Chondrogenic differentiation was performed using MSC at P1 which were seeded into 6-well plates (Sarstedt) at 2 × 105 cells per well in DMEM with 15% knockout serum (Gibco, Invitrogen), 5 mg/mL ascorbic acid, 6 μg/mL transferrin, 10 μM dexamethasone, 1 × 10−7 M retinoic acid (all form Sigma–Aldrich), and 1 ng/mL recombinant human transforming growth factor-β3 (TGF-β3) (ProSpec-Tany TechnoGene; Deltaclon). Adipogenic and osteogenic differentiation were performed with MSCs at P1 seeded in 6-well plates (Sarstedt) at 2 × 105 cells per well in adipogenic or osteogenic commercial medium (Cambrex, Lonza), following the manufacturer's instructions, to assess the mesodermal differentiation potential. Control MSC were grown under DMEM with 10% knockout serum (Gibco, Invitrogen), 1% penicillin, and 1% streptomycin (Sigma–Aldrich). All the mediums were changed every 3 days.
Knockout serum
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Knockout serum is a laboratory reagent used to supplement cell culture media. It is designed to support the growth and maintenance of embryonic stem cells and other pluripotent cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (16 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Glutamax, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Matrigel, by Corning (7 mentions)
β mercaptoethanol, by Merck Group (6 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Glutamine, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (4 mentions)
Accumax, by Merck Group (4 mentions)
Mtesr1 medium, by STEMCELL (3 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Mitomycin c, by Merck Group (3 mentions)
Accutase, by Thermo Fisher Scientific (3 mentions)
54 protocols using knockout serum
1
Mesenchymal Stem Cell Differentiation
2
Inducible Usp26-driven Differentiation of ESCs
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Human embryonic kidney (HEK) 293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) with 10% fetal bovine serum (Valley Biomedical) and 1% antibiotic–antimycotic solution (Gibco). ESCs were maintained in ES culture medium (DMEM supplemented with 10% Knockout serum, 2 mM L-glutamine, 100 μM non-essential amino acids (Gibco), 0.1 mM ß-mercaptoethanol, and 50 ng ml−1 LIF). For Usp26-induced differentiation, ES cells were transduced with lentiviruses expressing Dox-inducible GFP-tagged Usp26 or empty vector (Day −3). After ESCs formed colonies, 0.1 µg ml−1 Dox was added for pre-selection (Day −1). Day 0 was defined as the day when pre-selected GFP-positive colonies were cultured in iSF1 medium with high-dose Dox (2 µg ml−1), and individual colonies were tracked and taken pictures on days 1, 2, 3, and 4. For RA-induced differentiation, Usp26 KO, Cbx4 KO, Cbx6 KO, or WT ES cells were cultured in ES differentiation medium (DMEM supplemented with 10% Knockout serum, 2 mM L-glutamine, 100 μM non-essential amino acids (Gibco), and 0.1 mM ß-mercaptoethanol) with 1 μM RA. Individual colonies were tracked and photographed over 8 days.
3
Maintenance and Passaging of Human Embryonic Stem Cells
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Human ESCs (KhES-1) were used in accordance with ‘The Guidelines for Derivation and Utilization of Human Embryonic Stem Cells’ of the Ministry of Education, Culture, Sports, Science and Technology of Japan after approval by the Institutional Review Board. hESCs were maintained and cultured as previously described (Sakaguchi et al., 2015 (link)). In brief, hESCs were maintained on a feeder layer of mouse embryonic fibroblasts inactivated by mitomycin C treatment in DMEM/F12 (Wako) supplemented with 20% knockout serum replacement (KSR, Invitrogen), 2 mM glutamine, 0.1 mM nonessential amino acids (Invitrogen), 5 ng ml−1 recombinant human basic fibroblast growth factor (bFGF) (Wako) and 0.1 mM 2-mercaptoethanol under 2% CO2. For passaging, hESC colonies were detached and recovered en bloc from the feeder layer by treating them with 0.25% (weight/vol) trypsin and 1 mg ml−1 collagenase IV in PBS containing 20% (vol/vol) KSR and 1 mM CaCl2 at 37°C for 7-8 min. The detached hESC clumps were broken into smaller pieces (several dozens of cells) by gentle pipetting. The passages were performed at a 1:4 to 1:6 split ratio every 4-5 days.
