繰り返す分岐形態形成能力を有するヒトiPS細胞由来尿管芽オルガノイドの作製と拡大培養

京都大学0048新制・課程博士博士(医学)甲第22879号医博第4673号新制||医||1047(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 川口 義弥, 教授 柳田 素子, 教授 小川 修学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Protocol for the generation and expansion of human iPS cell-derived ureteric bud organoids

The ureteric bud (UB) is a kidney precursor tissue that repeats branching morphogenesis and gives rise to the collecting ducts (CDs) and lower urinary tract. Here, we describe protocols to generate iUB organoids from human iPSCs; iUB organoids repeat branching morphogenesis. We describe how to expand iUB-organoid-derived tip colonies and how to induce CD progenitors from iUB organoids. These organoids can be used to study CD development and potentially as a model of kidney and urinary tract diseases

In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells

ヒトiPS細胞から作製した腎前駆細胞に未分化な細胞が残存していないことを確認する方法の開発. 京都大学プレスリリース. 2022-11-16.A new sensitive method to detect for minute amounts of contaminating undifferentiated iPS cells. 京都大学プレスリリース. 2022-11-21.Cell therapies using human induced pluripotent stem cell (hiPSC)-derived nephron progenitor cells (NPCs) are expected to ameliorate acute kidney injury (AKI). However, using hiPSC-derived NPCs clinically is a challenge because hiPSCs themselves are tumorigenic. LIN28A, ESRG, CNMD and SFRP2 transcripts have been used as a marker of residual hiPSCs for a variety of cell types undergoing clinical trials. In this study, by reanalyzing public databases, we found a baseline expression of LIN28A, ESRG, CNMD and SFRP2 in hiPSC-derived NPCs and several other cell types, suggesting LIN28A, ESRG, CNMD and SFRP2 are not always reliable markers for iPSC detection. As an alternative, we discovered a lncRNA marker gene, MIR302CHG, among many known and unknown iPSC markers, as highly differentially expressed between hiPSCs and NPCs, by RNA sequencing and quantitative RT-PCR (qRT-PCR) analyses. Using MIR302CHG as an hiPSC marker, we constructed two assay methods, a combination of magnetic bead-based enrichment and qRT-PCR and digital droplet PCR alone, to detect a small number of residual hiPSCs in NPC populations. The use of these in vitro assays could contribute to patient safety in treatments using hiPSC-derived cells

Expansion of Human iPSC-Derived Ureteric Bud Organoids with Repeated Branching Potential

腎集合管のもとになる組織を大量に作製することに成功. 京都大学プレスリリース. 2020-08-03.Ureteric bud (UB) is the embryonic kidney progenitor tissue that gives rise to the collecting duct and lower urinary tract. UB-like structures generated from human pluripotent stem cells by previously reported methods show limited developmental ability and limited branching. Here we report a method to generate UB organoids that possess epithelial polarity and tubular lumen and repeat branching morphogenesis. We also succeed in monitoring UB tip cells by utilizing the ability of tip cells to uptake very-low-density lipoprotein, cryopreserving UB progenitor cells, and expanding UB tip cells that can reconstitute the organoids and differentiate into collecting duct progenitors. Moreover, we successfully reproduce some phenotypes of multicystic dysplastic kidney (MCDK) using the UB organoids. These methods will help elucidate the developmental mechanisms of UB branching and develop a selective differentiation method for collecting duct cells, contributing to the creation of disease models for congenital renal abnormalities

A Modular Differentiation System Maps Multiple Human Kidney Lineages from Pluripotent Stem Cells

ヒトiPS細胞から別個に分化させた複数の腎前駆細胞から腎組織を再生することに成功. 京都大学プレスリリース. 2020-04-09.Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs, and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here, we create a culture system in which these mesoderm-like cell types and paraxial and lateral plate mesoderm-like cells are separately generated from hPSCs. We recapitulate nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which are subsequently differentiated into glomeruli, renal tubules, and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and disease and also supply cell sources for regenerative therapies

Successful Kidney Transplantation for End-Stage Renal Disease in Marfan's Syndrome

