Upstroke time is a more useful marker of atherosclerosis than percentage of mean arterial pressure for detecting coronary artery disease in subjects with a normal ankle-brachial index.

Upstroke time (UT) and percentage of mean arterial pressure (%MAP) at the ankle have been shown to serve as atherosclerotic markers. The purpose of this study was to directly compare the diagnostic accuracy of UT with that of %MAP for clinical coronary artery disease (CAD) in subjects with a normal ankle-brachial index (ABI) in both legs. We measured UT and %MAP in 1953 subjects with a normal ABI. The optimal cutoff values of UT and %MAP derived from a receiver operating characteristic (ROC) curve to diagnose CAD were 148 ms and 40.4%, respectively. Multivariable analyses revealed that both UT ≥ 148 ms (odds ratio [OR], 2.72; p < 0.001) and %MAP ≥ 40.4% (OR, 1.28; p = 0.003) were significantly associated with CAD. When the subjects were divided into four groups according to the cutoff values of UT and %MAP, there was no significant difference in the risk of CAD between subjects with UT ≥ 148 ms and %MAP < 40.4% and those with UT ≥ 148 ms and %MAP ≥ 40.4% (OR, 1.45; p = 0.09). ROC curve analyses revealed that the area under the curve value of UT was significantly higher than that of %MAP (0.69 vs. 0.53, p < 0.001). The addition of UT to traditional risk factors significantly improved the diagnostic accuracy for CAD (0.82 to 0.84, p = 0.004), whereas the addition of %MAP to traditional risk factors did not improve the diagnostic accuracy for CAD (0.82 to 0.82, p = 0.84). UT is more useful than %MAP for identifying individuals with CAD among those with a normal ABI.

A Body Shape Index as a Simple Anthropometric Marker of Abdominal Obesity and Risk of Cardiovascular Events.

BACKGROUND: A Body Shape Index (ABSI) has been reported to have associations with cardiovascular risk factors. However, there is no information on the association between ABSI and incidence of cardiovascular events. METHODS: We investigated the associations between ABSI and first major cardiovascular events (death from cardiovascular disease, nonfatal acute coronary syndrome, and nonfatal stroke) in 1857 subjects from the database of Flow-Mediated Dilation Japan registry and from Hiroshima University Vascular Function registry. RESULTS: The areas under the curves of ABSI to predict the first major cardiovascular events were superior to BMI (men: P=0.032, women: P=0.015) and waist circumference in women (men: P=0.078, women: P=0.002). The subjects were divided into two groups based on the cutoff value of ABSI for predicting first major cardiovascular events: a low ABSI group (<0.0822 in men and <0.0814 in women) and a high ABSI group (≥0.0822 in men and ≥0.0814 in women). During a median follow-up period of 41.6 months, 56 subjects died (23 from cardiovascular causes), 16 had nonfatal acute coronary syndrome, and 14 had nonfatal stroke. The Kaplan-Meier curves for first major cardiovascular events were significantly different between the two groups (men, P<0.001; women, P<0.001). Multivariate analysis revealed that high ABSI remained an independent predictor of first major cardiovascular events (men: hazard ratio, 2.33; 95% CI, 1.07 to 5.06; P=0.033; women: hazard ratio, 8.33; 95% CI, 1.06 to 65.49; P=0.044). CONCLUSIONS: High ABSI is independently associated with incidence of cardiovascular events. ABSI calculation should be performed for evaluation of risk of cardiovascular events.

Association of cumulative low-density lipoprotein cholesterol exposure with vascular function.

