Highly parallel production of designer organoids by mosaic patterning of progenitors.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

Nephron progenitors rhythmically alternate between renewal and differentiation phases that synchronize with kidney branching morphogenesis.

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo-Yap, retinoic acid (RA), and other pathways. We then find in human stem-cell derived nephron progenitor organoids that Wnt/ß-catenin-induced differentiation is converted to a renewal signal when it temporally overlaps with YAP activation. Similar experiments using RA activation indicate a role in setting nephron progenitor exit from the naive state, the spatial extent of differentiation, and nephron segment bias. Together the data suggest that nephron progenitor interpretation of consistent Wnt/ß-catenin differentiation signaling in the niche may be modified by rhythmic activity in ancillary pathways to set the pace of nephron formation. This would synchronize nephron formation with ureteric bud branching, which creates new sites for nephron condensation. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues.

Highly-parallel production of designer organoids by mosaic patterning of progenitors.

Human organoids are a promising approach for disease modeling and regenerative medicine. However, organoid variability and limited control over morphological outcomes remain significant challenges. Here we extend a DNA 'velcro' cell patterning approach, precisely controlling the number and ratio of human stem cell-derived progenitors contributing to nephron and mosaic nephron/ureteric bud organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints.

Rho/ROCK activity tunes cell compartment segregation and differentiation in nephron-forming niches.

Controlling the time and place of nephron formation in vitro would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches in vitro. The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.