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

Abstract

Controlling the time and place of nephron formationin vitrowould 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 nichesin 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.

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

“…4D , Note S5 ). Whether stromal intercalation during the post-branching phase causes or results from nephron progenitor differentiation and segregation from the cap mesenchyme is a point for future study 38 .…”

“…While requiring validation, this explanation is attractive given the rhythmic variation in cell-cell/cell-matrix adhesion-related transcripts in our cap mesenchyme GeoMx data including Tnc , Itga8 , and Tgfbi 36,47,48,50,51,100 . Alternatively or in conjunction, life-cycle-correlated intercalation of stromal cells via protrusion into the niche may confer contact-dependent pro-differentiation signaling in nephron progenitors 20,23,38,52,62,101 .…”

“…4D, Note S5). Whether stromal intercalation during the post-branching phase causes or results from nephron progenitor differentiation and segregation from the cap mesenchyme is a point for future study 38 . Turning to cell renewal, our GSEA and curated-gene analyses suggested that nephron progenitors in postbranching niches may be more proliferative (Fig.…”

“…1C,D), potentially through the effect of its curvature on Wnt9b concentration gradients 35 , although this is debated 30 . Indeed, any life-cycle-dependent change in nephron progenitor contact area with the ureteric bud or stroma 20 , 'mixing' state among these compartments [36][37][38] , or niche mechanical microenvironment 29 are reasonable hypotheses yet to be disproven. renderings of the mouse embryonic kidney ureteric bud tubule tree at embryonic day 14 (E14) and example niches at its tips at E17.…”