BACKGROUND: The relationship between cumulative low-density lipoprotein cholesterol (LDL-C) exposure and progression of atherosclerosis remains uncertain. OBJECTIVE: The aim of this study was to determine the relationship between cumulative LDL-C level and flow-mediated vasodilation (FMD), nitroglycerine-induced vasodilation (NID) and the presence of plaque in the common carotid artery (CCA). METHODS: This was a cross-sectional study. We measured FMD in 8208 subjects, NID in 1822 subjects, and CCA plaque in 591 subjects who were not taking lipid-lowering drugs. The subjects were divided into four groups based on cumulative LDL-C exposure: <4000 mg·year/dL, 4000-4999 mg·year/dL, 5000-5999 mg·year/dL, and ≥6000 mg·year/dL. RESULTS: The odds ratio of the lower quartile of FMD in the cholesterol-year-score <4000 mg·year/dL group was significantly higher than the odds ratios in the other groups. The odds ratio of the lower quartile of NID in the <4000 mg·year/dL group was significantly higher than the odds ratios in the 5000-5999 mg·year/dL and ≥6000 mg·year/dL groups. The odds ratio of the prevalence of CCA plaque in the <4000 mg·year/dL group was significantly higher than that in the ≥6000 mg·year/dL group. CONCLUSIONS: Endothelial dysfunction occurred from cumulative LDL-C exposure of 4000 mg·year/dL, vascular smooth muscle dysfunction occurred from cumulative LDL-C exposure of 5000 mg·year/dL, and prevalence of CCA plaque occurred from cumulative LDL-C exposure of 6000 mg·year/dL. CLINICAL TRIAL REGISTRY INFORMATION: http://www.umin.ac.jp (UMIN000012950, UMIN000012951, and UMIN000012952, UMIN000003409).

Remote audit practice for inspection of structural and equipment standards for cell processing facilities under the act on the safety of regenerative medicine in Japan.

Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources.

Seasonal variations in endothelium-dependent flow-mediated vasodilation and endothelium-independent nitroglycerine-induced vasodilation.

Cardiovascular mortality has been shown to vary seasonally. However, it has not been determined whether vascular function is affected by the season. The purpose of this study was to investigate the associations of vascular function with season and outdoor temperature. Between April 2007 and March 2022, measurements of flow-mediated vasodilation (FMD) of the brachial artery as an index of endothelial function and nitroglycerine-induced vasodilation (NID) as an index of endothelium-independent vasodilation were performed in 2190 subjects. There was no significant seasonal difference in FMD (spring, 3.9 ± 3.1%; summer, 3.5 ± 3.0%; fall, 3.7 ± 3.0%; winter, 3.6 ± 3.2%; P = 0.14). There was no significant correlation between FMD and daily mean outdoor temperature (r = -0.02, P = 0.25). Multivariate analyses revealed that neither season (ß = -0.020, P = 0.31) nor outdoor temperature (ß = 0.005, P = 0.81) was significantly associated with FMD after adjustment for other confounding factors. There were significant seasonal differences in NID (spring, 12.8 ± 6.3%; summer, 12.0 ± 6.1%; fall, 11.7 ± 6.1%; winter, 12.3 ± 5.9%; P = 0.02). However, multivariate analysis revealed that there was no significant association between season and NID after adjustment for other confounding factors (ß = -0.012, P = 0.56). There was no significant correlation between NID and daily outdoor mean temperature (r = -0.03, P = 0.17). Multivariate analysis revealed that outdoor temperature was not significantly associated with NID (ß = -0.006, P = 0.78). There was no significant association of FMD or NID with season or outdoor temperature, suggesting that it is not necessary to take into account the effects of season and outdoor temperature on vascular function when interpreting the results of FMD and NID measurements. Public trials registry number: UMIN000039512.

Percentage of mean arterial pressure as a marker of atherosclerosis for detecting patients with coronary artery disease.

