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

Retinoic acid regulates erythropoietin production cooperatively with hypoxia-inducible factors in human iPSC-derived erythropoietin-producing cells

Erythropoietin (EPO) is a crucial hormone for erythropoiesis and produced by adult kidneys. Insufficient EPO production in chronic kidney disease (CKD) can cause renal anemia. Although hypoxia-inducible factors (HIFs) are known as a main regulator, the mechanisms of EPO production have not been fully elucidated. In this study, we aimed to examine the roles of retinoic acid (RA) in EPO production using EPO-producing cells derived from human induced pluripotent stem cells (hiPSC-EPO cells) that we previously established. RA augmented EPO production by hiPSC-EPO cells under hypoxia or by treatment with prolyl hydroxylase domain-containing protein (PHD) inhibitors that upregulate HIF signals. Combination treatment with RA and a PHD inhibitor improved renal anemia in vitamin A-depleted CKD model mice. Our findings using hiPSC-EPO cells and CKD model mice may contribute to clarifying the EPO production mechanism and developing efficient therapies for renal anemia

iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome

ヒトiPS細胞から作製した腎オルガノイドを用いたアルポート症候群病態モデルの開発. 京都大学プレスリリース. 2023-09-28.iPSC-derived kidney organoids to model a lifelong renal disease. 京都大学プレスリリース. 2023-10/17.Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS

Elucidation of HHEX in pancreatic endoderm differentiation using a human iPSC differentiation model

ヒトiPS細胞分化モデルを用いた膵内胚葉分化におけるHHEXの役割の解明. 京都大学プレスリリース. 2023-06-09.Identification of HHEX as a crucial factor in pancreatic endoderm differentiation using a human iPS cell differentiation model. 京都大学プレスリリース. 2023-06-15.For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX. HHEX knockdown suppressed the expression of another pancreatic endoderm marker gene, PTF1A, as well as NKX6.1, independently of PDX1, a known regulator of NKX6.1 expression. In contrast, the overexpression of HHEX upregulated the expressions of NKX6.1 and PTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such as NKX6.1, PTF1A, ONECUT1 and ONECUT3, in HHEX knockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation

Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD

腎集合管オルガノイドを用いた多発性嚢胞腎モデルの作製 iPS創薬により治療薬候補を発見、治験開始へ. 京都大学プレスリリース. 2023-12-01.Developing more advanced renal organoids to model polycystic kidney disease. 京都大学プレスリリース. 2023-12-01.In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated in vitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1−/− hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress in vitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model in vivo. Therefore, our in vitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD

ヒト多能性幹細胞から腎臓へ分化しうる中間中胚葉への分化のモニタリングと高効率な分化誘導法の開発

京都大学0048新制・課程博士博士(医学)甲第18148号医博第3868号新制||医||1002(附属図書館)31006京都大学大学院医学研究科医学専攻(主査)教授 山下 潤, 教授 柳田 素子, 教授 斎藤 通紀学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Apparent digestibility of selected feedstuffs by mud crab, Scylla serrata

A feeding experiment was conducted to determine apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD), crude fat (ACFD), crude fiber (ACFbD), nitrogen-free extract or NFE (ANFED), and crude ash (AAD) of selected feed ingredients for mud crab, Scylla serrata. The nine feed ingredients were Peruvian fish meal, squid meal, Acetes sp., meat and bone meal, copra meal, wheat flour, rice bran, corn meal, and defatted soybean meal. A reference diet (RF) and test diets (consisted of 70% RF diet and 30% of the feedstuff) were used with Cr2O3 as external indicator. The ADMD of the RF and test diets were high except for diet with meat and bone meal. Crude protein, crude fiber, and ash of feedstuffs were digestible in mud crab. Nutrients in squid meal, corn meal, and defatted soybean meal were digested well (ACFbD>95%; ANFED>92%; AAD>71%) compared with nutrients in the meat and bone meal. The AAD of copra meal, wheat flour, rice bran, and meat and bone meal were similar. The ACFD in carbohydrate-rich plant feedstuffs were significantly higher than that in protein-rich animal feedstuffs. For this species, the relative amounts of dietary protein and NFE in feedstuffs had an effect on the ACFD but not on ADMD.The authors would like to acknowledge the commendable technical assistance of Ms. Ellen Flor Doyola and Mr. Hernando Alcalde

Differentiation and isolation of iPSC-derived remodeling ductal plate-like cells by use of an AQP1-GFP reporter human iPSC line

Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) β2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1⁺ cells in vitro. By confirming that the isolated AQP1⁺ cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9⁺CK19⁺AQP1⁺ remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation

Development of new method to enrich human iPSC-derived renal progenitors using cell surface markers

An Author Correction to this article was published on 18 July 2019Cell therapy using renal progenitors differentiated from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) has the potential to significantly reduce the number of patients receiving dialysis therapy. However, the differentiation cultures may contain undifferentiated or undesired cell types that cause unwanted side effects, such as neoplastic formation, when transplanted into a body. Moreover, the hESCs/iPSCs are often genetically modified in order to isolate the derived renal progenitors, hampering clinical applications. To establish an isolation method for renal progenitors induced from hESCs/iPSCs without genetic modifications, we screened antibodies against cell surface markers. We identified the combination of four markers, CD9⁻CD140a⁺CD140b⁺CD271⁺, which could enrich OSR1⁺SIX2⁺ renal progenitors. Furthermore, these isolated cells ameliorated renal injury in an acute kidney injury (AKI) mouse model when used for cell therapy. These cells could contribute to the development of hiPSC-based cell therapy and disease modeling against kidney diseases