Abstract: FR-OR096
Dissecting Lineage Relationships in Kidney Organoids
Session Information
- Stem Cells to Organoids: How We Get There
November 03, 2017 | Location: Room 385, Morial Convention Center
Abstract Time: 04:42 PM - 04:54 PM
Category: Developmental Biology and Inherited Kidney Diseases
- 402 Stem Cells
Authors
- Howden, Sara E., Murdoch Childrens Research Institute, Parkville, New South Wales, Australia
- Vanslambrouck, Jessica May, Murdoch Childrens Research Institute, Parkville, New South Wales, Australia
- Wilson, Sean, Murdoch Childrens Research Institute, Parkville, New South Wales, Australia
- Little, Melissa H., Murdoch Childrens Research Institute, Parkville, New South Wales, Australia
Background
The capacity to create a model of the developing human kidney in vitro provides a unique
opportunity to better understand nephrogenesis at the molecular level in human cells. Here we begin to characterise lineage relationships during human nephrogenesis through the generation of complex 3D kidney organoid cultures from human pluripotent stem cells (PSCs) that have been genetically modified to permit fate-mapping of specific cell lineages in real-time.
Methods
Human PSCs lines suitable for fate-mapping studies were generated by inserting a dual
fluorescence cassette (loxP-flanked EGFP and adjacent mCherry reporter genes) within the “safe-harbor” GAPDH locus (GAPDHdual). Upon expression of Cre recombinase, cells switch from constitutive EGFP (green) expression to constitutive mCherry (red) expression. To interrogate the nephron lineage within human kidney organoids and validate the utility of GAPDHdual PSCs for fate-mapping studies, we used CRISPR/Cas9 to insert the Cre recombinase gene within the endogenous SIX2 locus of GAPDHdual PSCs (GAPDHdual:SIX2Cre). Kidney organoids were generated from GAPDHdual:SIX2Cre PSCs and monitored for mCherry expression.
Results
Following the derivation of kidney organoids from GAPDHdual:SIX2Cre PSCs, mCherry-expressing
cells arise from day 10 of differentiation, coinciding with endogenous SIX2 expression. The proportion of mCherry+ cells increased steadily thereafter with a corresponding loss of EGFP-expressing cells. Importantly, subsequent analysis by immunofluorescence revealed that mCherry+ cells predominantly contributed to ECAD+ epithelial structures characteristic of the developing nephron. In constrast, mCherry+ cells were excluded from GATA3+ collecting duct. This is consistent with the lineage relationships previously demonstrated in murine kidney development.
Conclusion
Our findings validate lineage relationships of nephrons during human development and
demonstrate the feasibility of using reporter/Cre-driver PSCs for fate-mapping studies in developing kidney organoids. We are currently using CRISPR/Cas9 mediated knock-out of specific genes in these GAPDHdual:SIX2Cre PSCs to interrogate the requirement for specific genes in the formation of human nephrons. We also propose to generate several other Cre-drivers to interrogate lineage relationships of other cellular compartments within the developing kidney that remain largely unknown.