The percentage of mean arterial pressure (%MAP) is the height of the mean arterial waveform divided by the peak amplitude of the waveform of pulse volume recording. The purpose of this study was to determine whether the cutoff value of 45% for %MAP at the ankle, which is recommended for the diagnosis of lower extremity artery disease, in combination with ankle-brachial index (ABI) is useful for detecting patients with clinical coronary artery disease (CAD) and investigate the optimal cutoff value of %MAP to diagnose patients with CAD.　We measured ABI and %MAP in 2213 subjects　(mean age: 61.2 ± 15.5 years). Multivariate analysis revealed that %MAP ≥ 45% was significantly associated with a higher risk of CAD after adjusting for traditional cardiovascular risk factors (odds ratio [OR], 2.14; 95% confidence interval [CI], 1.43-3.21; p < 0.001). However, the association was no longer significant after adjusting for ABI (OR, 1.39; 95% CI, 0.83-2.33; p = 0.21), whereas ABI was significantly associated with CAD (OR, 0.98; 95% CI, 0.97-0.99; p = 0.005). The optimal cutoff value of %MAP derived from a receiver operating characteristic curve to diagnose CAD was 40.3%. Multivariate analysis revealed that %MAP ≥ 40.3% was significantly associated with a higher risk of CAD (OR, 1.63; 95% CI, 1.19-2.24; p = 0.002) independent of ABI (OR, 0.98; 95% CI, 0.97-0.99; p = 0.002). The cutoff value of 40.3%, but not 45%, for %MAP may be useful for detecting patients with advanced atherosclerosis and for cardiovascular risk assessment independent of ABI. REGISTRATION INFORMATION: http://www.umin.ac.jp (University Hospital Medical Information Network Clinical Trials Registry) (UMIN000039512).

Effect of exposure to radiation caused by an atomic bomb on endothelial function in atomic bomb survivors.

Background: The purpose of this study was to evaluate the effects of exposure to radiation caused by an atomic bomb in atomic bomb survivors on vascular function and vascular structure and to evaluate the relationships of radiation dose from the atomic bomb with vascular function and vascular structure in atomic bomb survivors. Methods: Flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) as indices of vascular function, brachial-ankle pulse wave velocity (baPWV) as an index of vascular function and vascular structure, and brachial artery intima-media thickness (IMT) as an index of vascular structure were measured in 131 atomic bomb survivors and 1,153 control subjects who were not exposed to the atomic bomb. Ten of the 131 atomic bomb survivors with estimated radiation dose in a cohort study of Atomic Bomb Survivors in Hiroshima were enrolled in the study to evaluate the relationships of radiation dose from the atomic bomb with vascular function and vascular structure. Results: There was no significant difference in FMD, NID, baPWV, or brachial artery IMT between control subjects and atomic bomb survivors. After adjustment of confounding factors, there was still no significant difference in FMD, NID, baPWV, or brachial artery IMT between control subjects and atomic bomb survivors. Radiation dose from the atomic bomb was negatively correlated with FMD (ρ = -0.73, P = 0.02), whereas radiation dose was not correlated with NID, baPWV or brachial artery IMT. Conclusion: There were no significant differences in vascular function and vascular structure between control subjects and atomic bomb survivors. Radiation dose from the atomic bomb might be negatively correlated with endothelial function.

Functional renal collecting ducts from human PSCs.

Collecting ducts in the kidney control the balance of water and electrolytes, as well as pH of the urine to maintain body homeostasis. Shi et al.1 recently reported in Nature Biotechnology a protocol to generate functional collecting duct cells from human pluripotent stem cells.

The prevalence of cardiovascular disease is higher in patients with bilateral low ankle-brachial index than in patients with unilateral low ankle-brachial index.

BACKGROUND AND AIMS: Ankle-brachial index (ABI) has been used as a vascular marker of atherosclerosis for cardiovascular risk assessment. However, it is unclear whether there is a difference in cardiovascular risk between patients with low ABI (<1.00) in one leg (unilateral low ABI) and patients with low ABIs in both legs (bilateral low ABI). Therefore, we investigated the associations of cardiovascular disease (CVD) with unilateral low ABI and bilateral low ABI to determine whether cardiovascular risk is higher in patients with bilateral low ABI than in patients with unilateral low ABI. METHODS: We measured ABI in 2226 subjects. RESULTS: The prevalence of CVD was higher in patients with bilateral low ABI than in individuals with normal ABI (1.00-1.40) and patients with unilateral low ABI (49.2%, 25.7% and 17.0%, respectively; p < 0.001). Multivariate analysis revealed that bilateral low ABI was significantly associated with an increased risk of CVD (OR, 2.30; 95% CI, 1.16 to 4.54; p = 0.02), whereas there was no significant association between unilateral low ABI and CVD (OR, 0.83; 95% CI, 0.47 to 1.46; p = 0.51). Propensity score matching analysis showed that the prevalence of CVD was significantly higher in patients with bilateral low ABI than in patients with unilateral low ABI (45.5% vs. 27.3%, p = 0.02). CONCLUSIONS: Cardiovascular risk may be higher in patients with bilateral low ABI than in patients with unilateral low ABI. More attention should be paid to whether a low ABI is present in one leg or in both legs for more precise cardiovascular risk assessment.

