Cell Culture and Lentiviral Transductions
Dermal fibroblasts derived from skin biopsies from three CGD patients after informed consent (ethical permission no. Zurich 2010-0077/2) and two unaffected individuals were cultured in tissue-culture-coated flasks (Iwaki T25) in Iscove's modified Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum (PAA Laboratories, Somerset, U.K.), 2 mM l-glutamine (PAA Laboratories), 0.1 mM nonessential amino acids (PAA Laboratories), and 100 units/ml penicillin (PAA Laboratories). Induction of pluripotency was performed using the four transcription factors OCT4, SOX2, LIN28, NANOG delivered as separated lentiviral particles (iPS-CGD1.1; Stemgent, San Diego, CA) or OCT4, SOX2, KLF4, and c-MYC as a single lentiviral particle carrying a polycistronic vector encoding all four transcription factors (Allele biotech, San Diego, CA). The lentiviral vectors were added to cultures of dermal fibroblasts in the log phase of growth according to manufacturer's instructions. Briefly, 2 × 105 fibroblasts were seeded onto one well of a 12-well plate and cultured in Iscove's Modified DMEM, 10% fetal calf serum (PAA Laboratories), 2 mM l-glutamine (PAA Laboratories), 0.1 mM nonessential amino acids (PAA Laboratories), and 100 units/ml penicillin (PAA Laboratories) for 24 hours. The cells were then infected with lentiviral vectors (multiplicity of infection (MOI) = 2) with the addition of 6 μg/ml polybrene. Six days after transduction, fibroblasts were disaggregated to single cells by trypsinization (0.05% trypsin; Invitrogen, Paisley, U.K.) and then plated onto feeder layers of mitotically inactivated mouse embryonic fibroblasts (MEFs) in human ESC (hESC) culture medium at a density of 8,000 cells per one well of a six-well plate. The feeder plates with lentiviral vector-treated cells were maintained at 37°C/5% CO2 in hESC medium (knockout DMEM [KO-DMEM], 20% knockout serum replacement, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 100 units/ml penicillin [PAA Laboratories], and 10 ng/ml human recombinant basic fibroblast growth factor [bFGF]) for 21 days or until colonies of cells with a morphology similar to hESC appeared. These were mechanically dissected into several pieces and plated onto fresh feeder cells to develop further colonies for characterization.
Culture and Differentiation of Patient-Specific and Unaffected iPSC Lines
CGD patient-specific and unaffected control hiPSC lines were cultured as discrete colonies on feeder layers of mitotically inactivated MEFs as previously described [21
] with hESC medium containing KO-DMEM (Invitrogen), 2 mM l
-glutamine (Invitrogen), 100 mM nonessential amino acids (Invitrogen), 20% serum replacement (Invitrogen), and 8 ng/ml FGF2 (Invitrogen). For monolayer differentiation, hESC colonies were released from the feeder cell layers by treatment with Collagenase IV (1 mg/ml) followed by mechanical disruption then transferred to six-well plates (Iwaki, Buckinghamshire, U.K.) precoated with 1% gelatin in differentiation medium (KO-DMEM, 20% fetal bovine serum (FBS), 2 mM l
-glutamine, and 100 mM nonessential amino acids (all from Invitrogen) as described in [22
]. Embryoid body (EB) differentiation was induced by releasing the hESC and iPSC from the feeder cells using Collagenase IV and transferring them to low attachment six-well plates (Costar, Arlington, U.K.) in differentiation medium as described above.
Total RNA was extracted using TRIzol reagent (Invitrogen, Paisley, U.K.) according to manufacturer's instructions. Following DNaseI treatment using RQ1 DNaseI (Promega, Mannheim, Germany), cDNA was synthesized using SuperScript Reverse Transcriptase (Invitrogen). Quantitative reverse transcript polymerase chain reaction (RT-PCR) analysis was carried out using SYBR Green PCR master mix (Sigma) and the primers are listed SOX2 total forward: AACCCCAAGATGCACAACTC; reverse: TCTCCGTCTCCGACAAAAGT; SOX2 endogenous forward: GGCGCTTTGCAGGAAGTTTG; reverse: GCAAGAAGCCTCTCCTTGAA.
