All iPSCs were cultured as described previously (Roberts et al., 2017 (link)). Untagged iPSCs (Coriell Institute for Medical Research; GM25256) and mEGFP-tagged LMNB1 iPSCs (Allen Institute for Cell Science, Coriell; AICS-0013 cl.210) were grown in mTeSR and mTeSR Plus in cell culture dishes coated with Matrigel matrix (Growth Factor Reduced; Corning; 354230, or phenol-red-free; Corning; 356237). Matrigel was applied at a concentration of 137–154 µg/ml dissolved in PBS or DMEM. Cells were dissociated using Gentle Cell Dissociation Reagent (Stem Cell Technologies; 100-0485) or Versene Solution (Thermofisher Scientific; 15040066) and ROCK Inhibitor Y27632 at a final concentration of 10 µM (ATCC; ACS-3030). iPSCs were differentiated using STEMdiff Trilineage Differentiation Kit (Stem Cell Technologies; 05230) or STEMdiff Mesoderm Induction Medium (Stem Cell Technologies; 05220). All cells were maintained at 37°C at 5% CO2. Pluripotency and differentiation were confirmed using Oct4, Nanog, HNF-3β, Otx-2, and Brachyury antibodies in immunofluorescence assays.
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iPSC Culture and Differentiation Protocol
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Matrigel matrix (Growth Factor Reduced;
Gentle Cell Dissociation Reagent
Versene Solution
STEMdiff Trilineage Differentiation Kit
STEMdiff Mesoderm Induction Medium
Antibodies
Assays immunofluorescence
Brachyury
Cell culture
Cells
Dishes
Growth factor
Ipscs
Lmnb1
Matrigel
Mesoderm
Ml 137
Oct4
Stem cell
Versene
Y27632
Corresponding Organization : University of Wyoming
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Variable analysis
independent variables
- Culture conditions of iPSCs (mTeSR and mTeSR Plus)
- Coating of cell culture dishes with Matrigel matrix
- Dissociation of cells using Gentle Cell Dissociation Reagent or Versene Solution
- Addition of ROCK Inhibitor Y27632
- Differentiation protocols (STEMdiff Trilineage Differentiation Kit or STEMdiff Mesoderm Induction Medium)
- Pluripotency and differentiation status of iPSCs, confirmed using Oct4, Nanog, HNF-3β, Otx-2, and Brachyury antibodies in immunofluorescence assays
- Cell line: Untagged iPSCs (Coriell Institute for Medical Research; GM25256) and mEGFP-tagged LMNB1 iPSCs (Allen Institute for Cell Science, Coriell; AICS-0013 cl.210)
- Incubation conditions: 37°C, 5% CO2
- Positive control: Not specified
- Negative control: Not specified
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