4
Lentiviral Induction of iNeurons from iPSCs
5
Culturing Human Embryonic Stem Cells
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Experiments with these cells were carried out in accordance with the guidelines and regulations of the National Health and Medical Research Council and with the approval of the Austin Health Human Research Ethics Committee (Approval number H2008/03194) and University of Melbourne Human Research Ethics Committee (Approval number 0605017).
H9 (WA-09, WiCell, Madison, Wisconsin, USA), human embryonic stem cells (hESC), were cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) in hESC medium consisting of high-glucose Dulbecco's modified Eagle's minimal essential medium (DMEM) without sodium pyruvate, supplemented with insulin/transferrin/selenium 1%, β-mercaptoethanol 0·1 mM, nonessential amino acids (NEAAs) 1%, glutamine 2 mM, penicillin 25 U/ml, streptomycin 25 μg/ml (all from Invitrogen, Victoria, Australia) and fetal calf serum (FCS) 20% (HyClone, Australia) or on mitomycin-C treated human foreskin fibroblasts (HFF; ATCC, CRL-2097) in KSR media consisting of DMEM/nutrient mixture F-12, supplemented with β-mercaptoethanol 0·1 mM, NEAAs 1%, glutamine 2 mM, penicillin 25 U/ml, streptomycin 25 μg/ml, and knockout serum replacement 20% (all from Invitrogen). All cells were cultured at 37°C in 5% CO2. Colonies were mechanically dissected every seven-days and transferred to freshly prepared MEFs or HFFs. Media was changed every second day.
H9 (WA-09, WiCell, Madison, Wisconsin, USA), human embryonic stem cells (hESC), were cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) in hESC medium consisting of high-glucose Dulbecco's modified Eagle's minimal essential medium (DMEM) without sodium pyruvate, supplemented with insulin/transferrin/selenium 1%, β-mercaptoethanol 0·1 mM, nonessential amino acids (NEAAs) 1%, glutamine 2 mM, penicillin 25 U/ml, streptomycin 25 μg/ml (all from Invitrogen, Victoria, Australia) and fetal calf serum (FCS) 20% (HyClone, Australia) or on mitomycin-C treated human foreskin fibroblasts (HFF; ATCC, CRL-2097) in KSR media consisting of DMEM/nutrient mixture F-12, supplemented with β-mercaptoethanol 0·1 mM, NEAAs 1%, glutamine 2 mM, penicillin 25 U/ml, streptomycin 25 μg/ml, and knockout serum replacement 20% (all from Invitrogen). All cells were cultured at 37°C in 5% CO2. Colonies were mechanically dissected every seven-days and transferred to freshly prepared MEFs or HFFs. Media was changed every second day.
6
Directed Differentiation of iPSCs to Neural Cells
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Induced-pluripotent stem cells were grown using mTeSR™1 medium (Stemcell Tech., 05850) on matrigel (Corning, 354277). Human iPS cells were detached from matrigel using ReLeSRTM (Stemcell Tech., 05872), resuspended in medium consisting of Dulbecco’s modified Eagle’s medium/ F12 supplemented with 10% knockout serum (Invitrogen, 10828-028), 1% N2 supplement (Gibco, 17502-048), 0.05% B27 (Gibco, 17504-044), 20 ng/ml epidermal growth factor (Sigma, E9644) and 10 ng/ml basic fibroblast growth factor (Gibco, PHG0024), and plated within a low-attachment petri dish to induce embryoid body (EB) formation for 4 days. The EBs were plated on polyornithine (Sigma, P3655)/laminin (Sigma, L2020)-coated dishes for additional 6 days to induce the formation of neural rosettes. Neural rosettes were then manually removed, dissociated with accutase (Stemcell Tech., 07920), plated on poly-L-ornithine/laminin-coated dishes, and then treated with 3 µM retinoic acid for 7 days. The medium was changed daily and cultures were passaged weekly by accutase and plated on matrigel-coated dishes in the above-mentioned neural medium.
