Primed-hiPSCs of both patients were maintained on irradiated mouse embryonic fibroblasts (MEFs) in DMEM/F12 GlutaMAX supplemented with 20% KSR (thermofisher), 1X non-essential amino acids, 50 µM β-mercaptoethanol (thermofisher), and 10 ng/ml of bFGF (thermofisher). Media were replaced every day. Cells were passaged as clumps every 4 to 6 days using trypsin and 10 µM of ROCK inhibitor (TEBU-Bio). Primed-like hiPSCs were converted into a naive state by cultivating them on MEFs feeders cells in knock-out DMEM medium (thermofisher) supplemented with 20% KSR (thermofisher), 2 mM L-Glutamine (thermofisher), 0.1 mM MEM NEAA (thermofisher) and 50 µM β-mercaptoethanol (thermofisher) and containing basic fibroblast growth factor (b-FGF, thermofisher), human leukemia inhibitory factor (LIF, Peprotech), transforming growth factor-β1 (TGFβ, TEBU-Bio), and four small inhibitory molecules (4i), 3 μM CHIR99021 (R&D systems), 1 μM PD0325901 (R&D systems), 5 μM SB203580 (R&D systems), and 5 μM SP600125 (R&D systems)27 (link). Cells were maintained in 4i hiPSC medium for two weeks replacing daily with fresh medium. The growing naive-hiPSCs colonies were picked up and passaged every 2–3 days as single cells with accutase. For each passage, 10 μM of ROCK inhibitor (TEBU-Bio) were used.
Knockout dmem medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
KnockOut DMEM medium is a basal medium designed for the culture of embryonic stem (ES) cells and other pluripotent stem cells. It provides the nutrients and growth factors required to maintain the undifferentiated state of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (9 mentions)
Glutamax, by Thermo Fisher Scientific (8 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (5 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Ko serum replacement, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Vitronectin, by Thermo Fisher Scientific (2 mentions)
Pd0325901, by Merck Group (2 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (2 mentions)
E8 supplement, by Thermo Fisher Scientific (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Esc fcs, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rock inhibitor, by Tebu-Bio (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Pd0325901, by R&D Systems (1 mentions)
24 protocols using knockout dmem medium
1
Primed-hiPSCs to Naive-hiPSCs Conversion
2
Murine Pluripotent Stem Cell Maintenance
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Murine ESCs and iPSCs (OG2 ESCs [Szabó et al., 2002 (link)], CD45.1 iPSCs clones 10.3, 1.1, and 10.4, miPAP 1 and 2, and iPSC lin-7 and lin-9 [Pfaff et al., 2012 (link)]) were maintained in Knockout DMEM medium (Thermo Fisher Scientific) supplemented with 15% FCS, 1 mM penicillin-streptomycin, 1 mM L-glutamine, 0.05 mM β-mercaptoethanol, 1 mM non-essential amino acids, and 103 U/ml LIF on C3H MEF as previously described (Ackermann et al., 2014 (link)).
3
Generation and Culture of iPSC-derived RPE
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Human iPSCs were generated from WT BJ fibroblasts (ATCC CRL2522) as previously described [49 (link)]. Briefly, to begin the spontaneous differentiation process, confluent iPSCs were cultured in Knockout DMEM medium (ThermoFischer Scientific) supplemented with 20% KO serum replacement (ThermoFischer Scientific), 1% GlutaMAX (ThermoFischer Scientific), 1% non-essential amino acids (ThermoFischer Scientific), 0.1% β-mercaptoethanol (ThermoFischer Scientific) and 1% penicillin-streptomycin (ThermoFischer Scientific). Approximately six weeks later, pigmented foci were manually dissected, pooled, dissociated with 0.25% trypsin, filtered through a 40-μm filter and seeded at a density of ~3 × 104 cells per 0.32 cm2 on a 1/30 dilution Corning Matrigel HESC-qualified matrix (Dominique Dutscher). All analyses were performed on iPSC-derived RPE at P3.
