Isolation and cell culture of MSCs
Human umbilical cords (UC) were provided from healthy, normal, full-term newborns, after obtatining written informed consent in accordance with guidelines approved by the Ethics Committee on the Use of Human Subjects at Asan Medical Center (IRB# : 2015-0303). Human UCs were collected and UC-MSCs were isolated as previously described [23]. Human UC-MSCs were maintained in alpha-MEM (Gibco, Franklin Lakes, NJ) supplemented with 10% fetal bovine serum (Gibco), 2% penicillin/streptomycin (Gibco).
Preparation of Cas9 protein and gRNAs
Recombinant Cas9 protein was purified as described previously [24]. Cas9 crRNAs and tracrRNAs were designed and synthesized by Integrated DNA Technology [25]. For single guide RNA system, sgRNAs were synthesized by in vitro transcription using T7 RNA polymerase and a template oligonucleotide as described previously [26]. Calf intestinal phosphatase (CIP) (New England Biolabs, Ipswich, MA, USA) was used to remove the 5′-triphosphate from gRNAs. In vitro transcribed gRNAs were treated with 250 units of CIP for 3 h at 37°C and the gRNAs were cleaned up using a miRNeasy Mini kit (Qiagen, Germantown, MD, USA). For plasmid DNA delivery, spCas9 (Addgene, #43945) and pRG2 (Addgene, #104174) were used. The gRNA target sequences are listed in Table S1.
Electroporation in MSCs
5 x 104 MSCs were mixed with the Cas9 RNPs with various molar ratios and incubated for 15 min at room temperature prior to electroporation. Two electroporation systems, Neon transfection system (Thermo Fisher Scientific, Waltham, MA, USA) and 4D-Nucleofector system (Lonza Bioscience, Basel, Switzerland), were used to optimize MSC engineering conditions. The RNP complexes and MSCs were electroporated using 24 different sets of parameters with the Neon system and two different programs (EW104 and FF104) using the 4D-Nucleofector system.
Analysis of mutation frequencies and off-target effects
Targeted deep sequencing was used to analyze mutation frequencies, as described previously [27]. Briefly, genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen Pty Ltd, Hilden, Germany) and the target regions were then amplified using Phusion DNA polymerase (New England Biolabs), in accordance with the manufacturer’s protocol. The PCR amplicons were subsequently purified and subjected to paired-end sequencing using the Illumina MiniSeq or iSeq 100 sequencing systems. The mutation frequencies were analyzed using the MAUND program [28]. The PCR primer sequences are listed in Table S2.
To determine the off-target effects of B2M-TS1 gRNA, the potential off-target sites were predicted by in silico analysis using Cas-OFFinder (http://www.rgenome.net/cas-offinder) and in vitro analysis using Digenome-seq [29, 30]. In the analysis using Digenome seq, MSC genomic DNA was cleaved in vitro by the addition of100 nM Cas9 proteins and 300 nM B2M-TS1 gRNA for 18 h in NEBuffer 3.1 (New England Biolabs). RNase A was also added to the reaction. The cleaved genomic DNA was then purified using DNeasy Blood & Tissue Kit (Qiagen) and the cleavage efficiencies were analyzed by real-time PCR. Both the cleaved and wild-type genomic DNA were sequenced using the Illumina HiSeq X Ten system and the resulting data were analyzed to identify potential off-target sites using the Digenome-seq web tool (http://www.rgenome.net/digenome-js) [31]. The potential off-target sites were then amplified using Phusion DNA polymerase and used for NGS library construction as mentioned above.
CPD (cumulative population doublings)
MSCs were cultured from passage 12 to 19. The number of cells was counted at each stage and 1 x 105 cells were seeded for the next passage. The number of newly grown cells during each passage was calculated, and a graph (CPD curve) was drawn using these counts as the y-axis of the curve.
