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 Ca2+ 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.

The results are expressed as the means ± SEM. CCh, carbachol; ND, not done; NA, not applicable.   Table S7. The results of the microarray analyses comparing the gene expression profiles of iPSC-derived vascular smooth muscle cells from ADPKD patients with intracranial aneurysms and those from ADPKD patients without ICAs, Related to Figure 5.
The gene expression profiles of the iPSC-derived vascular smooth muscle cells from four ADPKD patients with ICAs (P1, P3, P4 and P7) were individually compared with those from three patients without ICAs (P2, P5 and P6). From these 12 comparisons, genes with an average fold change in expression of more than two were selected.

ECs SMCs
Fold Change (with ICA / without ICA)

Generation of Patient-specific iPSCs
Fibroblasts derived from ADPKD patients were maintained and expanded in Dulbecco's Modified Eagle Medium (DMEM, Nacalai Tesque) containing 10% fetal bovine serum (FBS, Japan Bioserum). The induction of iPSCs was performed as described previously. 4 In brief, the patients' fibroblasts were seeded in six-well plates at 1.0 × 10 5 cells/well. The next day, the cells were infected with Slc7a1 lentiviruses with 4 g/ml polybrene (Nacalai Tesque). Then, the patients' fibroblasts expressing the mouse Slc7a1 gene were seeded in six-well plates at 1.0 × 10 5 cells/well one day before transduction.
Equal amounts of three or four retrovirus-containing supernatants were mixed and supplemented with 4 g/ml polybrene. Six days after transduction, the fibroblasts were replated onto mitomycin C-treated SNL feeder cells. 5 Thirty days after transduction, iPSC colonies were selected for expansion.

RT-PCR and Real-time Quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's recommendations, followed by cDNA synthesis using standard protocols. Briefly, 1 g of total RNA was treated with DNase I (Qiagen) for 15 min, and the cDNA was synthesized using ReverTra Ace (TOYOBO for 30 s. As recommended by the manufacturer, the threshold cycle method was used to analyze the data for the gene expression levels and was normalized to those of the housekeeping gene, -ACTIN. The PCR reactions were performed in triplicate for each sample. The primer sequences are listed in Table S10.

Short Tandem Repeat (STR) Analysis and Karyotyping
The STR analyses were performed at BEX CO. LTD., Japan, and the chromosomal G-band analyses were performed at the Nihon Gene Research Laboratories, Japan.

Bisulfite Sequencing
Sodium bisulfite conversion of genomic DNA (1 g) was performed using the EZ DNA methylation kit (ZYMO Research), according to the manufacturer's instructions. The promoter regions of the human OCT4 and NANOG genes were amplified by PCR as described previously. 4 The PCR products were subcloned into the pCR4-TOPO TA vector (Thermo Fisher Scientific) and were sequenced.

Embryoid Body (EB) Formation
For EB formation, a 10 cm plate containing human iPSCs was rinsed with PBS and EBs were transferred to gelatin-coated plates and cultured in the same medium for another 8 days.

Teratoma Formation
The iPSCs were harvested using CTK solution, collected into tubes and centrifuged, and the pellets were resuspended in DMEM/F12 (Thermo Fisher Scientific). One quarter of the cells from a confluent 100 mm dish was injected into the testes of a NOD-SCID mouse (CREA). Nine to twelve weeks after injection, the tumors were dissected and fixed with PBS containing 4% paraformaldehyde (PFA). Paraffin-embedded tissues were sliced and stained with hematoxylin and eosin.

Mutational Analysis
Mutational analyses of the three families with ADPKD (P4, P6 and P7) were performed as described previously, with some modifications. 1  To elucidate the 2 bp deletion mutation of the family of patient P7, genotyping was performed using the GeneScan TM device (Thermo Fisher Scientific), which can detect an allele two bases shorter by semiautomatic electrophoresis of the PCR-amplified products using fluorescently-labeled primers. For patient P7, the 2 bp deletion in exon 15 of PKD1 was amplified by LR-PCR, followed by nested-PCR. The primer sets used were reported previously. 5 Briefly, in the LR-PCR, a 3378 bp product was amplified with the F26 and R2LR primers (Table S10) and then the product was diluted to 10 -5 . The diluted mixture was amplified with the nested primer set of PKDeX15.14F and PKDeX15.14R ( Table S10). The expected size of the patient allele (7024del AC) was 202, while that of normal size was 204. The difference in size between the mutated allele and the wild allele was identified by the GeneScan TM system using the fluorescently-labeled reverse primer (PKDex15.14R).
The mutational analyses of the remaining four ADPKD patients (P1, P2, P3 and P5) were performed by whole-exome sequencing and amplicon-sequencing.

Whole-exome Sequencing
Exon capture and Illumina's library preparation were performed using the SeqCap EZ v3 (Roche) and a TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA) as reported previously. 11 Paired-end sequencing for 2 X 101 cycles was performed using a TruSeq SBS Kit v3 and a HiSeq2500 instrument (Illumina). Processing of the raw data, mapping and SNV/indel calling were performed as previously reported. 11

Amplicon Sequencing
PCR was performed using KAPA HiFi Hot Start Ready (KAPA Biosystems) for exon 1 of PKD1 or Prime STAR GXL DNA polymerase (TaKaRa, Japan) for the other regions.
The PCR conditions when using KAPA HiFi Hot Start was 1 cycle of 95˚C for 5 min, followed by 25 cycles of 98˚C for 20 sec, 65˚C for 15 sec and 72˚C for 3 min. The PCR using Prime STAR GXL was 30 cycles of 98˚C for 10 sec and 68˚C for 30 min. The primer pairs used to amplify the PKD1 genomic fragments are shown in Table S10.
PCR products were then purified using a QIAquick PCR Purification Kit (QIAGEN) and Illumina's libraries were prepared using a TruSeq PCR-free Kit (Illumina).
Paired-end sequencing for 2 X 151 cycles was performed using a MiSeq device

Tube Formation Assay
Tube formation assay was performed as described previously. 2 In brief, vascular endothelia derived from iPSCs (4.0 × 10 4 cells/well) were seeded onto matrigel-coated (Becton Dickinson) 24-well plates. Cells were incubated for 24 h, and digital images of the tubes that formed were captured. Total tube length was calculated using BZ-II analyzer software (KEYENCE) after three randomly selected fields were examined in each well of a 24-well plate. The total tube length was calculated as the average for the three fields.

Western Blot Analysis
Immunoblotting was performed for 10 µg of cell lysate extracted with radioimmune