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RNA sequencing. For RNA sequencing, total RNA was extracted as described
above. The samples preserved at −80 °C were shipped and analyzed by DNAFORM. The quality of total RNA was evaluated by a Bioanalyzer (Agilent) to
ensure over 8.0 RIN (RNA integrity number) or by electrophoresis waveforms.
Double-stranded cDNA libraries (RNA-seq libraries) were prepared using a
SMART Seq Stranded Kit (Clontech) according to the manufacturer’s protocols.
RNA-seq libraries were sequenced using paired-end reads (50 nt of read 1 and 25
nt of read 2) on a NextSeq 500 (Illumina). Obtained reads were mapped to the
human GRCh38 genome analyzed by STAR (version 2.7.3a). Annotated reads were
counted using featureCounts (version 2.0.1) and RSEM (version 1.3.1). FPKM
values were calculated from mapped reads by normalizing to total counts. The gene
expression heatmap was drawn based on the log2 fold change (log2FC) compared
with day 13 + 4 kidney organoid samples using FPKM.
Flow cytometry. Kidney organoids were dissociated in TrypLE Select Enzyme
(Thermo Fisher Scientific) for approximately 30 min at 37 °C. The cell suspension
was incubated with primary antibodies in 2% fetal bovine serum (FBS; Wako)/PBS
for 30 min on ice. After washing, the cell suspension was incubated with secondary
antibodies for 30 min on ice. Antibody-labeled cells were resuspended with 2% FBS/
PBS containing 4’,6-diamidino-2-phenylindole (DAPI; Sigma). Flow cytometry was
performed using a BD FACSAria II (BD Biosciences). The FACS Diva (BD) software program was used to analyze the data. The cells stained with isotype control
and secondary antibodies were used as a negative control. Gating was set such that
DAPI(−) live negative control cells had a positive fraction of less than 1%.
10
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Acknowledgements
The authors thank Drs. Keiko Okamoto-Furuta and Haruyasu Kohda, Division of
Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University, for technical support with the electron microscopy, Dr. Kanae
Mitsunaga, CiRA, Kyoto University, for the flow cytometry analysis, Dr. Naoki Kojima,
Taisho Pharmaceutical Co., Ltd., for helpful discussions, and Dr. Peter Karagiannis for
critically reading and revising the manuscript. This study was supported by Taisho
Pharmaceutical Co., Ltd., the Japan Agency for Medical Research and Development
(AMED) under Grant numbers JP23bm1123002 and JP22bm0804013 to K. Osafune, and
the iPS Cell Research Fund.
Author contributions
R.H. carried out all primary analyses and wrote and edited the paper. K.W. and A.H.
generated the mutation-corrected iPSC line and wrote parts of the paper. K.T., T.O., T.A.,
and S.I.M. performed some experiments and analyzed the data. T.H., T.Y., K.I., and K.N.
provided PBMCs to the AS patients. K. Okita established the patient’s iPSCs. K. Osafune
supervised the project and wrote and edited the paper. All authors discussed the results
and commented on the manuscript.
Competing interests
R.H. is an employee of Taisho Pharmaceutical Co., Ltd. T.O. is an employee of Ono Pharmaceutical Co., Ltd. K. Osafune is a founder and member of the scientific advisory boards of
iPS Portal, Inc., and a founder and chief scientific advisor of RegeNephro Co., Ltd. T.A. is a
founder and scientific advisor of RegeNephro Co., Ltd. S.I.M. is a scientific advisor of RegeNephro Co., Ltd. R.H., T.A. and K. Osafune are the inventors of the iPSC-based disease model
for AS (patent application 2021–184043). The other authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s42003-023-05203-4.
Correspondence and requests for materials should be addressed to Kenji Osafune.
Peer review information Communications Biology thanks Ryuji Morizane and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work. Primary
Handling Editors: Simona Chera and Manuel Breuer.
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