Relationship of Daily Coffee Intake with Vascular Function in Patients with Hypertension.

We evaluated the relationship of daily coffee intake with endothelial function assessed by flow-mediated vasodilation and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation in patients with hypertension. A total of 462 patients with hypertension were enrolled in this cross-sectional study. First, we divided the subjects into two groups based on information on daily coffee intake: no coffee group and coffee group. The median coffee intake was two cups per day in the coffee group. There were significant differences in both flow-mediated vasodilation (2.6 ± 2.8% in the no coffee group vs. 3.3 ± 2.9% in the coffee group, p = 0.04) and nitroglycerine-induced vasodilation (9.6 ± 5.5% in the no coffee group vs. 11.3 ± 5.4% in the coffee group, p = 0.02) between the two groups. After adjustment for confounding factors, the odds ratio for endothelial dysfunction (OR: 0.55, 95% CI: 0.32-0.95) and the odds ratio for vascular smooth muscle dysfunction (OR: 0.50, 95% CI: 0.28-0.89) were significantly lower in the coffee group than in the no coffee group. Next, we assessed the relationship of the amount of daily coffee intake with vascular function. Cubic spline curves revealed that patients with hypertension who drank half a cup to 2.5 cups of coffee per day had lower odds ratios for endothelial dysfunction assessed by flow-mediated vasodilation and vascular smooth muscle dysfunction assessed by nitroglycerine-induced vasodilation. Appropriate daily coffee intake might have beneficial effects on endothelial function and vascular smooth muscle function in patients with hypertension.

Generation of the organotypic kidney structure by integrating pluripotent stem cell-derived renal stroma.

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.

Impaired NEPHRIN localization in kidney organoids derived from nephrotic patient iPS cells.

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. We previously reported NEPHRIN and SD abnormalities in the podocytes of kidney organoids generated from patient-derived induced pluripotent stem cells (iPSCs) with an NPHS1 missense mutation (E725D). However, the mechanisms underlying the disease may vary depending on the mutations involved, and thus generation of iPSCs from multiple patients is warranted. Here we established iPSCs from two additional patients with different NPHS1 mutations and examined the podocyte abnormalities in kidney organoids derived from these cells. One patient had truncating mutations, and NEPHRIN was undetectable in the resulting organoids. The other patient had a missense mutation (R460Q), and the mutant NEPHRIN in the organoids failed to accumulate on the podocyte surface to form SD precursors. However, the same mutant protein behaved normally when overexpressed in heterologous cells, suggesting that NEPHRIN localization is cell context-dependent. The localization of another SD-associated protein, PODOCIN, was impaired in both types of mutant organoids in a cell domain-specific manner. Thus, the new iPSC lines and resultant kidney organoids will be useful resources for dissecting the disease mechanisms, as well as for drug development for therapies.

Molecular detection of maturation stages in the developing kidney.

Recent advances in stem cell biology have enabled the generation of kidney organoids in vitro, and further maturation of these organoids is observed after experimental transplantation. However, the current organoids remain immature and their precise maturation stages are difficult to determine because of limited information on developmental stage-dependent gene expressions in the kidney in vivo. To establish relevant molecular coordinates, we performed single-cell RNA sequencing (scRNA-seq) on developing kidneys at different stages in the mouse. By selecting genes that exhibited upregulation at birth compared with embryonic day 15.5 as well as cell lineage-specific expression, we generated gene lists correlated with developmental stages in individual cell lineages. Application of these lists to transplanted embryonic kidneys revealed that most cell types, other than the collecting ducts, exhibited similar maturation to kidneys at the neonatal stage in vivo, revealing non-synchronous maturation across the cell lineages. Thus, our scRNA-seq data can serve as useful molecular coordinates to assess the maturation of developing kidneys and eventually of kidney organoids.

