Abstract: FR-OR104
Defining Kidney Organoid Cell Diversity by scRNA-Seq
Session Information
- Stem Cells to Organoids: How We Get There
November 03, 2017 | Location: Room 385, Morial Convention Center
Abstract Time: 06:18 PM - 06:30 PM
Category: Developmental Biology and Inherited Kidney Diseases
- 402 Stem Cells
Authors
- Wu, Haojia, Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
- Uchimura, Kohei, Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
- Donnelly, Erinn L, Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
- Morris, Samantha A, Department of Developmental Biology, Washington University in St. Louis, Saint Louis, Missouri, United States
- Humphreys, Benjamin D., Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
Background
Kidney organoids differentiated from pluripotent stem cells hold great promise for understanding organogenesis, disease modeling and ultimately as a source of replacement tissue. Realizing this potential requires a comprehensive evaluation of organoid cell diversity and differentiation state.
Methods
We generated kidney organoids from human BJFF iPS cells (Little protocol). Proper organoid differentiation was confirmed by histology and immunofluorescence. Day 26 organoids were processed for single cell RNA-Sequencing (scRNA-Seq) using DropSeq. We sequenced 4958 cells to a final read depth of 9086 mapped reads/cell with 2137 transcripts and 1185 unique genes detected per cell. Single cell data was visualized by an unsupervised approach combining dimension reduction and graph-based clustering embedded in Seurat. Clusters were annotated by examining known markers and gene ontology analysis. Gene correlation was visualized after data transformation by diffusion based imputation.
Results
Unsupervised clustering revealed 12 separate cell types present in d26 organoids. Kidney cell types made up 61% of all cells: podocytes, proximal tubule (PT), Loop of Henle (LOH), distal convoluted tubule, myofibroblasts and fibroblasts. The epithelial cell types were immature. For example, PT cells expressed terminal differentiation markers SLC3A1, SLC34A1 but coexpressed anterior intermediate mesoderm markers LHX1 and EMX2. LOH also expressed differentiation markers (SLC12A1) and developmental markers (ID1, ID2, and ID4). Surprisingly, many known neural markers (e.g. CRABP1, MAP2, PCP4 and CNTNAP2) were uniquely expressed in four cell clusters and were absent from all kidney cell clusters, indicating distinct neuronal populations. In fact neuronal cell types made up 20.5% of the cell total. Re-analysis of the bulk RNA-seq data in Takasato et al confirmed the presence of these neuron-specific genes suggesting that neural differentiation may be a common outcome using this differentiation protocol.
Conclusion
We provide the first comprehensive scRNA-Seq analysis of 4958 kidney organoid cells. We reveal diverse kidney cell types that are immature and the unexpected emergence of neuronal identity with the kidney organoid.
Funding
- NIDDK Support