Human dermal fibroblast nucleofector kit

A Nucleofector™ 2b device (Lonza) was used for nucleofection of human primary cells. IMR90 cells, human myoblasts and human CD34⁺ hematopoietic cells were nucleofected with the Cell Line Nucleofector™ Kit R (Lonza, catalog # VCA-1001, program X-001), the Human Dermal Fibroblast Nucleofector™ Kit (Lonza, catalog # VPD-1001, program P-022) and the Human CD34⁺ Cell Nucleofector™ Kit (Lonza, catalog # VPA-1003, program U-008), respectively. Cell numbers for each nucleofection were 2 × 10⁵. We used 4.5 μg target plasmid DNA (expressing sgRNA/Cas9 or sgRNA/Cas9–Klenow) and 0.5 μg GFP-expressing plasmid DNA (CmiR0001-MR03, GeneCopoeia, Inc.) for each nucleofection, where the GFP-expressing plasmid DNA was used as an indicator for nucleofection efficiency. Transfected cells were checked for similar proportions of GFP-positive cells under a fluorescent microscope before further experiments.

6 Primary RA FLS cell lines (highest ranked by RARα PPR: RA6, RA1, RA4 (CL1) and lowest ranked by RARα PPR: RA10, RA11, RA9 (CL2)) were cultured (at 5% CO2, 37 °C) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine, gentamicin, penicillin/streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) as previously described^{41 (link)}. Optimal RARα knockdown by siRNA transfection was performed as previously described^{42 (link)} and assessed in time course experiments. Cells were transfected with ON-TARGETplus SMARTpool siRNA targeting human RARα (Dharmacon) or ON-TARGET plus Nontargeting Pool siRNA (Dharmacon) using the Human Dermal Fibroblast Nucleofector Kit (Lonza) and cultured in 10%FCS for 3 days. These pools include multiple targeting or matched control siRNA sequences. Cells were serum starved for 18 h in 0.1%FCS/DMEM and subsequently treated with IL-1 (2 ng/ml) for 6 h. RNA was extracted using the RNeasy kit (Qiagen) and qPCR was performed using a cell-based standard approach⁴³ . Ct (threshold cycle) values were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH).

A mixture of four siRNA duplexes (siGENOME SMARTPool siRNA) specific for IL17RA (NM_014339), IL17RB (NM_018725) and IL17RC (NM_032732) were purchased from Dharmacon (Lafayette, LA, USA). RA synoviocytes were used at 80–90% confluence. Cells were transfected with control siRNAs (siGENOME Non-Targeting Control siRNA #4) or target siRNAs by nucleofection using Amaxa technology (Lonza, Basel, Switzerland) according to the manufacturer’s instructions (program U23; Human Dermal Fibroblast Nucleofector kit). Dose- and time-response experiments were performed to determine the best time point and the lowest suitable concentration of siRNA duplexes needed for efficacious RNA silencing. Thus, 5 × 10⁵ cells were nucleofected with 0.5 µg of IL17RA, IL17RB, or IL17RC siRNA duplexes alone or in combination. Forty-eight hours post-transfection, part of the cells was stimulated as explained above in the Section “Cell Culture and Experimental Design” and another part was dedicated to siRNA efficacy control by qRT-PCR analysis of IL17RA, IL17RB, and IL17RC gene expression.

Adult human Type-I diabetic (T1D) donor fibroblasts obtained with patient informed consent, were purchased from DV Biologics, and cultured in fibroblast culture medium (I-Gro medium, DV Biologics). Episomal expression of SOX2, OCT4, KLF4, c-MYC, NANOG, LIN28, SV40LT was performed by nucleofection of 1x10⁶ diabetic fibroblast cells with 2 μg each of three plasmids, pCEP4-EO2S-EN2L, pCEP4-EO2S-ET2K, and pCEP4-EO2S-EM2K^{27 (link),28 (link)}. Single fibroblast cells were obtained with Accutase, and nucleofected using the human dermal fibroblast nucleofector kit (Lonza, VPD-1001) and Amaxa nucleofector program U-023. Nucleofected cells were transferred onto irradiated MEF in fibroblast growth medium supplemented with 10 μM Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y27362 (Stemgent). The next day, 2 mL of DMEM/F-12 supplemented with 20% KOSR, 0.1 mM MEM NEAA, 1 mM L-Glutamine, 0.1 mM β-mercaptoethanol, 50 ng/mL bFGF, 10 μM Y27362, 5 μg/mL ascorbic acid, and 3 μM CHIR99021 was added. Half of the medium was replaced with fresh medium without Y27362 every other day, until hiPSC colonies appeared. Individual hiPSC colonies were manually isolated, expanded onto vitronectin-coated plates in E8 medium, or further expanded and cryopreserved.

Cell culture media (RPMI 1640, M199 and DMEM), fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, amphotericin B, TRIzol reagent and DiOC₆ (3,3′-Dihexyloxacarbocyanine Iodide) were from Invitrogen (Cergy-Pontoise, France). LPS from Salmonella abortus equi and Propidium Iodide (PI) solution was obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). Synthetic bacterial lipopeptide Pam₃CSK₄ (BLP) was obtained from EMC Microcollections GmbH (Tuebingen, Germany). Polyinosine-Polycytidylic acid (Poly(I:C)) was obtained from InvivoGen (Toulouse, France). iScript cDNA Synthesis Kit and SsoAdvanced SYBR Green Supermix from Bio-Rad (Marnes-la-Coquette, France). The miScript System, miRNA mimc and Allstars negative control siRNA were obtained from Qiagen (Courtabeuf, France). miR-30a-3p antagonists were from Fisher scientific (Illkirch Cedex, France). Human Dermal Fibroblast Nucleofector kit was from Lonza (Cologne, Germany). The enzyme immunoassay kits for human BAFF, APRIL and IL-6 detection and recombinant IFN-γ were from R&D systems (Lille, France).