We carried out DNA fingerprinting to confirm that CGD patient-specific and unaffected hiPSC were of identical origin to the respective patient dermal fibroblasts. Total genomic DNA was extracted from all five samples and amplified with 11 microsatellite markers: D3S1358, vWA, D16S539, D2S1338, amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, and FGA and analyzed on an ABI 377 sequence detector using Genotype software (Applied Biosystems, Foster City, CA).
Validation of Known CGD Patient Mutations in iPSCs
To validate the mutations in iPSC-CGD1.1 and iPSC-CGD1.2 found in the patient's peripheral blood DNA, sequences were amplified using the primers ATGAGGTGTTCAGAGTGGTGACAG and CCATGCCCAGCTCGCAT and the long range PCR kit (Qiagen, Hombrechtikon, Switzerland). PCR products were subcloned into TOPO XL (Invitrogen AG, Basel, Switzerland) and sequenced using a ABI Prism Genetic Analyzer (Applied Biosystems Deutschland GmbH, Darmstadt, Germany). To confirm the mutation in iPSC-CGD2 the primers GTCATACTGGTGGAGGGAAAGC and GCTCCAACCTGCCCTTCC, and for iPSC-CGD3 the primers TTCTGTCAGGTTTGCCATGT and GCCTCCTTGTCTTTTGTGTTC were used for PCR amplification prior to subcloning and sequencing.
Cells were fixed in 4% (wt/vol) paraformaldehyde for 20 minutes. An additional permeabilization step (0.2% (vol/vol) Triton X-100 in phosphate-buffered saline [PBS]) was performed prior to staining with antibodies for internal cell markers. Blocking step was performed by incubation in 1% (wt/vol) bovine serum albumin (BSA) or alternatively in 10% (vol/vol) goat serum. Cells were incubated with primary antibodies for 1 hour and secondary antibodies for 30–60 minutes. Primary antibodies used in this study are anti-OCT-4, clone 7F9.2 (1:100; Millipore, Watford, U.K.), anti-SSEA-4, clone MC813-70 (1:100; BD Pharmingen), anti-TRA-1-60 (1:100; Millipore), anti-CD31 (1:100; PECAM1) (1:100; BD Pharmingen), anti-β-III-Tubulin (TUJ1) (1:100; Covance, NJ), anti-α-fetoprotein (AFP) (1:100; Sigma), and anti-mouse IgG-fluorescein isothiocyanate (FITC) conjugated (1:200; Sigma). The nuclei were counterstained with 10 μg/ml Hoechst 33342 (Molecular Probes [Invitrogen]). The bright-field and fluorescent images were obtained using a Zeiss microscope and the AxioVision software (Carl Zeiss, Jena, Germany).
Alkaline Phosphatase Staining
Alkaline phosphatase (AP) staining was carried out using the AP Detection kit according to manufacturer's instructions (Chemicon, Temecula, CA). The bright-field images were obtained using a Zeiss microscope and AxioVision software (Carl Zeiss).
Karyotype Analysis of CGD Patient-Specific and Unaffected hiPSC
Karyotypes were determined by Standard G-Banding Procedure.
To evaluate the developmental potential of hiPSC, approximately 5 × 105
hiPSCs were injected subcutaneously into the right flank of in adult severe combined immunodeficient (SCID) mice and maintained for 6–12 weeks [23
]. Similarly, for a positive control, approximately 5 × 105
hESCs (H9 cell line, reviewed in [24
]) were injected subcutaneously in adult SCID male mice. All cells were cotransplanted with 50 μl Matrigel (BD Biosciences) to enhance teratoma formation. Four to six animals were injected in each group. After 6–12 weeks, mice were sacrificed, tissues were dissected, fixed in Bouins overnight, processed and sectioned according to standard procedures, and counterstained with either hematoxylin and eosin or Massons Trichrome stain. Sections (5–8 μm) were examined using bright-field light microscopy and photographed as appropriate.
