Human urine-derived iPSC reprogramming

Urine-derived iPSCs from controls were kindly provided by the iPSC core facility of Nantes Université supported by Biogenouest and IBiSA.

Isolation of ERCs (epithelial renal cells) and reprogramming. Urine samples were collected and ERCs isolated by the iPSC core facility (INSERM, CNRS, UNIV Nantes, CHU Nantes, France) from urine samples and cultures, as previously described [39]. Briefly, cells were seeded on Matrigel-coated wells on day − 1 in their usual culture media and transfected daily, from day 0 to day 10, with 625 ng of an mRNA cocktail (38% Oct4, 11.4% Sox2, 12.7% Klf4, 10.1% Lin28, 12.7% Myc, 10.1% Nanog, 5.1% nGFP, Milenyi Biotec) in Pluriton media (Reprocell, Glasgow, G20 0XA UK) supplemented with 4 ng/ml FGF2 (Peprotech, Neuilly sur Seine, France) and 200 ng/ml B18R (eBioscience, ThermoFisher scientific). From day 11, cells were cultured in Pluriton media supplemented with 4 ng/ml FGF2. Colonies were picked and expanded on feeders in KSR + FGF2 media or directly on Matrigel-coated dishes in TeSR1 or iPS Brew. The expression of pluripotency genes (Oct4, Sox2, Nanog) was verified by qPCR (Additional file 2: Figure SD2 and Additional file 3: Table SD3). Assays were performed to assess the ability of each iPSC clone to differentiate into the three germ line lineages [39]. The karyotype of each iPSC cell line was normal.

iPSC culture

Human urine-derived iPSCs were initially cultured in feeder-free KSR medium (DMEM/F-12, 20% KnockoutTM serum replacement, 1% non-essential amino acids, 1% Glutamax, 50 µM 2-mercaptoethanol, and 10 ng/ml fibroblast growth factor 2 [Peprotech Neuilly sur Seine, France]) at the iPSC core facility (INSERM, CNRS, UNIV Nantes, CHU Nantes, France). They were mechanically passaged by cutting colonies with a needle. All cells were cultured at 37 °C under 20% O2 and 5%CO2. Then, iPSCs were cultured at the Institute of Cardiometabolism and Nutrition (ICAN, F-75013 Paris, France). Colonies were picked and expanded in mTeSR1 (Stemcell technologies, Vancouver, BC, Canada) on Matrigel matrix-coated plates. The iPSCs were passed manually once a week using a Lynx microscope (Vision Engineering, New Milford, CT, USA), and the culture medium was changed daily. The cells were cultured at 37 °C under 5% CO2 and 5% O2.

Chondrogenic differentiation

Chondrogenic differentiation of iPSCs was performed using a published protocol [40]. Briefly, iPSCs were subjected to differentiation by changing the medium to a mesendodermal differentiation medium (DMEM/F12 with 10 ng/ml Wnt3a [R&D Systems], 10 ng/ml Activin A [R&D], 1% ITS [Invitrogen], 1% FBS [Invitrogen]) (day 0). At day 3, the medium was changed to basal medium (DMEM with 1% ITS and 1% FBS) supplemented with 50 μg/ml ascorbic acid (Nacalai), 10 ng/ml BMP-2 (Osteapharma), 10 ng/ml GDF5, 10 ng/ml TGFβ (Peprotech), and 10 ng/ml FGF-2. Fourteen days after starting the differentiation of the iPSCs (day 14), the cartilaginous nodules were physically separated from the bottom of the dishes to form particles, which were then transferred to a suspension culture in 3.5-cm petri dishes. To increase proliferation, 50 μg/ml ascorbic acid (Nacalai), 10 ng/ml BMP-2 (Osteapharma), 10 ng/ml GDF5, and 10 ng/ml TGFβ (Peprotech) was added to the chondrogenic medium from day 3 to day 14. The medium was changed to conventional medium (DMEM with 10% FBS) on day 42. The medium was changed every 2 to 7 days.

Human fibroblast-derived iPSCs and PBMC-derived iPSC reprogramming

Fibroblast- or PMBC-derived iPSCs were kindly provided by the Institute of Cardiometabolism and Nutrition (ICAN, F-75013 Paris, France) or the iPSC core facility (INSERM, CNRS, UNIV Nantes, CHU Nantes, France) after reprogramming by various methods (episomal vector, mRNA transfection) as previously described [39, 41, 42].

Peripheral blood mononuclear cell (PBMC) culture and reprogramming with ascorbic acid

After the collection of PBMCs from control (obtained through the “Établissement Français du Sang” (EFS) according to the current ethical rules) and SRS patient (Comité de Protection des Personnes 18/56), they were allowed to proliferate for 7 days in blood medium (StemPro34 SFM medium with 100 µg/mL SCF, 100 µg/mL FLT3, 100 µg/mL IL3, 100 µg/mLIL6, and 54 U/µL EPO). For reprogramming, 2 × 105 cells were cultured in 24-well plates with a virus cocktail using the Sendaï 2.0 CytoTuneiPS reprogramming kit (Life Technologies) in either 5% CO2, 20% O2, and 75% N2 (normoxia condition) or 5% CO2, 5% O2, and 90% N2 (hypoxia condition). The virus was removed 24 h later (centrifugation at 300 × g for 7 min) and the cells transferred to 12-well plates in 1 ml blood media. After 3 days in culture, the cells were transferred to matrix gel-coated 6-well plates. At 7 days post-transduction, the cells were cultured in epiPS™ medium (mTeSR1 medium completed with ascorbic acid (50 µg/ml)) and half-was renewed daily. Fifteen days post-transduction, the iPSC clones appeared, were picked, and cultured in epiPS™ medium.

