical School, Singapore, Singapore
  • Ouyang, John F., Duke-NUS Medical School, Singapore, Singapore
  • Rackham, Owen J l, Duke-NUS Medical School, Singapore, Singapore
  • Tryggvason, Karl, Duke-NUS Medical School, Singapore, Singapore
Background

The steady rise of chronic kidney diseases has caused a major burden for the healthcare systems worldwide. Yet, most of their molecular pathomechanisms are understudied due to the lack of functional model systems. In vitro modeling of kidney diseases and nephrotoxicity drug testing mainly employ immortalized kidney cell lines that do not truly represent their in vivo counterparts, or rely on limited sources of mouse and human primary kidney cells that de-differentiate in culture. hESC-derived NPCs, in contrast, may provide unlimited cell source for the generation of functional kidney disease models. Most current protocols differentiate pluripotent cells to various kidney cell types that aggregate into kidney organoids. These systems utilize undefined matrices and xenogenic products with high variability, making it difficult to study molecular disease mechanisms. The objective of this study was to generate a pure population of NPCs from hESCs in a chemically defined system through the identification of NPC markers.

Methods

hESCs were differentiated on human recombinant laminin toward mesoderm, metanephric mesenchyme and NPC lineage in the presence of relevant Wnt signaling molecule.

Results

By day 30, we observed 82% WT1+ in total cell population via FACS analysis. Quantitative RT-PCR showed significant increases in mRNA expression of kidney lineage markers, in particular those of podocytes (OSR1, WT1, SYNPO, and NPHS1), and significant decreases in early metanephric mesenchymal markers from day 6 to day 30 (CD133 and CD24). We performed single-cell RNA sequencing on hESC-derived cells at different time points to establish signature surface markers that could allow us to purify and enrich for NPC population. From the analysis of the single-cell transcriptome, we have identified a set of genes whose gene expression profiles are highly correlated with those of podocyte lineage genes. Among those, we have selected potential surface markers for NPC enrichment, such as CD83, PLP2, and TM7SF2.

Conclusion

Our highly reproducible protocol allows efficient production of hESC-derived NPCs in monolayer in a chemically defined, xeno-free culture. These cells promise to provide an unlimited source to construct kidney disease models for analyzing drug clearance and toxicity, and for studying molecular disease mechanisms.

Funding

  • Government Support - Non-U.S.