Kidney Week

Abstract: FR-OR097

Ureteric Bud Organoids Derived from Human Pluripotent Stem Cells Facilitate Self-Organization of Nephron Organoids

Session Information

• Stem Cells to Organoids: How We Get There
  November 03, 2017 | Location: Room 385, Morial Convention Center
  Abstract Time: 04:54 PM - 05:06 PM

Category: Developmental Biology and Inherited Kidney Diseases

• 402 Stem Cells

Authors

• Gupta, Navin R., Brigham and Women's Hospital, Boston, Massachusetts, United States

Navin R. Gupta, MD

• Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States

Joseph V. Bonventre,

• Morizane, Ryuji, Brigham and Women's Hospital, Boston, Massachusetts, United States

Ryuji Morizane,

Background

Renal epithelia arise from the reciprocal induction between two populations of cells, the ureteric bud (UB) of the anterior intermediate mesoderm (aIM) and nephron progenitor cells (NPCs) of the posterior intermediate mesoderm (pIM). We previously generated nephron organoids through the efficient induction of SIX2+ NPCs from human pluripotent stem cells (hPSCs). By design the nephron organoids were optimized for formation from pIM and hence lacked the organized collecting duct system derived from the UB, an outpouching of the Wolffian duct (WD).

Methods

A 4-step directed differentiation protocol over 7 days was designed to mimic mammalian UB development in vivo. Differentiation efficiency was evaluated by immunostaining at key intermediate cell stages. Quantification of aIM induction efficiency was performed using quantitative morphometric analysis of protein expression. hPSC-GFP lines, generated from the transduction with a CMV-GFP lentiviral construct, permitted lineage tracing in co-culture experiments with distinction between UB and nephron organoids.

Results

Following induction of mesendoderm, addition of retinoic acid, FGF2, and BMP4 induced aIM expressing up to 95% GATA3, 88% HOXB4, 78% PAX2, 74% WT1, and 71% LHX1 in both human embryonic stem cells and induced pluripotent stem cells. Subsequent treatment of aIM with noggin generated CDH1⁺PAX2⁺GATA3⁺ tubular epithelia consistent with putative Wolffian Duct (WD). Ensuing treatment of WD with GDNF and retinoic acid to induce ret signaling upregulated SALL4 in tubular epithelial structures, consistent with ureteric bud (UB) induction. Using an hESC-GFP line, 3D culture of aIM-GFP cells treated with noggin and FGFs (2, 7,10) generated presumptive WD organoids. Co-culture of WD-GFP and nephron organoids induced GFP⁺CDH1⁺GATA3⁺ tubular epithelial structures connected in series with CDH1⁺GFP^- distal segments of MM organoids, consistent with the addition of an in-series distal tubule-collecting duct structural linkage. Moreover, UB organoids provided Wnt signaling to the nephron organoids, negating the need for a transient CHIR pulse.

Conclusion

hPSC-derived UB organoids, whether alone or in co-culture with nephron organoids, will provide a human tissue tool for the study of kidney development and human disease modeling with implications for drug discovery and regenerative medicine.

Funding

• Other NIH Support