Human LQTS iPSCs (iPSCORE_2_145 (link)) and human ESCs (H1 and H946 (link)) were maintained on Matrigel (Corning)-coated surfaces in mTeSR1 (STEMCELL Technologies) as previously decribed47 (link). Cardiac progenitor differentiation was performed as described previously22 . Briefly, hPSCs were singularized with Accutase (Thermo Fisher Scientific) and plated onto Matrigel-coated plates at a density of 3.0×105 cells/cm2 in mTeSR1 supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Selleckchem) on day −2. The differentiation was initiated with Wnt activation by 8 μM CHIR99021 (Selleckchem) on day 0 and sequential Wnt inhibition by 5μM IPW2 on day 3. Cardiac progenitors were ready on day 6 for CS generation.
Mtesr1
Manufactured by Selleck Chemicals
Sourced in United States
MTeSR1 is a feeder-free and serum-free medium for the maintenance and growth of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in an undifferentiated state. The medium is optimized to support the long-term culture of stem cells without the need for a feeder layer or serum.
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1, by STEMCELL (6 mentions)
Matrigel, by Corning (5 mentions)
Y 27632, by Selleck Chemicals (4 mentions)
Accutase, by Thermo Fisher Scientific (3 mentions)
Chir99021, by Selleck Chemicals (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Thiazovivin, by Selleck Chemicals (2 mentions)
Matrigel, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Selleck Chemicals (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Cytodex 1, by GE Healthcare (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
7 protocols using mtesr1
1
Cardiac Progenitor Differentiation from hPSCs
2
Culturing Healthy Human iPSCs
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The fully characterized hiPSC line is derived from dermal fibroblasts of a healthy male as previously published (Shinnawi et al., 2015 (link)). Informed consent was obtained after approval by the IRB committee of Amsterdam Medical Center. Colonies of hiPSCs were cultured in mTeSR-1 (StemCell Technologies) on plates coated with growth factor-reduced Matrigel (1:200 dilution, Corning). Cells were collected using 0.5 mM EDTA (Invitrogen), passaged every 4–6 days, and replated in mTeSR-1 supplemented with 2 μM thiazovivin (Selleck Chemicals). mTeSR-1 medium was replaced daily, except for the first day after passaging.
3
Culturing hiPSCs in mTeSR-1 medium
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hiPSCs were cultured in mTeSR-1 (STEMCELL Technologies, Vancuver, BC, Canada, 85850) on plates coated with 1:500 diluted growth factor-reduced Matrigel (Corning, Bedford, MA, USA, FAL356231). Cells were passaged every 4–6 days via dissociation with 0.5 mM EDTA (Invitrogen, Grand Island, NY, USA, 15575-038) and seeded in mTeSR-1 supplemented with 2 μM Thiazovivin (Selleck Chemicals, Burlington, ON, Canada, S1459). Between passages, the mTeSR-1 medium was replaced every day, except for the first day after passaging.
4
Maintenance of hPSCs in mTeSR1 medium
5
Microcarrier Coating for Stem Cell Culture
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Method for microcarrier coating have been reported earlier by our group [57 (link), 58 (link)]. Briefly, 20 mg/mL of Cytodex 1 (GE Healthcare, USA) was coated with (1:30 v/v) Geltrex® in DMEM/F12 medium (Thermo Fisher Scientific) overnight at 4 °C. Prior to use, pre-warmed (37 °C for 45 min) Cytodex 1 was centrifuged at 2000 rpm for 5 min. The supernatant was replaced with mTeSR™1 supplemented with 10 μM ρ-activated kinase inhibitor Y-27632 (Selleckchem, USA).
6
Cardiomyocyte Differentiation of hPSCs
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Human H9 and H13 embryonic stem cells51 (link), human IMR90-4 and 19-9-11 induced pluripotent stem cells52 (link), and NKX2.5EGFP/+ hPSCs36 (link) were maintained on Matrigel (Corning)-coated surfaces in mTeSR1 (STEMCELL Technologies) as previously described53 (link). CM differentiation was performed as described previously11 (link). A step-by-step protocol describing the differentiation method can be found at Protocol Exchange (DOI: 10.21203/rs.3.pex-1571/v1). Different cell seeding densities and different concentrations of CHIR99021 were applied to manipulate differentiation efficiency. Briefly, hPSCs were singularized with Accutase (Thermo Fisher Scientific) and plated onto Matrigel-coated plates at a density ranging from of 2.9 × 104 cells/cm2 to 5.7 × 105 cells/cm2 (1.0 × 105 cells to 2.0 × 106 cells per well of a 12-well plate) in mTeSR1 supplemented with 10 µM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Selleckchem) 2 days before initiating differentiation. Differentiation was initiated by Wnt signaling activation with 8–12 µM CHIR99021 (Selleckchem) on day 0, followed by inhibition of Wnt signaling with 5 µM IPW2 on day 3.
7
Directed Cardiac Differentiation of hPSCs
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Human H9 embryonic stem cells 42 , human IMR90-4 induced pluripotent stem cells 43 , and NKX2.5 EGFP/+ hPSCs 31 were maintained on Matrigel (Corning)-coated surfaces in mTeSR1 (STEMCELL Technologies) as previously described 44 . CM differentiation was performed as described previously 11 . Different cell seeding densities and different concentrations of CHIR99021 were applied to manipulate differentiation efficiency. Briefly, hPSCs were singularized with Accutase (Thermo Fisher Scientific) and plated onto Matrigel-coated plates at a density ranging from of 2.9 × 10 4 cells/cm 2 to 5.7 × 10 5 cells/cm 2 (1.0 × 10 5 cells to 2.0 × 10 6 cells per well of a 12-well plate) in mTeSR1 supplemented with 10 µM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Selleckchem) 2 days before initiating differentiation.
Differentiation was initiated by Wnt signaling activation with 8 µM to 12 µM CHIR99021 (Selleckchem) on day 0, followed by inhibition of Wnt signaling with 5µM IPW2 on day 3.
Differentiation was initiated by Wnt signaling activation with 8 µM to 12 µM CHIR99021 (Selleckchem) on day 0, followed by inhibition of Wnt signaling with 5µM IPW2 on day 3.
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