7
Feeder-dependent and Feeder-free hiPSC and hESC Culture
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Feeder-dependent human induced pluripotent stem cells (hiPSCs) (Systems Biosciences, cat. no. SC101A-1) were obtained from System Biosciences with pluripotent verification and contamination-free. Feeder-dependent hiPSCs were cultured with irradiated mouse embryonic fibroblast (MEF) feeder cells (MTI-GlobalStem, cat. no. 6001G) on gelatin coated (0.1% gelatin in PBS) 6-well culture plates using human embryonic stem cell (hESC) medium: DMEM/F12 (Invitrogen) containing 20 ng/mL bFGF (produced by IMBA institute Molecular Biology Service core facility), 3.5µL/500mL media of 2-mercaptoethanol, 20% KnockOut Serum (Invitrogen), 1% GlutaMAX (Invitrogen), 1% MEM-NEAA (Sigma), and 3% FBS). Feeder free H9 human embryonic stem cells (hESCs) were obtained from WiCell with verified normal karyotype and contamination-free. Feeder-free hESCs were cultured on hESC-qualified Matrigel (Corning cat. no. 354277) coated 6-well plates using mTeSR1 (Stemcell Technologies). All stem cells were maintained in a 5% CO2 incubator at 37°C. Standard procedures were used for culturing and splitting hPSCs as explained previously12 (link). All cell lines were routinely tested for contamination and confirmed mycoplasma-negative.
8
Generating Human-Induced Pluripotent Stem Cells
9
Pluripotency Evaluation of Stem Cells
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The pluripotency of siPSCs was examined by in vitro differentiation from EBs. To form EBs, siPSCs were trypsinised into single cells and cultured in suspension on low-adhesion plates (Corning) in mESC medium containing 15% knockout serum (Invitrogen), 4.5 g L−1 L-glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, 50 units ml−1 penicillin, 50 μg ml−1 streptomycin, and 100 U ml−1 LIF. Media were refreshed every 3 days, and EBs were allowed to grow for 8 days in suspension and then replated onto a 0.1% gelatin-coated tissue culture dish. Spontaneous differentiations of siPSCs into cells of mesodermal, endodermal, and ectodermal lineages were then detected by RT-PCR (primers are listed in Supplementary Table 2 ) and with appropriate markers by immunofluorescence. Directed differentiation of siPSCs to neurons, CMs, and endothelial cells followed previously published protocols6 (link)31 (link).
10
Induced Neuronal Differentiation from iPSCs
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Singularized iPSCs were plated on Matrigel (Corning) coated 6-well plates at a density of 2.50 × 105 cells per well, in 2 mL of mTeSR (STEMCELL Technologies) containing 10 µM Y-27632 (STEMCELL Technologies). The following day, they were changed into 2 mL of fresh mTeSR containing 10 µM Y-27632 and 1 µg/mL polybrene, as well as Ngn2 and rtTA lentiviruses which had been titered and an MOI of 1 was used for all transductions. The virus-containing media was replaced with fresh mTeSR at 24 h post infection. The cells were allowed to recover to approximately 70–80% confluency before being passaged into Matrigel-coated 6-well plates. At their next 70–80% confluency, cells were either frozen (in media containing 50% knockout serum (Invitrogen), 40% mTeSR, and 10% DMSO), or singularized for induction into iNeurons. All infected iPSCS were used within five passages of infection to maintain induction efficiency.
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