Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however , confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts , we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor ␤ 1 treatment. However , using either red fluorescent protein or ␤-galactosidase as fate markers , we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus , although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis , FoxD1-positive( ؉ ) mesenchymal cells give rise to adult CD73 ؉ , platelet derived growth factor receptor ␤ ؉ , smooth muscle actin-negative interstitial pericytes , and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin ؉ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease. (Am J Pathol

SUMMARY Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process is unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell-types of the main body of the nephron, during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell-autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) would enable organ regeneration, disease modeling, and drug screening in vitro. We established an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-pattern into nephron structures. In both 2D and 3D culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.