Immunofluorescence staining

iPSCs were cultured in four-well culture slides (Corning) for 3 days and then fixed in paraformaldehyde (4%) and permeabilized in blocking/permeabilization buffer (2% BSA, 0.5% Triton-X-100 in PBS) for 45 min and then incubated overnight at 4 °C with primary antibodies diluted in blocking/permeabilization buffer. The cells were washed three times in PBS and incubated with Alexa-conjugated secondary antibodies and DAPI, both diluted 1:1000 in blocking/permeabilization buffer, for 45 min at room temperature. The images were acquired using an epifluorescence microscope (Eclipse TE300, Nikon, Amsterdam, the Netherlands). The following antibodies were used: rabbit anti-Nanog (#4903S, 1:200, Cell Signaling-Ozyme, Beverly, MA, USA), rabbit anti-Oct4 (#3576-100, 1:200, Biovision, Cliniscience, Mountain View, CA, USA), rabbit anti-Sox2 (#AB5603, 1:200, Millipore, Ballerica, MA, USA), mouse anti-Tra-1-60 (#MAB4360, 1:100, Millipore), mouse anti-Tra-1-81 (#MAB4381, 1:100, Millipore), and mouse anti-SSEA4 (#sc-21704, 1:100, Santa Cruz, Dallas, TX, USA) (Figure SD2, in Additional file 2).

Karyotype analysis

Conventional cytogenetic studies on cell cultures at passage 20 were performed by Trousseau Hospital to verify chromosomal integrity. Fifteen metaphases were analyzed (Figure SD2, in Additional file 2).

Embryoid body formation and scorecard

Generated iPSCs were grown on Matrigel, dispensed into small clumps with a cell scraper, and cultured in suspension for 10 days in TeSR-E6 medium (Stemcell). At day 8, RNA was isolated from the embryoid bodies (Ambion). A scorecard kit (Life Technologies) was used to evaluate the gene expression of the three germ layers. The scorecard plate was analyzed using a StepOneplus™ system real-time PCR (Life Technologies) (Figure SD2, in Additional file 2).

Alkaline phosphatase

Alkaline phosphatase activity was measured using a detection kit (Sigma-Aldrich) and performed following the manufacturer's instructions (Figure SD2, in Additional file 2).

RNA extraction and reverse transcription

RNA was extracted from iPSCs before and on days 7, 14, 21, 28, and 74 of chondrogenic differentiation. Total RNA was extracted using the NucleoSpin miRNA Kit for the isolation of small and large RNA (Macherey–Nagel, France) with DNase treatment. Both DNA and RNA were quantified using a DS-11 spectrophotometer (DeNovix). cDNA was synthesized from long RNA using the miScript PCR System (Qiagen, France) and used for quantitative PCR.

Quantitative expression of chondrogenic differentiation-specific markers

Quantitative expression of chondrogenic differentiation-specific markers (Aggrecan, COL2A1, COL10A1, SOX9) was assessed for all samples using SyBR Select Master Mix (Applied Biosystems, ThermoFisher Scientific) and a Light Cycler LC480 instrument (Roche LifeSciences). The primers are listed in Table SD4 in Additional file 4.

DNA extraction

DNA was extracted from iPSCs before and on days 7, 14, 21, 28, and 74 of chondrogenic differentiation using an in-house protocol after cell lysis by a salting-out procedure, as previously described [43, 44].

Bisulfite treatment of DNA

Sodium bisulfite treatment of DNA converts all unmethylated cytosine residues to uracil residues. The methylated cytosine residues are unaffected. This process generates C/T polymorphisms, which can be used to distinguish between the methylated and unmethylated allele. Genomic DNA (400 ng) was treated with sodium bisulfite using the EZ DNA Methylation Lighting kit (Zymo Research, USA), according to the manufacturer’s instructions. Genomic DNA was eluted using 40 μl RNase-free H2O and conserved at − 20 °C.

TaqMan allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR) and methylation analysis.

The methylation status of seven imprinted loci (seven DMRs: 6q24 PLAGL1:alt-TSS-DMR, 7q12 GRB10:DMR, 7q32 MEST promoter DMR, 11p15 H19/IGF2:IG-DMR, 11p15 KCNQ1OT1:TSS-DMR, 14q32 MEG3/DLK1:IG-DMR, 15q11 PWS/AS:DMR) was assessed by ASMM RTQ-PCR, as previously described [14]. The methylation index (MI) at each locus was assigned by calculating the ratio between the methylated and unmethylated alleles as follows: (amount of methylated allele/sum of both methylated and unmethylated alleles) × 100. The ASMM RTQ-PCR primers and probes sequences are provided in Supplementary Methods (Table SD5, in Additional file 5).

gDNA and cDNA sequencing

To assess the mono or biallelic expression of imprinted genes, two single nucleotide polymorphisms from the H19 (rs217727) and DLK1 (rs1802710) gDNA and cDNA were sequenced by standard Sanger sequencing by Eurofins Genomics (Germany). The primers used for gDNA and cDNA amplification and sequencing are provided in Table SD6, in Additional file 6). The sequencing products were then analyzed using Sequencing Analysis 5.2. Generated and available iPSCs lines and sex of donors are resumed in Table SD7, in Additional file 7. All iPSCs lines have been derived from somatic cells of male individuals.

Statistics

Data in the figures are presented as means. All graphs were generated and statistical analysis performed using GraphPad Prism 6 (USA).