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

Successful management of refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis by vincristine adriamycin dexamethasone chemotherapy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation is rarely reported and has a poor prognosis in general (a median survival of 1.6 months). Moreover, the optimum treatment for this condition is still undecided. This is the first report on the successful use of vincristine, adriamycin and dexamethasone chemotherapy for refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis without cardiac decompensation.</p> <p>Case presentation</p> <p>We report the case of a 68-year old Japanese male with systemic immunoglobulin light chain amyloidosis presenting with bilateral pleural effusion (more severe on the right side) in the absence of cardiac decompensation that was refractory to diuretic therapy. The patient was admitted for fatigue, exertional dyspnea, and bilateral lower extremity edema. He had been receiving intermittent melphalan and prednisone chemotherapy for seven years. One month before admission, his dyspnea had got worse, and his chest radiograph showed bilateral pleural effusion; the pleural effusion was ascertained to be a transudate. The conventionally used therapeutic measures, including diuretics and thoracocentesis, failed to control pleural effusion. Administration of vincristine, adriamycin, and dexamethasone chemotherapy led to successful resolution of the effusion.</p> <p>Conclusion</p> <p>Treatment with vincristine, adriamycin, and dexamethasone chemotherapy was effective for the refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation and appears to be associated with improvement in our patient's prognosis.</p

Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

Abstract Of late, numerous microphysiological systems have been employed to model the renal proximal tubule. Yet there is lack of research on refining the functions of the proximal tubule epithelial layer—selective filtration and reabsorption. In this report, pseudo proximal tubule cells extracted from human-induced pluripotent stem cell-derived kidney organoids are combined and cultured with immortalized proximal tubule cells. It is shown that the cocultured tissue is an impervious epithelium that offers improved levels of certain transporters, extracellular matrix proteins collagen and laminin, and superior glucose transport and P-glycoprotein activity. mRNA expression levels higher than those obtained from each cell type were detected, suggesting an anomalous synergistic crosstalk between the two. Alongside, the improvements in morphological characteristics and performance of the immortalized proximal tubule tissue layer exposed, upon maturation, to human umbilical vein endothelial cells are thoroughly quantified and compared. Glucose and albumin reabsorption, as well as xenobiotic efflux rates through P-glycoprotein were all improved. The data presented abreast highlight the advantages of the cocultured epithelial layer and the non-iPSC-based bilayer. The in vitro models presented herein can be helpful in personalized nephrotoxicity studies

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

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

<div><p>The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.</p></div

The Small Molecule Method Functions via RARβ Receptors.

<p>(A) Results of the flow cytometric analyses showing induction of OSR1⁺ cells by the small molecule method, when AM580 or TTNPB was replaced by all-trans retinoic acid (ATRA), adapalene, and CD1530. (B) Effects of adding RAR antagonists, BMS493, LE135, and MM11253, on the induction of OSR1⁺ cells by the TTNPB method. (C) The knockdown efficiency of siRNAs targeting <i>RARB</i>, and (D and E) effects on expression levels of <i>OSR1</i> and the induction of OSR1⁺ cells by the TTNPB method. (F) Effects of adding a pan-RXR agonist (SR11237) or a pan-RXR antagonist (UVI3003) to the induction efficiency of OSR1⁺ cells by the TTNPB method. (G) The induction of OSR1⁺ cells by the small molecule method, when SR11237 replaced AM580 or TTNPB. (H) Results of qRT-PCR analyses showing <i>OSR1</i> expression activated by the AM580 and TTNPB methods, and when ATRA, adapalene, or CD1530 were used instead of AM580 or TTNPB. OSR1-GFP knock-in hiPSCs prior to treatments were used to normalize the data. The data in (A–H) are presented as the mean±SD on culture day 6 of three independent experiments (n = 3).</p

The Small Molecule Method Can Produce IM Cells without the Mesendoderm Step.

<p>(A) Induction of BRACHYURY⁺ mesendoderm cells generated from OSR1-GFP knock-in hiPSCs (3D45) after 24, 48, 72, and 96 hrs of treatment with CHIR99021 and TTNPB or activin A. (B) Results of the qRT-PCR analyses showing mRNA expression of the mesendoderm markers, <i>BRACHYURY</i>, <i>GOOSECOID</i>, and <i>MIXL1</i>, in differentiation cultures from the growth factor and TTNPB methods. (C) Effects of adding a RAR inhibitor, BMS493, to differentiation cultures of 3D45 cells treated with CHIR99021 and activin A, analyzed by anti-BRACHYURY immunostaining. (D) Induction of OSR1⁺ cells on culture day 11 of the growth factor method, with or without addition of BMS493 at various concentrations. (E) Results of qRT-PCR analyses showing expression of <i>RARB</i> mRNA. 3D45 cells on day 1, before treatment, were used to normalize the data shown in (B) and (E). The data in (A–E) are means±SD of three independent experiments (n = 3). Scale bars, 100 m.</p

The Small Molecule Method for Differentiating hiPSCs into IM Cells.

<p>(A) The results of the flow cytometric analyses comparing the induction efficiency of OSR1⁺ cells on culture days 6, 8, and 11 for various treatments with CHIR99021 and AM580 or TTNPB, and the growth factor method (CHIR99021 and activin A during Stage 1, CHIR99021 and BMP7 during Stage 2). (B) Results of flow cytometric analyses of OSR1⁺ cell induction on days 3 and 6 of the AM580, TTNPB, and growth factor methods. (C) Numbers of OSR1⁺ and total cells generated by the TTNPB and growth factor methods. (D) Results of the flow cytometric analyses showing the differentiation of OSR1⁺ cells on day 6 using the serum-free small molecules methods. (E) Results of RT-PCR analyses showing mRNA expression of IM and non-IM marker genes in undifferentiated hiPSCs before treatment on day 1, and in OSR1⁺ cells on day 6 after induction by the small molecule method. (F) Immunostaining for SALL1, WT1, LIM1, and PAX2 in the OSR1⁺ cells on day 6 of induction by the TTNPB method. Scale bars, 100 m. The data in (A–D) are means±SD of three independent experiments (n = 3). The data in (E) and (F) are representative of three independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084881#pone.0084881.s002" target="_blank">Figure S2</a> for additional data.</p

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