Bisulfite Sequencing of the OCT4 and NANOG Promoters
Briefly, genomic DNA was isolated from CGD patient-specific and unaffected hiPSCs (DNEasy kit, Qiagen), denatured, and converted by reaction with sodium bisulfite using the EpiTect bisulfite kit (Qiagen, Crawley, U.K.). The bisulfite-treated DNA was used as template to amplify regions of the OCT4 and NANOG promoter sequences using the following primers sets: OCT4 Promoter forward CCTAAACTCCCCTTCAAAATCTA, reverse GGATGTTATTAAGATGAAGATAG; NANOG Promoter forward TGG TTAGGTTGGTTTTAAATTTT, reverse AACCCACCCTTATAAATTCTCAA. The resulting PCR products were subcloned into TOPO vector, and 10 clones of each cell population were sequenced. The sequence data were analyzed using the biQ Analyzer software (Max Planck Institut Informatik, Universität des Saarlandes, Germany).
Telomerase Activity Measurement
Telomeric repeat amplification protocol (TRAP) reactions were carried using the Telo TAGGG
Telomerase PCR enzyme-linked immunosorbent assay (ELISA) Plus (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions and as described previously [25
Differentiation to Monocytes and Macrophages
iPS lines and a control hESC line HUES2 [26
] (work on this cell line was reviewed and approved by the U.K. Stem Cell Bank Steering Committee) were differentiated to monocytes and thence to macrophages, as described previously [20
]. Briefly, colonies were mechanically lifted from MEFs and patches were transferred into six-well ultra-low adherence plates (Corning) in hES culture medium and cultured for 3 days. For differentiation, 20 EBs were transferred into one well of a six-well tissue culture plate in 3 ml medium. Two-thirds of the medium was replaced every 5 days. Culture medium consisted of Advanced DMEM (Invitrogen) and 10% fetal calf serum (Biosera, Ringmer, U.K.), supplemented with 100 ng/ml M-CSF (R&D Systems Inc., Minneapolis, MN), 25 ng/ml IL-3 (R&D), 2 mM glutamax (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen AG, Basel, Switzerland), and 0.055 mM β-mercaptoethanol (Invitrogen). Monocytes emerging into the supernatant were harvested and counted, and fresh medium added to the culture for subsequent monocyte production. Harvested monocytes were differentiated into macrophages at a density of 1.5 × 105
cells per square centimeter in regular tissue culture plates. Culture medium consisted of RPMI (Invitrogen) and 10% fetal calf serum (Biosera), supplemented with 100 ng/ml M-CSF (R&D), 2 mM glutamax (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Medium was changed every 3–4 days and cells were used after 1 week of differentiation.
Isolation of Blood Monocytes
Adult human blood was obtained in accordance with Local Institutional Review Board policy from anonymous donors through the U.K. National Blood Bank Service and was tested negative for HIV-1, hepatitis B/C, and syphilis. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia-Amersham) density-gradient centrifugation from heparinized buffy coats. Monocytes were isolated by CD14-positive selection using anti-CD14 magnetic beads (Miltenyi Biotec) by following manufacturer's instructions. Monocytes were plated as for iPS-derived monocytes described above, for differentiation to macrophages.
Cells were washed and stained in fluorescence-activated cell sorting (FACS) buffer consisting of PBS, human IgG (10 μg/ml, Sigma), 1% fetal calf serum (Biosera), and 0.01% sodium azide. Macrophages were detached using cold PBS containing 5 mM ethylenediaminetetraacetic acid, washed, and stained for surface markers on ice for 30 minutes. Cells were washed three times before acquisition on a Becton Dickinson FACs Calibur, and all antibodies were compared with the appropriate isotype-matched control at the same concentration from the same manufacturer. Antibodies used were Immuno Tools allophycocyanin (APC)-conjugated mouse IgG1 anti-human CD14, mouse IgG1 anti-human CD45, and IgG1 isotype control. Data were analyzed using FlowJo software and presented as histograms with antibody staining (black line) relative to isotype-matched control (gray fill).
