Human Ureteric Bud Organoids Derived From Pluripotent Stem Cells Recapitulate Embryonic Development and Differentiate Into Collecting Duct Cell Types With Functional Ion Transport
- Genetics, Development, and Therapies
November 05, 2022 | Location: W308, Orange County Convention Center‚ West Building
Abstract Time: 05:24 PM - 05:32 PM
Category: Development‚ Stem Cells‚ and Regenerative Medicine
- 500 Development‚ Stem Cells‚ and Regenerative Medicine
- McCracken, Kyle, Boston Children's Hospital, Boston, Massachusetts, United States
- Patel, Ankit B., Brigham and Women's Hospital, Boston, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States
The ability to derive functional ureteric and collecting duct epithelia from human pluripotent stem cells (hPSCs) will be an essential component of kidney regenerative medicine. Here we describe highly efficient methods to guide the differentiation of ureteric bud (UB) organoids and derive functional collecting duct (CD) cells from these organoids.
Directed differentiation of hPSCs was performed using sequential growth factor manipulations to recapitulate normal developmental stages including pronephric intermediate mesoderm (IM), nephric duct (ND), and UB. At day 3 IM cells were aggregated into spheroids, which at day 7 were then embedded into a 3D matrix to support branching morphogenesis. Cells from the organoids were functionally interrogated using 2D transwell culture and Ussing chambers.
hPSCs were induced into PAX2/GATA3+ pronephric IM progenitor cells at 90% efficiency, which then generated spheroids that underwent spontaneous organization and adopted a molecular phenotype consistent with the ND. In 3D culture, the spheres developed into UB organoids that exhibited branching morphogenesis and spatial organization comparable to the fetal UB. At later stages the UB organoids differentiated into CD organoids, which contained >95% CD cell types as estimated by scRNA-seq. Cells isolated from the organoids generated robust ENaC-mediated vectorial Na+ transport that was further induced by stimulation with mineralocorticoid signaling. We also showed that ectopic FOXI1 induced formation of intercalated cells (ICs), which in 2D exhibited V-type ATPase-mediated H+ secretion that produced a transepithelial pH gradient.
These methods result in efficient and consistent differentiation of UB organoids, making them accessible to the kidney research community. The organoids exhibit developmental properties including branching morphogenesis, which will enable their use in catalyzing novel regenerative medicine approaches. The derivation of both functional principal cells and ICs that exhibit Na+ and H+ transport, respectively, is the first demonstration of renal cells derived from hPSCs with advanced ion transport and electrophysiologic properties and presents an unprecedented model for interrogating CD physiology and disease.
- NIDDK Support