Activin Is Superior to BMP7 for Efficient Maintenance of Human iPSC-Derived Nephron Progenitors.

Kidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFß signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8+/PDGFRA-/SIX2-GFP+ NPCs by 5-fold per week at 80%-90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening.

PAX2 is dispensable for in vitro nephron formation from human induced pluripotent stem cells.

The kidney is formed by reciprocal interactions between the nephron progenitor and the ureteric bud, the former of which gives rise to the epithelia of nephrons consisting of glomeruli and renal tubules. The transcription factor PAX2 is essential for this mesenchymal-to-epithelial transition of nephron progenitors, as well as ureteric bud lineage development, in mice. PAX2 mutations in humans cause renal coloboma syndrome. We previously reported the induction of nephron progenitors and three-dimensional nephron structures from human induced pluripotent stem (iPS) cells. Here we generate iPS cells lacking PAX2, and address the role of PAX2 in our in vitro induction protocol. While PAX2-null human nephron progenitors were properly formed, they unexpectedly became epithelialised to form glomeruli and renal tubules. However, the mutant glomerular parietal epithelial cells failed to transit to the squamous morphology, retaining the shape and markers of columnar epithelia. Therefore, PAX2 is dispensable for mesenchymal-to-epithelial transition of nephron progenitors, but is required for morphological development of glomerular parietal epithelial cells, during nephron formation from human iPS cells in vitro.

Non-muscle myosin II deletion in the developing kidney causes ureter-bladder misconnection and apical extrusion of the nephric duct lineage epithelia.

In kidney development, connection of the nephric duct (ND) to the cloaca and subsequent sprouting of the ureteric bud (UB) from the ND are important for urinary exit tract formation. Although the roles of Ret signaling are well established, it remains unclear how intracellular cytoskeletal proteins regulate these morphogenetic processes. Myh9 and Myh10 encode two different non-muscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases. Here we report that ND/UB lineage-specific deletion of Myh9/Myh10 in mice caused severe hydroureter/hydronephrosis at birth. At mid-gestation, the mutant ND/UB epithelia exhibited aberrant basal protrusion and ectopic UB formation, which likely led to misconnection of the ureter to the bladder. In addition, the mutant epithelia exhibited apical extrusion followed by massive apoptosis in the lumen, which could be explained by reduced apical constriction and intercellular adhesion mediated by E-cadherin. These phenotypes were not ameliorated by genetic reduction of the tyrosine kinase receptor Ret. In contrast, ERK was activated in the mutant cells and its chemical inhibition partially ameliorated the phenotypes. Thus, myosin II is essential for maintaining the apicobasal integrity of the developing kidney epithelia independently of Ret signaling.

Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.

Sall1 in renal stromal progenitors non-cell autonomously restricts the excessive expansion of nephron progenitors.

The mammalian kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud, the former of which contains nephron progenitors. The third lineage, the stroma, fills up the interstitial space and is derived from distinct progenitors that express the transcription factor Foxd1. We showed previously that deletion of the nuclear factor Sall1 in nephron progenitors leads to their depletion in mice. However, Sall1 is expressed not only in nephron progenitors but also in stromal progenitors. Here we report that specific Sall1 deletion in stromal progenitors leads to aberrant expansion of nephron progenitors, which is in sharp contrast with a nephron progenitor-specific deletion. The mutant mice also exhibited cystic kidneys after birth and died before adulthood. We found that Decorin, which inhibits Bmp-mediated nephron differentiation, was upregulated in the mutant stroma. In contrast, the expression of Fat4, which restricts nephron progenitor expansion, was reduced mildly. Furthermore, the Sall1 protein binds to many stroma-related gene loci, including Decorin and Fat4. Thus, the expression of Sall1 in stromal progenitors restricts the excessive expansion of nephron progenitors in a non-cell autonomous manner, and Sall1-mediated regulation of Decorin and Fat4 might at least partially underlie the pathogenesis.

Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.

Recapitulating three-dimensional (3D) structures of complex organs, such as the kidney, from pluripotent stem cells (PSCs) is a major challenge. Here, we define the developmental origins of the metanephric mesenchyme (MM), which generates most kidney components. Unexpectedly, we find that posteriorly located T(+) MM precursors are developmentally distinct from Osr1(+) ureteric bud progenitors during the postgastrulation stage, and we identify phasic Wnt stimulation and stage-specific growth factor addition as molecular cues that promote their development into the MM. We then use this information to derive MM from PSCs. These progenitors reconstitute the 3D structures of the kidney in vitro, including glomeruli with podocytes and renal tubules with proximal and distal regions and clear lumina. Furthermore, the glomeruli are efficiently vascularized upon transplantation. Thus, by reevaluating the developmental origins of metanephric progenitors, we have provided key insights into kidney specification in vivo and taken important steps toward kidney organogenesis in vitro.

Islet1 deletion causes kidney agenesis and hydroureter resembling CAKUT.

Islet1 (Isl1) is a transcription factor transiently expressed in a subset of heart and limb progenitors. During studies of limb development, conditional Isl1 deletion produced unexpected kidney abnormalities. Here, we studied the renal expression of Isl1 and whether it has a role in kidney development. In situ hybridization revealed Isl1 expression in the mesenchymal cells surrounding the base of the ureteric bud in mice. Conditional deletion of Isl1 caused kidney agenesis or hypoplasia and hydroureter, a phenotype resembling human congenital anomalies of the kidney and urinary tract (CAKUT). The absence of Isl1 led to ectopic branching of the ureteric bud out from the nephric duct or to the formation of accessory buds, both of which could lead to obstruction of the ureter-bladder junction and consequent hydroureter. The abnormal elongation and poor branching of the ureteric buds were the likely causes of the kidney agenesis or hypoplasia. Furthermore, the lack of Isl1 reduced the expression of Bmp4, a gene implicated in the CAKUT-like phenotype, in the metanephric region before ureteric budding. In conclusion, Isl1 is essential for proper development of the kidney and ureter by repressing the aberrant formation of the ureteric bud. These observations call for further studies to investigate whether Isl1 may be a causative gene for human CAKUT.