2. MATERIALS AND METHODS
2.1 M ATERIALS
2.1.1 Cell culture media, supplements and small molecule inhibitors
Reagents are further specified in the respective sections of the experimental procedures where they were applied. Reagents and supplies routinely used in cell culture are listed in Table M1 below.
Table M1. Media, supplements and equipment used in cell culture experiments. cat. #, catalogue number.
Reagent cat. # Supplier
2-Mercaptoethanol 31350-010 Life Technologies
Accutase A6964-100ML Sigma-Aldrich
B27 Supplement 17504044 Life Technologies
BMP-2 (recombinant) 0120-02-10 PeproTech
Bovine Serum Albumin (BSA), Cell Culture Grade 10773877 Thermo Fisher Scientific
Cell scraper sc-213230 Santa Cruz
CHIR99021 4953/50 R&D Systems
Collagenase, type IV 17104019 Life Technologies
Cryotubes 10577391 Thermo Fisher Scientific
DMEM 21969035 Life Technologies
DMEM/F12 11320074 Life Technologies
DMSO D5879-100ml Sigma-Aldrich
Dorsomorphin 3093 Tocris
Doxycycline hydrochloride D9891-1G Sigma-Aldrich
EGF E9644-.2MG Sigma-Aldrich
Fibronectin from human plasma F1056-1MG Sigma-Aldrich
Gelatin, powdered, pure Ph. Eur., NF A1693.0500 AppliChem
Geltrex Basement Membrane Matrix A1413302 Life Technologies
Gentle Cell Dissociation Reagent 7174 STEMCELL technologies
GlutaMAX, 100X 35050038 Life Technologies
HyClone™ Fetal Bovine Serum (South America), Research
Grade SV30160.03
LOT #RZB35918 GE Healthcare
Insulin 12585014 Life Technologies
Knockout-Serum Replacement (KSR) 10828028 Thermo Fisher Scientific
Matrigel-Matrix 354230 Corning
MEM Non-Essential Amino Acids Solution (NEAA, 100X) 11140050 Thermo Fisher Scientific
mFreSR™ 5855 STEMCELL technologies
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone,
33 mm, gamma sterilized SLGP033RS Merck Millipore
mTeSR1 medium 5850 STEMCELL technologies
N2 Supplement 17502048 Life Technologies
Neurobasal Medium 21103049 Life Technologies
Phosphate-buffered Saline (PBS), 1x 14190094 Life Technologies
Penicillin-Streptomycin 15070063 Life Technologies
Recombinant Human FGF-basic (154 a.a.; bFGF/FGF-2) 100-18B Peprotech
StemMACS iPS-Brew XF 130-104-368 Miltenyi Biotec
StemMACS SB431542 in Solution 130-106-543 Miltenyi Biotec
Trypsin-EDTA (0.25%), phenol red 25200056 Thermo Fisher Scientific
Y-27632 dihydrochloride (ROCKi) 1254/10 R&D Systems
µ-Slide 8 Well, ibidi-treat 80826 Ibidi
2.1.2 Cell lines
Two different human embryonic stem cell lines, H9 (WA09) and HUES9 iCas9190 were used in this work as control lines and for genetic engineering purposes as outlined in the respective methods sections below. The iCas9 line bears a genomic integration of the endonuclease Caspase 9 (Cas9), expression of which can be induced by administration of doxycycline (DOX) to the culture medium190 .
For comparison to the BOS patient-derived iPSC that were generated during this study (see section 2.2.2), two control hiPSC lines were applied. These were generated by E.
Rusha and Dr. Pertek at the hiPSC Core Facility of the Helmholtz Center Munich, Germany via reprogramming of fibroblasts from a healthy 2-year-old donor using modified mRNA (for details on the reprogramming method, see191), and via reprogramming of B-lymphocytes derived from a healthy 12-year-old donor via 4-in-1-mini-intronic plasmids17.
2.1.3 Chemicals
All chemicals were of reagent grade; routinely used reagents are denoted in Table M2 below.
Table M2. Chemicals, reagents and solutions routinely used in this study.
