Abstract: PO0636
Synthetic Peptide Hydrogels as Support Matrices for the Generation of Distinct Cell Populations Within Induced Pluripotent Stem Cell-Derived Kidney Organoids
Session Information
- Development, Stem Cells, and Regenerative Medicine
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Treacy, Niall, University College Dublin School of Biomolecular and Biomedical Science, Dublin, Ireland
- Clerkin, Shane, University College Dublin School of Biomolecular and Biomedical Science, Dublin, Ireland
- Davis, Jessica L., University College Dublin School of Biomolecular and Biomedical Science, Dublin, Ireland
- Kennedy, Ciarán, University College Dublin School of Biomolecular and Biomedical Science, Dublin, Ireland
- Wychowaniec, Jacek K., University College Dublin School of Chemistry and Chemical Biology, Dublin, Ireland
- Brougham, Dermot F., University College Dublin School of Chemistry and Chemical Biology, Dublin, Ireland
- Crean, John, University College Dublin School of Biomolecular and Biomedical Science, Dublin, Ireland
Background
Kidney organoids that display improved maturity with reduced variability are needed to produce models that faithfully mirror the in vivo organ. We propose that organoid development will benefit from the biophysical support provided by a tunable, fully defined microenvironment. Synthetic self-assembling peptide hydrogels are ideal systems to support organoid development that more accurately mimics the in vivo environment due to the simplicity of the structure formed at the molecular level, their low immunogenicity, cell-retention, biodegradability, and tuneable mechanical properties.
Methods
Peptide hydrogel properties were investigated via transmission electron microscopy and rheology. Organoids were characterized by immunofluorescence and single cell RNA sequencing using the 10x Genomics platform.
Results
The self-assembling peptide hydrogels (SAPHs) were comprised of a fibrous structural architecture similar to that of natural polymer networks. The mechanical properties of the SAPHs were dynamic with Alpha4 increasing in G’ stiffness over time while Alpha5 reached a peak G’ on day 3 of media conditioning. Monolayer differentiation saw the loss of pluripotency markers, transient expression of primitive streak marker brachyury, and high levels of intermediate mesoderm markers PAX2 and HOXD11 by day 7. Suspension culture for 48 hours resulted in compacted pellets that allowed for encapsulation on day 9. By day 24 organoids displayed high levels of viability and were shown to have functionality through dextran uptake. Immunostaining confirmed the generation of key cell types of the maturing nephron. Organoids contained WT1+ve podocytes, LTL+ve proximal and ECAD+ve distal tubules, laminin+ve basement membrane and meis1/2/3+ve interstitial cells. scRNA-seq revealed distinct clusters comprising nephron and stromal compartments. Interestingly, growth within the SAPHs resulted in varying proportions between these two cell types particularly with increased stromal cells seen in Alpha4 organoids.
Conclusion
We propose that peptide hydrogels due to their defined and tunable nature provide an alternative to animal-derived support matrices. These results further support the use of designer matrices that will improve iPSC differentiation towards renal cell fate trajectories.
Funding
- Commercial Support –