Human iPSCs were maintained on matrigel-coated dishes in mTeSR™1 (STEMCELL Technologies, Temecula, CA, USA) or PluriSTEM™ (EMD Millipore, Vancouver, BC, Canada) medium. Human iPSC colonies were treated with alphazyme (1 ml/well in a six-well plate; PAA, Linz, Austria) for 5 to 10 minutes to obtain single cells. Then 100,000 to 200,000 cells were seeded in each well of a six-well plate having mTeSR™1 or PluriSTEM™ medium with 10 μM Rock inhibitor (Y27632; Sigma-Aldrich) to prevent the cell apoptosis. Twenty-four hours later, the medium was changed to mTeSR™1 or PluriSTEM™ containing TAT–Cre (catalogue number SCR508; EMD Millipore) with different concentrations of 0.5 μM, 1 μM and 2 μM. Cells were incubated with TAT-Cre recombinant protein for 5 hours. Cells were grown for 1 week and colonies were expanded either monoclonally or polyclonally, and then polymerase chain reaction (PCR) was performed to assess transgene deletion. Transgene-deleted clones were expanded and characterized further by immunostaining and differentiation.
Mtesr1
Manufactured by Merck Group
Sourced in United States
MTeSR1 is a chemically defined, feeder-free culture medium that supports the growth and maintenance of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). It provides the necessary components for the expansion and differentiation of these cell types while maintaining their pluripotency.
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Lab products found in correlation
Mtesr1, by STEMCELL (12 mentions)
Accutase, by Merck Group (6 mentions)
Blebbistatin b, by Merck Group (5 mentions)
Y 27632, by Merck Group (4 mentions)
Matrigel, by Corning (3 mentions)
Gfr matrigel, by BD (3 mentions)
Tat cre, by Merck Group (2 mentions)
Pluristem, by Merck Group (2 mentions)
Rock inhibitor y 27632, by Merck Group (2 mentions)
Matrigel, by Merck Group (2 mentions)
Matrigel mg gfr, by Corning (2 mentions)
Advanced rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Chir99021, by ReproCELL (1 mentions)
Advanced rpmi 1640, by R&D Systems (1 mentions)
Noggin, by R&D Systems (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Activin a, by R&D Systems (1 mentions)
Six well plate, by Corning (1 mentions)
Coated sixwell plates, by Corning (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
12 protocols using mtesr1
1
Efficient Transgene Deletion in Human iPSCs
2
Culturing IMR90.4 Stem Cells under Hypoxia
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The IMR90.4 cell line was obtained from WiCell (Madison, Wisconsin) and grown at UC San Diego with IRB approval. The cells were maintained in antibiotic free media on 1% (vol/vol) Matrigel-GFR (#354230; BD Biosciences) coated dishes at 37°C under hypoxic conditions (10% CO2/5% O2) in mTeSR1 (StemCell Technologies). These were passaged every 4 to 6 days with Accutase (#A6964; Sigma) for 8 to 10 min, dissociated to single, quenched in mTeSR1 plus 5 µM (-) blebbistatin (B; #B0560; Sigma), pelleted at 80 g for 5 min, and resuspended in mTeSR1 + B and plated at 5,000 cells per 35 mm dish as described earlier (15 (link), 16 (link), 21 (link)).
3
Maintenance of Pluripotent Stem Cells
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Stem cells were maintained antibiotic-free on 1% (vol/vol) Matrigel (MG)-GFR™ (#354230; Corning, New York, NY, USA) extracellular matrix (ECM) coated dishes at 37 °C under hypoxic conditions (10% CO2/5%O2) in mTeSR1 (Stem Cell Technologies, Vancouver, Canada) as previously described [19 (link),20 (link),21 (link),22 (link)]. Cells were passaged every 4–6 days, with Accutase (#A6964; Sigma-Aldrich, St. Louis, MO, USA) for 8–10 min, dissociated into single cells, quenched with mTeSR1 plus 5 μM blebbistatin (B; #B0560; Sigma-Aldrich, St. Louis, MO, USA), pelleted at 80× g for 5 min, resuspended in mTeSR1+B and plated at 2000 cells per single well of a 12-well plate [23 (link)]. After 48 h, cells were fed with mTeSR1 alone. To ensure cell quality, chromosomal integrity was evaluated by copy number variation (CNV) analysis using an Infinium HumanCore-24 v1.1 BeadChip (Illumina, San Diego, CA, USA).
4
Maintenance of Stem Cells in Hypoxia
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Stem cells were maintained antibiotic-free on 1% (vol/vol) Matrigel (MG)-GFR™ (#354230; Corning) extracellular matrix (ECM) coated dishes at 37 °C under hypoxic conditions (10% CO2/5%O2) in mTeSR1 (Stem Cell Technologies) as previously described65 (link),66 (link). Cells were passaged every 4–6 days, with Accutase (#A6964; Sigma) for 10–12 min, dissociated into single cells, quenched with mTeSR1 plus 5 μM blebbistatin (B; #B0560; Sigma), pelleted at 80 × g for 5 min, resuspended in mTeSR1+B and plated at 2000 cells per single well of a 12-well plate. After 48 h, cells were fed with mTeSR1 alone.