Reagent cat. # Supplier
16% Formaldehyde (w/v), Methanol-free 10321714 Thermo Fisher Scientific
2-Mercaptoethanol M3148 Sigma-Aldrich
Albumin from bovine serum (BSA) A9647-10G Sigma-Aldrich
Ampicillin sodium salt A-166 Sigma-Aldrich
Biozym LE Agarose 840004 Biozym
Boric acid, electrophoresis grade 15166.02 Serva Electrophoresis
DAPI Nuclear Staining Dye 1351303 Bio-Rad Laboratories
DNA Gel loading dye, 6x R0611 Thermo Fisher Scientific
Dynabeads(R) Protein A for Immunoprecipitation 10001D Life Technologies
EDTA Dinatriumsalz Dihydrat >99% X986.1 Carl Roth
EGTA 3054.1 Carl Roth
Ethanol, 99.8% 9065.2 Carl Roth
Ultra pure glycerol 15514011 Thermo Fisher Scientific
Glycine 23391.02 Serva Electrophoresis
HEPES, 1M Buffer Solution 15630122 Life Technologies
Isopropanol 6752.2 Carl Roth
Potassium hydroxide (KOH) 6751 Carl Roth
Lithium chloride (LiCl) 62480-500G-F Sigma
Lithium Acetate dihydrate, 98% 15157442 Thermo Fisher Scientific
Methanol 45631.02 Serva Electrophoresis
Magenesium chloride (MgCl2) KK36 Carl Roth
Na-deoxycholate D6750-10G Sigma-Aldrich
NaHCO3 S5761 Sigma-Aldrich
Tergitol type NP-40 70% solution NP40S Sigma-Aldrich
Nuclease-free water (H2O) AM9932 Life Technologies
Phenylmethylsulfonylfluorid (PMSF) 6367,1 Carl Roth
Powder Milk, blotting grade T145.1 Carl Roth
Protease Inhibitor Cocktail Set III, EDTA-Free 539134 Merk Millipore Ribonucleic acid, transfer from bakers yeast (tRNA) R5636-1ML Sigma-Aldrich
SDS Solution, 20 % 20768.02 Serva Electrophoresis
Sodium Chloride (NaCl) P029.2 Carl Roth
SYBR® Safe DNA Gel Stain 5001208 Life Technologies
TE buffer, pH 7.4, RNAse free 93302 Sigma-Aldrich
TRIS PUFFERAN® 5429.3 Carl Roth
Triton™ X-100 X100-500ML Sigma-Aldrich
Tween(R)-20 P9416 Sigma-Aldrich
2.1.4 Kits
Kits that were routinely used in this study are listed in Table M3 below.
Table M3. Kits routinely used in this study.
Kit cat. # Supplier
GeneJET Plasmid Miniprep Kit K0502 Fermentas
P3 Primary Cell 4D-Nucleofector® X Kit V4XP-3024 Lonza
PureLink HiPure Plasmid Filter Maxiprep Kit K210017 Life Technologies
QIAprep Spin Miniprep Kit 27104 Qiagen
QIAquick PCR Purification Kit 28104 Qiagen
QIAquick Gel Extraction Kit 28704 Qiagen
RNA 6000 Pico kit 5067-1513 Agilent
RNeasy MinElute cleanup kit 74204 Qiagen
RNeasy Mini Kit 4104 Qiagen
2.1.5 Enzymes
Unless otherwise noted, enzymes were used in the provided buffers, and are either denoted in Table M4 below or in the respective sections of the experimental procedures where they were applied.
