Transcriptional Analysis of Intravenous Immunoglobulin Resistance in Kawasaki Disease Using an Induced Pluripotent Stem Cell Disease Model.

BACKGROUND: Approximately 10-20% of Kawasaki disease (KD) patients are resistant to intravenous immunoglobulin (IVIG) treatment. Further, these patients are at a particularly high risk of having coronary artery abnormalities. The mechanisms of IVIG resistance in KD have been analyzed using patient leukocytes, but not patient vascular endothelial cells (ECs). The present study clarifies the mechanisms of IVIG resistance in KD using an induced pluripotent stem cell (iPSC) disease model.Methods and Results:Dermal fibroblasts or peripheral blood mononuclear cells from 2 IVIG-resistant and 2 IVIG-responsive KD patients were reprogrammed by the episomal vector-mediated transduction of 6 reprogramming factors. KD patient-derived iPSCs were differentiated into ECs (iPSC-ECs). The gene expression profiles of iPSC-ECs generated from IVIG-resistant and IVIG-responsive KD patients were compared by RNA-sequencing analyses. We found that the expression ofCXCL12was significantly upregulated in iPSC-ECs from IVIG-resistant KD patients. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that gene sets involved in interleukin (IL)-6 signaling were also upregulated. CONCLUSIONS: The first iPSC-based model for KD is reported here. Our mechanistic analyses suggest thatCXCL12, which plays a role in leukocyte transmigration, is a key molecule candidate for IVIG resistance and KD severity. They also indicate that an upregulation of IL-6-related genes may be involved in this pathogenesis.

The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n=3, 61.0 ± 8.2%) compared with wild-type subjects (n=6, 13.1 ± 7.7%; p<0.01). Aneuploidy was observed more frequently in fibroblasts (p<0.01) and induced pluripotent stem cells (iPSCs) (p<0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p<0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p<0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.

Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm.

There has been increasing success with the generation of pancreatic cells from human induced pluripotent stem cells (hiPSCs); however, the molecular mechanisms of the differentiation remain elusive. The purpose of this study was to reveal novel molecular mechanisms for differentiation to PDX1+NKX6.1+ pancreatic endoderm cells, which are pancreatic committed progenitor cells. PDX1+ posterior foregut cells differentiated from hiPSCs failed to differentiate into pancreatic endoderm cells at low cell density, but Rho-associated kinase (ROCK) or non-muscle myosin II (NM II) inhibitors rescued the differentiation potential. Consistently, the expression of phosphorylated myosin light chain 2 and NM IIA was downregulated in aggregation culture. Notably, the soluble factors we tested were substantially effective only with ROCK-NM II inhibition. The PDX1+NKX6.1+ cells induced with NM II inhibitors were successfully engrafted and maturated in vivo. Taken together, these results suggest that NM IIs play inhibitory roles for the differentiation of hiPSCs to pancreatic endoderm cells.

Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models.

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca(2+) entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.