5
Culturing Antibiotic-Free Stem Cells in Hypoxic Conditions
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Stem cells were maintained antibiotic free on 1% (vol/vol) Matrigel-GFR (#354230; BD Biosciences) coated dishes at 37°C under hypoxic conditions (10% CO2/5%O2) in mTeSR1 (Stem Cell Technologies) (Ludwig et al., 2006 (link); Yao et al., 2006 (link); Chen et al., 2011 (link); Wahlin et al., 2017 (link)). Cells were passaged every 4–6 days, with Accutase (#A6964; Sigma) for 8–10 min, dissociated to single cells, quenched with mTeSR1 plus 5 μM (-) blebbistatin (B; #B0560; Sigma), pelleted at 80 × g for 5 min, resuspended in mTeSR1 + B and plated at 5,000 cells per 35 mm dish (Walker et al., 2010 (link)). After 48 h, cells were fed without B.
6
Optimal Stem Cell Aggregation for Differentiation
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Stem cells were routinely maintained in either mTeSR1 or Essential 8 (E8). However, for all cell aggregation experiments, cells were maintained on 1% (vol/vol) Matrigel-GFR™ (BD Biosciences) coated dishes at 37 °C under hypoxic conditions (10% CO2/5%O2) in mTeSR1 (Stem Cell Technologies) prior to reaggregation28 (link), 51 (link), 71 (link). Cells were passaged with Accutase (Sigma) for 8–10 minutes, dissociated to single cells, quenched with mTeSR1 plus 5 μM blebbistatin (B; Sigma), pelleted at 80 × g for 5 minutes, resuspended in mTeSR1 + B and plated at 5,000 cells per 35 mm well32 (link). After 48 hours, cells were fed without B. To minimize cell stress, no antibiotics were used52 (link).
7
Directed Differentiation of Human iPSCs to Kidney Progenitors
8
Maintenance of human iPSC and H9 hESC lines
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Human iPSC line WTC11 (a kind gift from Bruce Conklin, Gladstone Institute of Cardiovascular Disease) was maintained in mTeSR1 (Stemcell Technologies) on Matrigel (Corning)-coated six-well plates (Corning) in a 37 °C incubator with 5% CO2. At 70–80% confluence, cells were dissociated with Accutase (Stemcell Technologies) and plated on Matrigel-coated six-well plates with mTeSR™1 containing 10 μM Rho kinase inhibitor, Y27632 (EMD Millipore). Y27632 was removed after the first 48 h and thereafter media was replaced with fresh mTeSR1 every day. Human H9 (purchased from Wicell) or H9-FP ubiquitously expressing miRFP703 fused to histone H2B (a kind gift from Dr. Andrew McMahon, University of Southern California) were maintained in StemFit (amsbio) with 100 ng/ml human FGF2 (R&D) on Geltrex (Thermo Fisher Scientific)-coated six-well plates. At 70–80% confluence, cells were dissociated with Accutase and plated on Geltrex-coated six-well plates with StemFit containing 100 ng/ml human FGF2 and 10 μM Y27632. After 48 h of culture, medium was replaced with fresh StemFit containing 50 ng/ml human FGF2. After the next 48 h, medium was replaced with fresh StemFit containing 25 ng/ml human FGF2.
9
Cardiac Differentiation of Human Embryonic Stem Cells
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Human embryonic stem cell (hESC)-derived cardiac differentiation was induced as previously described [9 (link)]. For cardiac lineage induction, H9 hESCs (WiCell, Madison, WI, USA) were plated onto Matrigel (Corning, New York, NY, USA)-coated 6 well plate at a density of 0.5 × 106 cells/well and cultured in mTeSR1 (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with cGMP for 2–3 days. Three days before cardiomyocyte differentiation, hESCs were plated on Matrigel-coated 6 well plate at a density of 0.6 × 106 cells/well and cultured in mTeSR1 supplemented with 5 μM Y27632 (Sigma-Aldrich, St. Louis, MO, USA). The culture medium was replaced with RPMI+B27 medium (RPMI1640 with B27 supplement without insulin; Gibco, Grand Island, NY, USA) supplemented with CHIR99021 (10 μM, S1263; Selleckchem, Houston, TX, USA) for 3 days. The culture medium was subsequently replaced with RPMI+B27 supplemented with IWP2 (5 μM; Tocris, Bristol, UK), followed by culture for 5 days. On day 8, the culture medium was replaced with RPMI+B27. The medium was changed every 1–2 days. Beating cardiomyocytes were observed on days 9–10. Cardiomyocytes were chemically sorted by lactate (Wako, Richmond, VA, USA).
10
Live Cell Staining of Embryoid Bodies
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For live cell staining, EBs were incubated with mTeSR1 plus calcein FM (Sigma-Aldrich) at 1 µM. After an incubation period for 30 min, EBs were washed twice with PBS and analyzed on an inverted fluorescence microscope.
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