Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation.

Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.

Generation of MBP-tdTomato reporter human induced pluripotent stem cell line for live myelin visualization.

Myelin basic protein (MBP) is a major component of the myelin sheaths of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system. Here we generated heterozygous fluorescent reporter of MBP gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock in fused tdTomato fluorescent protein and EF1 alpha promoter-driven Bleomycin (Zeocin) resistance gene to the translational MBP C-terminal region. The resulting line, MBP-TEZ, showed tdTomato fluorescence upon oligodendrocyte differentiation. This reporter hiPSC line provides a precedential opportunity for monitoring human myelin formation and degeneration and purifying MBP-expressing cell lineages.

Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations.

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.

Generation of human induced pluripotent stem cell lines derived from four DiGeorge syndrome patients with 22q11.2 deletion.

DiGeorge syndrome (22q11.2 deletion syndrome, or CATCH22 syndrome), caused by hemizygous deletion of chromosome 22q11.2, results in the poor development of multiple organs. Here we have generated DiGeorge syndrome-specific human induced pluripotsnt stem cells (hiPSCs) derived from four patients. These established hiPSC lines showed self-renewal and pluripotency and carried a hemizygous deletion in 22q11.2. Since the molecular pathogenesis of DiGeorge syndrome caused by the 22q11.2 deletion is largely unknown, these cell resources will be useful for recapitulating disease phenotypes and for developing new therapies for DiGeorge syndrome.

Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing.

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.