Poster BB2 - RHYTHMIC NEPHRON PROGENITOR RENEWAL AND DIFFERENTIATION INFORMS KIDNEY TISSUE ENGINEERING STRATEGIES

Introduction: Nephrons filter blood, removing waste to the ureteric bud (UB)-derived collecting ducts. Limited endogenous nephron regeneration leaves millions of people prone to chronic kidney disease each year. However, both dialysis and kidney transplantation affect quality of life and introduce adverse health outcomes. Engineering autologous replacement tissue is an emerging alternative strategy. Bioengineering efforts have begun addressing requirements for controlled nephron induction, maturation, and connection to a collecting duct network, however nephron generation falls substantially short of tissue replacement needs.

Differentiation protocols lack repeated nephron induction that occurs during kidney development. In vitro, nephrogenesis occurs as a single event with remaining nephron progenitor (NP) pools diminishing over time. In vivo, NP pools sit adjacent to UB tips in the cap mesenchyme (CM). A portion of each CM undergoes nephrogenesis approximately every UB tip branching cycle while remaining NPs expand and divide among the daughter UB tips for future nephrogenesis. Exponential duplication of nephrogenic niches through UB branching and balanced NP renewal and differentiation determine final nephron number, however the mechanisms balancing NP fate are not fully resolved.

Due to the apparent synchronization of UB branching with nephrogenesis, we used spatial transcriptomics to evaluate correlations between UB branching lifecycle and NP state. Then we perturbed phase-specific pathways in human induced pluripotent stem cell (iPSC)-derived NPs to evaluate the balance of NP differentiation and renewal. Engineering rhythmic control over NP fate may enable successive nephrogenesis for kidney replacement tissues, finally contributing a third arm to therapy beyond dialysis and transplantation.

Materials and

Methods: Spatial transcriptomics: We profiled 47 disparate CM from an embryonic day (E)17 kidney using NanoString GeoMx Digital Spatial Profiler (GeoMx). CM were qualitatively pseudo-aged relative to UB lifecycle (pre-, mid-, and post-branching). GeoMx data was analyzed in R using code modified from Bioconductor and NanoStringNCTools, GeomxTools, and GeoMxWorkflows libraries for differential gene expression analyses.

Organoid culture: iPSCs were plated at 5,200 cells/cm2 in mTeSR plus media (D-1). Media was switched to TeSR-E6 plus (TeSR-E6+) with 7 μM CHIR99021 (media A) from D0-D5. From D5-D10, media was switched to TeSR-E6+ with 1 μg/mL heparin and 200 ng/mL FGF9 (media B). On D10, cells were lifted and spotted in 1 μl droplets (~3x105 cells) onto a 0.4 μm polyester transwell membrane. Organoids were ‘pulsed’ with media A for 2 hr. Then they were ‘chased’ with media B for 2 hr. Pulse and chase media included 0.1 μM TTNPB (retinoic acid activation), 4 μM TRULI (YAP activation), or equivolume DMSO. Media was switched to media B post chase until D12 and finally switched to TeSRE6+ until D25.

Immunofluorescence: Organoids were fixed at D12 or D25. Organoids were permeabilized and blocked at room temperature but incubated in antibody solutions overnight at 4°C. D12 organoids were counterstained with 1μg/mL DAPI.

Imaging and analysis: Z-stacks were acquired on a spinning-disk confocal microscope. A single, central z-plane for each organoid was manually thresholded in FIJI for percent area quantification. One- way ANOVA and Tukey’s post-hoc tests were performed in R.

Results, Conclusions, and Discussions:

Results: Our GeoMx analysis revealed elevated WNT, retinoic acid (RA), JAK-STAT, and TGFb signaling in pre-/mid- branching CM and elevated YAP, FGF, and NOTCH signaling in post-branching CM. Whole mount immunofluorescence of E17 kidneys showed similar trends at the protein level. Using a published whole kidney single cell RNA-seq dataset, mean normalized expression of post-branching GeoMx hits largely associated with uncommitted NP clusters while pre/mid- branching GeoMx hits associated with differentiating NP clusters. We then evaluated how phase specific pathways (YAP or RA) influenced NP fate in response to inductive cues using iPSC-derived NP organoids (iNP). Transient b-catenin activation with a GSK3b inhibitor, CHIR99021 (CHIR), was used to induce iNPs. YAP activation significantly increased the NP (SIX2+) area when overlapped with CHIR and reduced both early nephron (JAG1+) and mature nephron (ECAD+, NPHS1+, GATA3+, and LTL+) area independent of timing. RA activation significantly reduced NP area but had no change in early or mature nephron area independent of timing.

Discussion: Our work suggests that NPs rhythmically fluctuate between phases of renewal and differentiation synchronized with UB branching. Our previous work suggests that UB branching induces mechanical stress in the nephrogenic niche9. Our current work suggests YAP signaling, a central mechanosensitive pathway, is activate in the post-branching niche and promotes maintenance of iNPs even in the presence of inductive cues. This suggests mechanical stress from UB branching rhythmically activates YAP signaling in the CM which may promote NP renewal. This is in line with our previous report that nephrogenesis pauses briefly post UB branching. Here, we found that RA activation decreased iNP maintenance and may promote nephron induction. Less is known about the role of RA signaling in NPs, but neighboring CM are separated by a stromal progenitor population that actively secrete RA10. This suggests UB branching may regulate stromal derived cues to influence NP fate.

Conclusion: This work provides new insight on the regulation of NP fate. Rhythmic activation of ancillary pathways may promote iPSC-derived nephron cultures capable of repeated nephron induction. This would massively increase nephron formation for kidney replacement tissue.

Acknowledgements (Optional): We thank members of the Hughes laboratory for helpful discussions and assistance with experimental protocols.