Fluorescent Zymosan Uptake
Saccharomyces cerevisiae ZymosanA BioParticles (Alexa Fluor 594 conjugated, Invitrogen) were reconstituted according to the manufacturer's instructions, using PBS with 2 mM sodium azide and sonication to disrupt particle aggregates. Zymosan particles were carefully counted and added to adherent macrophages at a ratio of two particles/cell, in serum-free RPMI. Phagocytosis was allowed to occur for 30 minutes at 37°C, followed by one wash with PBS, one wash with 250 μg/ml trypan blue in PBS to quench particles bound to the outside of the cell, and a further wash with PBS. Cells were then incubated with 0.25% trypsin/0.5 mM EDTA (in Hanks' balanced saline solution (HBSS); Sigma) at 4°C for 1 hour to detach the cells. Detached macrophages were centrifuged at 400g and fixed with 4% formaldehyde in PBS. Uptake of zymosan was quantified using a Becton-Dickinson FACS Calibur flow cytometer and data were analyzed using FlowJo software. Fluorescence negative cells (not fed zymosan) were used to establish a threshold for quantifying the percentage of positive cells having taken up one or more zymosan particle.
Proteome Profiler Human Cytokine Array
Media from 7-day differentiated macrophages (either unactivated or activated for 18 hours with 20 ng/ml IFNγ (R&D) and 100 ng/ml lipopolysaccharide (LPS; Sigma) (added into the existing medium) were spun down at 400g to remove cells and the supernatant was frozen at −20°C until analysis. Proteome Profiler Human Cytokine Array (Panel A, R&D Systems) was used to detect the presence of 36 different cytokines using a membrane antibody array, with biotinylated detection antibodies followed by streptavidin conjugated to horse radish peroxidase and development with a chemiluminescent substrate (Pierce; performed according to the manufacturer's instructions). The pixel density of spots on the image (captured with Gene Snap software) was quantified using Gene Tools software, with background subtracted, and individual cytokine pixel density was expressed as a percentage of the mean positive control spot density.
Chemiluminescence assay was carried out in 96-well plates with 50,000 cells per plate. Cells were kept in HBSS with Ca/Mg (Invitrogen AG) and 0.5% human albumin (ZLB Bioplasma AG, Bern, Switzerland). Monocytes were stimulated with 200 ng/ml phorbol myristic acid (PMA; Sigma-Aldrich, Buchs, Switzerland). Macrophages were stimulated with 200 ng/ml PMA and 0.1 nmol/ml formyl-Met-Leu-Phe (fMLP, Sigma). Chemiluminescence signal was amplified by 50 μM luminol (Sigma-Aldrich) and analyzed on a Mithras LB940 Chemiluminescence reader (Berthold Technologies, Regensdorf, Switzerland).
Nitro Blue Tetrazolium Assay
Monocytes were cultured over night in 96-well plates in 100-μl volume. For the visualisation of reactive oxygen species (ROS) production, 100 μl of nitro blue tetrazolium (NBT; Sigma-Aldrich) 1 mg/ml in HBSS with Ca/Mg containing 1 μg/ml PMA was added. Macrophages were analyzed after 8 days of differentiation. For priming, cells were incubated with 1 ng/μl LPS (Sigma, L4391) for 3 hours prior to stimulation with 0.5 μg/ml PMA and 0.5 nmol/ml fMLP in presence of NBT. Cells were incubated at 37°C for 1 hour and fixed in PBS (Invitrogen AG, Basel, Switzerland), containing 2% formaldehyde (Merck [Schweiz] AG, Zug, Switzerland). Assay was documented using a Leica DM-IL inverse microscope equipped with a DFC480 digital camera (Leica Microsystems Ltd., Heerbrugg, Switzerland).
NBT and Dihydrorhodamine Assay of Blood Granulocytes
Both assays were conducted according to standard protocols.