PKD1-Dependent Renal Cystogenesis in Human Induced Pluripotent Stem Cell-Derived Ureteric Bud/Collecting Duct Organoids.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease leading to renal failure, wherein multiple cysts form in renal tubules and collecting ducts derived from distinct precursors: the nephron progenitor and ureteric bud (UB), respectively. Recent progress in induced pluripotent stem cell (iPSC) biology has enabled cyst formation in nephron progenitor-derived human kidney organoids in which PKD1 or PKD2, the major causative genes for ADPKD, are deleted. However, cysts have not been generated in UB organoids, despite the prevalence of collecting duct cysts in patients with ADPKD. METHODS: CRISPR-Cas9 technology deleted PKD1 in human iPSCs and the cells induced to differentiate along pathways leading to formation of either nephron progenitor or UB organoids. Cyst formation was investigated in both types of kidney organoid derived from PKD1-deleted iPSCs and in UB organoids generated from iPSCs from a patient with ADPKD who had a missense mutation. RESULTS: Cysts formed in UB organoids with homozygous PKD1 mutations upon cAMP stimulation and, to a lesser extent, in heterozygous mutant organoids. Furthermore, UB organoids generated from iPSCs from a patient with ADPKD who had a heterozygous missense mutation developed cysts upon cAMP stimulation. CONCLUSIONS: Cysts form in PKD1 mutant UB organoids as well as in iPSCs derived from a patient with ADPKD. The organoids provide a robust model of the genesis of ADPKD.

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.

Reconstitution of the embryonic kidney identifies a donor cell contribution to the renal vasculature upon transplantation.

The kidney possesses a highly organised vasculature that is required for its filtration function. While recent advances in stem cell biology have enabled the in vitro generation of kidney tissues, at least partially, recapitulation of the complicated vascular architecture remains a huge challenge. Herein we develop a method to reconstitute both the kidney and its vascular architecture in vitro, using dissociated and sorted mouse embryonic kidney cells. Upon transplantation, arteriolar networks were re-established that ran through the interstitial space between branching ureteric buds and eventually entered glomeruli. Using this system, we found that donor-derived endothelial cells significantly contributed to the arterioles and glomerular capillaries formed after transplantation. Unexpectedly, the near-complete depletion of canonical endothelial cells from the donor embryonic kidney suggested the existence of unidentified donor-derived endothelial precursors that were negative for canonical endothelial markers, but still contributed significantly to the vasculature in the transplants. Thus, our protocol will serve as a useful platform for identification of renal endothelial precursors and induction of these precursors from pluripotent stem cells.

Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells.

BACKGROUND: Previous research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selectively controlling differentiation of NPCs to podocytes has been a challenge. METHODS: We investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes. RESULTS: Mouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf-ß signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf-ß signaling supported domination of the RV proximal domain. Inhibition of Tgf-ß signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot process-like and slit diaphragm-like structures), and showed functional responsiveness to drug-induced injury. CONCLUSIONS: Elucidation of signals that induce podocytes during the nephron-patterning process enabled us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and offer a new resource for disease modeling and nephrotoxicity testing.

Organoids from Nephrotic Disease-Derived iPSCs Identify Impaired NEPHRIN Localization and Slit Diaphragm Formation in Kidney Podocytes.

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. However, methods for SD reconstitution have been unavailable, thereby limiting studies in the field. In the present study, we established human induced pluripotent stem cells (iPSCs) from a patient with an NPHS1 missense mutation, and reproduced the SD formation process using iPSC-derived kidney organoids. The mutant NEPHRIN failed to become localized on the cell surface for pre-SD domain formation in the induced podocytes. Upon transplantation, the mutant podocytes developed foot processes, but exhibited impaired SD formation. Genetic correction of the single amino acid mutation restored NEPHRIN localization and phosphorylation, colocalization of other SD-associated proteins, and SD formation. Thus, these kidney organoids from patient-derived iPSCs identified SD abnormalities in the podocytes at the initial phase of congenital nephrotic disease.

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.