Abstract: FR-OR102

Defining Cellular Ontologies in Inducible Pluripotent Stem Cell (iPSC)-Derived Kidney Organoids by Mapping into Human Nephrogenesis via ssRNAseq

Session Information

Category: Developmental Biology and Inherited Kidney Diseases

  • 402 Stem Cells

Authors

  • Harder, Jennifer L., University of Michigan, Ann Arbor, Michigan, United States
  • Otto, Edgar A., University of Michigan, Ann Arbor, Michigan, United States
  • Menon, Rajasree, University of Michigan, Ann Arbor, Michigan, United States
  • Cebrian Ligero, Cristina, University of Michigan, Ann Arbor, Michigan, United States
  • Zou, Jizhong, NHLBI, NIH, Bethesda, Maryland, United States
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
  • Nelson, Robert G., National Institutes of Health, Phoenix, Arizona, United States
  • Kretzler, Matthias, University of Michigan, Ann Arbor, Michigan, United States
Background

Generation of kidney organoids from iPSCs offers a novel method to study genetic effects on kidney cell phenotypes, and may provide useful models of kidney development and disease. Techniques to address the variable cell heterogeneity of these organoids will enhance their value as model systems.

Methods

iPSCs were derived from peripheral blood mononuclear cells from individuals with diabetic kidney disease and kidney organoids were generated using a Matrigel sandwiching technique. Droplet-based single cell RNA-Seq (ssRNAseq) was performed on dissociated cells and sequencing data were aligned to the human genome. Downstream clustering and further analyses were performed using various R statistical packages (Seurat, SC3, Limma). Organoid single cell gene expression was compared to human fetal kidney ssRNAseq data and to publically available expression datasets.

Results

SsRNAseq signatures of cells from iPSC-derived organoids separated into 3 clusters with distinct patterns of differential gene expression reflecting differentiated kidney cell lineages: podocyte (PODXL, NPHS1, NPHS2, CLIC5), tubular (IGFBP7, SLC3A1, VILLIN1, CUBN, CLDN16), and stromal/mesangial (COL3A1, TAGLN, ACTA2). Gene expression of these clusters mapped to cell clusters from ssRNAseq analysis of early second trimester human fetal kidney. When compared to bulk RNA expression patterns of human fetal and adult tissues, iPSC-derived kidney organoid clusters overlapped prominently with microdissected developing and adult murine kidney tissues and showed kidney specificity.

Conclusion

Human iPSC-derived kidney organoid cultures contain subsets of cells with unique gene expression patterns that recapitulate distinct tissue states of developing and adult human kidney. Identification of individual cells with specific markers of interest provides both the ability to analyze co-expression of genes within an individual cell in a heterogeneous environment and a method to isolate cells of interest within organoid experiments. These results highlight the benefits of modeling human kidney disease with human iPSCs in a kidney organoid culture system and provide strategies to address the system's inherent cellular heterogeneity.

Funding

  • NIDDK Support