Antibody staining and flow cytometry
MSCs were detached with trypsin-EDTA, collected and 1.0 x 104 aliquots of cells were incubated with antibodies (antibody 1 μl/PBS 500 μl) for 15 minutes at 4°C. The cells were then washed with cold PBS and fixed with 4% paraformaldehyde. Antibodies attached to specific cell surface proteins were detected with a FACS Canto II machine (Becton Dickinson Biosciences, Bedford, MA, USA) and analyzed using FlowJo 10.4 software (FlowJo). The following antibodies were used: FITC anti-human HLA-ABC, FITC anti-human CD45, PerCP anti-human CD73, FITC anti-human CD34, PE anti-human CD90, FITC anti-mouse IgG, and PE anti-mouse IgG (all sourced from BioLegend, San Diego, CA, USA).
MSC and T cell co-cultures
Human T cells were obtained from healthy, normal donors after obtatining written informed consent in accordance with guidelines approved by the Ethics Committee on the Use of Human Subjects at Asan Medical Center (IRB# : 2015-0303). Aliquots of 1.0 x 104 MSCs were incubated in RPMI 1640 with or without IFN-γ (20ng/ml). The following day, 1.0 x 105 T cells were stained with CellTrace CFSE (Carboxyfluorescein succinimidyl ester) kit (Thermo Fisher Scientific) for 1 h and co-cultured with MSCs at a 10:1 ratio and IL-2 (10 ng/ml). After incubation for 6 days, the T cells were harvested, incubated with CD4- and CD8-conjugated antibodies (BioLegend), and analyzed by flow cytometry. The MSCs were also harvested and incubated with CCK-8 (Cell Counting kit-8) (Dojindo Laboratories, Kumamoto, Japan) for 4 h and then assayed by measuring the absorbance at 450nm using a microplate reader.
Quantitative real-time reverse transcription (RT)-PCR
Total RNA was isolated from MSC using RNA extraction kit (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. cDNA was then synthesized from these RNA preparations using a reverse transcriptase mixture (Thermo Fisher Scientific). Quantitative real-time PCR was subsequently performed on a Bio-Rad (CFX ConnectTMOptics Module) using the TOPrealTMqPCR 2x premix (SYBR Green with low ROX) (Enzynomics, Daejeon, Korea) as per the manufacturer’s instructions. The sequences of the primers used are listed in Table S2.
Western blotting
The MSC cultures to be analyzed by immunoblotting were washed with cold PBS and lysed in RIPA buffer (Thermo scientific) containing a protease inhibitor cocktail for 30 min. The resulting cell lysates were centrifuged at 3000g for 20 min and the supernatants were collected. The protein concentrations in the supernatants were subsequently analyzed using a BCA protein assay kit (Thermo scientific). Samples for each MSC culture containing 20 µg of protein were mixed with 2x SDS-sample buffer, boiled for 5 min and loaded onto a 8%–12% gradient sodium dodecyl sulfate reducing gel. The separated proteins were subsequently transferred onto nitrocellulose membranes using a transblot system. The membrane blots were first blocked for 1 h at room temperature in Tris-buffered saline containing 5% skim milk and washed three times with TBST (TSB containing 0.1% Tween 20). The membranes were then incubated at 4ºC overnight with primary antibodies at a 1:3000 dilution in TBST containing 3% skim milk. The primary antibodies used were raised against IDO-1 (Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). The membranes were further incubated at room temperature for 1 hour with secondary antibodies at a 1:6000 dilution in TBST containing 3% skim milk. The secondary antibodies used were anti-mouse IgG HRP-linked antibody and anti-rabbit IgG HRP-linked antibody (Cell Signaling, Danvers, MA). The signal was visualized by enhanced chemiluminescence (Advansta, San Jose, CA, USA) and detected by ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).
Quantification of immunomodulatory molecules
Aliquots of 1.0 x 105 MSCs were seeded into 6 well plates. After an initial 24 h incubation, the culture media was changed and after a further 24 h of culture, the supernatant was harvested. The sampled supernatants were then analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits for PGE2 (MyBioSource, San Diego, CA, USA), C-C motif chemokine ligand 2 (CCL2/MCP-1), and IL-6 (R&D Systems Inc., Minneapolis, MN, USA). For IFN-γ, TNF-α, IL-1β, chemokine (C-X-C motif) ligand (CXCL) 9, CXCL10 and C-C motif chemokine ligand 3 (CCL3), multiplex ELISA were performed through LABISKOMA company (Seoul, Korea) using human premixed multi-analyte kit and markers (R&D system inc., Minneapolis, MN, USA).