Table M4. Enzymes or enzyme master-mixes routinely used in this study. NEB, New England Biolabs
Enzyme/ Enzyme Mix cat. # Supplier
BsaI R0535 S NEB
EcoRI-HF R3101 NEB
Gibson Assembly Master-Mix E2611 L NEB
Pfu DNA Polymerase, recombinant EP0501 Thermo Fisher Scientific Power SYBR® Green PCR Master Mix 4367659 Thermo Fisher Scientific
Proteinase K Solution 20 mg/ml AM2546 Life Technologies
Q5® High-Fidelity 2X Master Mix M0492 NEB
Quick Ligation™ Kit M2200 L NEB
RNase A (20 mg/ml) 12091021 Life Technologies
SuperScript® III First-Strand Synthesis System for RT-PCR 18080051 Life Technologies
T4 DNA Polymerase (T4) M0203 NEB
Taq PCR Master Mix Kit 201445 Qiagen
TaqMan® Gene Expression Master Mix 4369016 Thermo Fisher Scientific
TopTaq Master Mix Kit 200403 Qiagen
TURBO DNase 2U/µl am2238 Life Technologies
XhoI (NEB) R0146S NEB
2.1.6 Oligonucleotides
Oligonucleotides used for cloning, CRISPR/Cas experiments, quantitative real-time PCR (qPCR) and reverse transcription PCR (RT-PCR) were purchased from
Sigma-Aldrich/Merck (St. Louis, USA), and sequences are listed in the respective sections of the experimental procedures. Primers were designed using the Primer3web software version 4.1.0192.
2.1.7 Antibodies
Table M5. Antibodies used in thus study. ICC, Immunocytochemistry; WB, Western Blot; ChIP, Chromatin immunoprecipitation; Co-IP, Co-immunoprecipitation.
Target/Clone Application Dilution cat. # Supplier
Oct4 ICC 1:400 2840 (C30A3) Cell Signaling
Sox2 ICC 1:200 2748 Cell signaling
Nanog ICC 1:200 ab21603 Abcam
Lin28a ICC 1:200 3978 (a177) Cell Signaling
TFAP2A ICC 1:200 sc-184X Santa Cruz
Sox9 ICC 1:100 - obtained from Prof. Dr.
Götz
SOX10 MAb (Cl 20B7) ICC 1:100 MAB2864 R&D
Ki67 ICC 1:500 - obtained from Prof. Dr.
Götz Anti-human CD57/HNK-1, Cl. VC1.1 ICC 1:1000 C6680-50TST Sigma
hNGF R/p75 NTR MAb (Cl 74902) ICC 1:100 MAB367 R&D
Alexa Fluor 594 anti-rat IgG ICC 1:1000 A-11007 Invitrogen
Alexa Fluor 488 anti-rabbit IgG ICC 1:1000 A-21206 Invitrogen Alexa Fluor 488 anti-mouse IgG ICC 1:1000 A-21202 Invitrogen Alexa Fluor 647 anti-mouse IgG ICC 1:1000 A-21235 Invitrogen Alexa Fluor 594 anti-rabbit IgG ICC 1:1000 A-21207 Invitrogen
ASXL1 (clone 12F9) ICC 1:2 - this study/E. Kremmer
ASXL1 (clone 4F6) WB 1:10 - this study/E. Kremmer
ZIC1 WB 1:400 AF4978-SP R&D
H2A WB 1:5000 GTX129418 GeneTex
Ubiquityl-Histone H2A (Lys119) WB 1:2000 8240 New England Biolabs
H3K27me3 ChIP and WB 1:750 C15410069 Diagenode
ACTIN WB 1:1000 3700 Cell Signaling
mouse anti-rat IgG (isotype IgG2a),
HRP-conjugated WB 1:1000 - Dr. Kremmer
Goat anti-mouse IgG
HRP-conjugated WB 1:30,000 115-035-003 Dianova/Jackson
Goat anti-rabbit antibody,
HRP-conjugated WB 1:30,000 111-035-045 Dianova/Jackson
rabbit IgG isotype control ChIP, Co-IP 5 µl/IP GTX35035 GeneTex
EZH2 Co-IP 10 µl/IP 39934 Active Motif
2.1.8 Software
Table M6. Software routinely used for analyses or visualization of results in this study.
Software Supplier
ApE - A plasmid Editor v2.0.46 M. Wayne Davis Genomatix Software Suite v3.10 Genomatix AG
R v3.3.2 The R foundation for Statistical Computing (R Core team 2014)
Image Lab v5.2.1 Bio-Rad Laboratories
Fiji/ImageJ v2.0.0 National Institutes of Health NIH, USA Excel (for mac 2011) v14.0.0 Microsoft Corporation
Integrative Genomics Viewer v2.3.98 Broad Institute, USA