Marfan’s syndrome is a systemic disorder of the connective tissue caused by mutations in the extracellular matrix protein fibrillin-1, with aortic dissection and aneurysm being its most life-threatening manifestations. Kidney transplantation for end-stage renal disease (ESRD) in patients with Marfan’s syndrome has not been reported in the literature, and the rate of the incidence of dissection or aneurysm in the iliac artery is unknown. Here, we present a patient with Marfan’s syndrome with ESRD due to severe renal ischemia caused by massive bleeding from thoracoabdominal aortic dissection leading to transplant surgery of a living kidney procured from the patient’s mother. After kidney transplantation, the renal function normalized without vascular complications, and stable graft function along with negative results for both microhematuria and proteinuria continued for two years. Also, vascular complication such as aneurysm or dissection of the iliac artery was not observed using ultrasonography during the follow-up period. ESRD patients with Marfan’s syndrome might be suitable for kidney transplantation, but long-term and careful observations are needed

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Combination assay using magnetic bead-based cell isolation and qRT-PCR analysis to detect hiPSCs intermingled in NPCs derived from a clinical-grade iPSC stock line.

(A) Flow cytometric analysis of the MACS positive fractions of 1:1,000 mixtures of GFP (+) hiPSCs and GFP (-) NPCs at three different concentrations of anti-TRA-1-60 antibody-conjugated beads (upper panels) and one concentration of anti-SSEA-4 antibody-conjugated beads (lower left panel), GFP (-) NPCs (without MACS selection; lower center panel), and a 1:1,000 mixture of GFP (+) hiPSCs and GFP (-) NPCs (without MACS selection; lower right panel). The bar graph in the right panel shows the mean ± SEM of GFP (+) cells from the flow cytometric analysis at various bead concentrations in staining reagent. PF: positive fraction; NF: negative fraction; 0.2TRA-1-60: 0.2 μL TRA-1-60 beads / 1 μL staining reagent; 0.02TRA-1-60: 0.02 μL TRA-1-60 beads / 1 μL staining reagent; 0.002TRA-1-60: 0.002 μL TRA-1-60 beads / 1 μL staining reagent; 0.2SSEA-4: 0.2 μL SSEA-4 beads / 1 μL staining reagent. (B) Scatter plots of the TBP-normalized gene expressions of MIR302CHG, CUZD1 and POU5F1 in NPCs (NPC), the MACS negative fraction of the mixture (NF), 0.0001% hiPSC/NPC mixtures (10−4% iPSC), the MACS positive fraction of the mixture (PF), and hiPSCs (iPSC) by qRT-PCR analysis. (C) Scatter plots of the TBP-normalized gene expressions of MIR302CHG, CUZD1, and POU5F1 in day 4 cells (D4C), NF, 10−4% iPSC, PF and iPSC by qRT-PCR. The dots and lines in the center of the scatter plots in (B) and (C) indicate the experimental data and mean values of the data, respectively. UD: undetermined. *p p <0.01 by Tukey-Kramer post hoc tests against the sample with NPCs. NS, not significant.</p

scRNA-seq analysis of purified OSR1(+)SIX2(+) induced NPCs for iPSC and NPC marker genes.

(A) Violin plots of the cells in hiPSC-derived metanephric and mesonephric NPC populations reported in Tsujimoto et al. (2020) with standard quality control parameters after filtering. (B) UMAP plots for hiPSC-derived metanephric and mesonephric NPCs. The number of cells in each NPC population: metanephric NPCs, 190; and mesonephric NPCs, 199. (C, D) Violin plots of representative NPC (C) and iPSC (D) markers of hiPSC-derived metanephric and mesonephric NPCs. (E) UMAP plots of representative NPC markers (SIX2, PAX2, PAX8 and WT1) in hiPSC-derived metanephric and mesonephric NPCs. (F) Scatter plots of iPSC markers (LIN28A, CNMD and SFRP2) and NPC markers (SIX2 and PAX2). Numbers above the plots are Pearson correlation coefficients. Mesonephric NPCs were induced from hiPSCs by the same protocol as the metanephric NPC induction except for removing activin A at Stage 4. MESO, mesonephric NPC; META, metanephric NPC. (TIF)</p