4
Efficient Hepatocyte Differentiation from Human iPSCs
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Human iPSC lines, TkDA3-4 was kindly provided by University of Tokyo (Dr. Koji Eto and Dr. Hiromitsu Nakauchi), and 1231A3, 1383D2, 1383D6, Ff-I01, and Ff-I01s04 were provided by Kyoto University (Dr. Shinya Yamanaka, Dr. Keisuke Okita, and Dr. Masato Nakagawa). All iPSC lines were maintained on Laminin 511-E8 fragment (iMatrix-511™, kindly provided by Nippi,Inc.)-coated dishes in StemFit® AK02N (Ajinomoto Co., Inc.). A detailed procedure for differentiating hepatocytes has been described previously15 (link). Briefly, the cells were incubated in RPMI 1640 (Thermo Fisher Scientific) supplemented with 2% B27, 50 ng/ml Wnt-3a, and 100 ng/ml activin A for six days to derive the DE. KnockOut DMEM medium (Thermo Fisher Scientific) supplemented with 20% KnockOut serum replacement (Thermo Fisher Scientific), 1% DMSO, 0.1 mM 2-ME, 0.5% L-glutamine, and 1% NEAAs was used to derive the HE and IHs. HBM (Lonza Bioscience) supplemented with the Single QuotesTM kit without EGF (HCM without EGF), 5% fetal bovine serum (FBS), dexamethasone, and OSM was used to derive MHs.
The use of human iPSC was approved by the ethical committee at Yokohama City University and the University of Tokyo.
The use of human iPSC was approved by the ethical committee at Yokohama City University and the University of Tokyo.
5
Derivation and Maintenance of Murine and Human ESCs
6
Dissociation and Hydrogel Attachment of Beating Cardiomyocytes
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After differentiation,
beating cardiomyocyte areas were cut with
a scalpel under a microscope and collected. Then, the aggregates were
partially dissociated to loosen the cell-to-cell bonds inside the
aggregate and to better allow the attachment on the hydrogel. Dissociation
was modified from the study of Ahola et al. 2014.11 (link) The enzymatic dissociation buffers were applied to the
cells incubated at 37 °C: First buffer for 45 min, second buffer
for 15 min, and third buffer for 10 min, but no mechanical dissociation
was done. The gentle dissociation treatment loosens the cardiomyocyte
aggregate and makes it more susceptible to attach on to the hydrogel
surface. Four aggregates were plated per well with all coating and
hydrogel preparations (2D and 3D), as described above for fibroblasts.
Cells were cultured with KnockOut-DMEM medium (Thermo Fisher Scientific,
USA) supplemented with 20% FBS, 1% nonessential amino acids (Cambrex,
NJ, USA), 2 mM GlutaMAX (Thermo Fisher Scientific, USA), and 50 U
mL–1 Pen/Strep. The medium was changed every 3 days,
always 1 day before analysis, and the cells were cultured for 7 days
maximum.
beating cardiomyocyte areas were cut with
a scalpel under a microscope and collected. Then, the aggregates were
partially dissociated to loosen the cell-to-cell bonds inside the
aggregate and to better allow the attachment on the hydrogel. Dissociation
was modified from the study of Ahola et al. 2014.11 (link) The enzymatic dissociation buffers were applied to the
cells incubated at 37 °C: First buffer for 45 min, second buffer
for 15 min, and third buffer for 10 min, but no mechanical dissociation
was done. The gentle dissociation treatment loosens the cardiomyocyte
aggregate and makes it more susceptible to attach on to the hydrogel
surface. Four aggregates were plated per well with all coating and
hydrogel preparations (2D and 3D), as described above for fibroblasts.
Cells were cultured with KnockOut-DMEM medium (Thermo Fisher Scientific,
USA) supplemented with 20% FBS, 1% nonessential amino acids (Cambrex,
NJ, USA), 2 mM GlutaMAX (Thermo Fisher Scientific, USA), and 50 U
mL–1 Pen/Strep. The medium was changed every 3 days,
always 1 day before analysis, and the cells were cultured for 7 days
maximum.
7
Generating Ogt floxed mESC lines
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Ogtfloxed Cre–ERT2KI/KI mice were crossed with Ogtfloxed Rosa26-YFPKI/KI mice to obtain OgtfloxedCre–ERT2+/KI Rosa26-LSL-YFP+/KI blastocysts (embryonic day 3.5) to generate male and female Ogt floxed mESC lines. Ogt deletion was then induced by the addition of 1 μM 4-hydroxytamoxifen (4-OHT) (TOCRIS, catalog number: 3412). Mouse ESCs were maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs; feeder cells) with LIF in knockout DMEM medium (ThermoFisher Scientific, catalog number: 10829018) supplemented with 15% KOSR (KnockOut Serum Replacement, ThermoFisher Scientific, catalog number: 10828028), 2 mM L-glutamine, 1 X MEM nonessential amino acids, and 50 μM β-mercaptoethanol. Cell numbers were counted by flow cytometry on a BD Accuri C6 (BD Biosciences).
8
Generating Human iPSC-Derived RPE for Virus Studies
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Human iPSCs were generated from wild type BJ fibroblasts (ATCC CRL2522) as previously described [83 (link)]. iPSCs were cultured in Knockout DMEM medium (ThermoFischer Scientific) supplemented with 20% KO serum replacement (ThermoFischer Scientific), 1% GlutaMAX (ThermoFischer Scientific), 1% non-essential amino acids (ThermoFischer Scientific), 0.1% β-mercaptoethanol (ThermoFischer Scientific) and 1% penicillin-streptomycin (ThermoFischer Scientific). Six weeks later, pigmented foci were manually dissected, pooled, dissociated with 0.25% trypsin, filtered through a 40-μm filter and seeded at a density of ~3x104 cells per 0.32 cm2 on a 1/30 dilution Corning Matrigel HESC-qualified matrix (Dominique Dutscher). Experiments were performed on iPSC-derived RPE at P3. USUV was added at the MOI of 0,2 in 200 μL of medium for 2h on an orbital shaker. 300 μL of new medium were then added and the inoculum removed.
9
Targeted siRNA Knockdown of Myosin 1C Isoforms
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Using the online tool https://rnaidesigner.thermofisher.com/rnaiexpress/ , siRNAs for knockdown of isoform A and for knockdown of all three isoforms of myosin 1C were written. The miRNA sequences are shown in Table 1 . The synthesis and annealing of sequences was carried out at DNK-Sintez (Russia). Transfection of siRNA was performed using the Turbofect transfection agent (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions at a siRNA concentration of 200 pmol/μL in knock-out DMEM medium (Thermo Fisher Scientific, United States). Physiological effects in cells were studied after 92 h of exposure. Cells were removed using a 0.05% trypsin-EDTA solution and culture medium and centrifuged at 750g for 5 min. During any experiment, part of the cells were taken for further verification of expression suppression.
10
Profiling Transcriptional Dynamics in Cell Lines
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HCT116 (CCL-247), HeLa (CCL-2), HEK293T (CRL-3216), LN-18 (CRL-2610), U-2 OS (HTB-96) cell lines were obtained from ATCC and cultured according to recommended protocols. Human iPSC WTC-11 (Coriell Institute) cells were cultured on Vitronectin (Thermo Fisher Scientific) coated 6-well plates or glass coverslips (for smRNA FISH purposes) in Essential 8 Flex medium (Thermo Fisher Scientific) with E8 supplement (Thermo Fisher Scientific), Rock inhibitor and 2.5% penicillin-streptomycin. iPS cells we passaged with EDTA in dPBS. Mouse embryonic stem cells (Harvard Stem cell institute) were cultured on top of gelatin (0.1%, EMD Millipore) coated plates or glass coverslips (for smRNA FISH purposes). Embryonic stem cell media was prepared as follows: KnockOut DMEM medium (Thermo Fisher) supplemented with ESC FCS (Millipore Sigma), non-essential amino acids (Thermo Fisher), GlutaMAX supplement (Thermo Fisher), penicillin-streptomycin (Thermo Fisher), 50 mM 2-mercaptoethanol, LIF, CHIR99021 and PD0325901 (Sigma-Aldrich).
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media. Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
Actinomycin D (Sigma-Aldrich) was used at final concentration of 5 μg/mL in full growth media. Cell pellets and coverslips were harvested at 0, 40 min, 2.5 h and 4.5 h after adding Actinomycin D, and processed for RNA extraction and smRNA FISH as described below.
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