Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning
Abstract
:1. Introduction
2. Results
2.1. High-Throughput Assessment of Hypoxia Protective Treatments Using 2D hiPSC-CMs
2.2. ECT Fabrication and Formation with Select Culture Conditions
2.3. Active Mechanical Properties of ECTs in Normoxia
2.4. Mechanical and Structural Changes of ECTs in Hypoxia
2.5. Metabolic Functional Changes of ECTs Occurring during Hypoxia
3. Discussion
4. Materials and Methods
4.1. Human Induced Pluripotent Stem Cell (hiPSC) Maintenance
4.2. HiPSC-CM Differentiation, Freezing/Thawing, and Expansion
4.3. In Vitro Treatment for 2D hiPSC-CM Hypoxia Experiments
4.4. Damage Induction
4.5. MTT Assay
4.6. LIVE/DEAD Assay and ImageJ Analysis
4.7. Cardiac Fibroblast (hCF) Maintenance and Freezing/Thawing
4.8. Polydimethylsiloxane (PDMS) Mold Fabrication
4.9. ECT Fabrication
4.10. In Vitro Treatment of ECTs
4.11. ECT Survival and Compaction Analysis during Culture to Assess Syncytium Formation
4.12. Immunohistochemical Staining and Imaging of ECTs to Assess Changes in Structure
4.13. Mechanical Testing of ECTs to Analyze Functional Changes in Stress Generation and Kinetics
4.14. Serum Lactate Assay for ECTs to Assess Functional Changes in Metabolism during Hypoxia
4.15. Calculation of Fold Change
4.16. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Metabolic conditioning | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After an overnight recovery period, hiPSC-CMs were switched to five different cell culture media for one week prior to hypoxia (n = 5 wells per condition). The media compositions were as follows: (1) RPMI/B27 (control; high glucose, 11.1 mM), (2) high glucose/high FA (RPMI/B27 + 0.5% w/v Albumax (Thermo Fisher Scientific, Waltham, MA, USA) + 2 mM L-Carnitine (Sigma Aldrich, St. Louis, MO, USA)), (3) low glucose/high FA (RPMI/B27–glucose + 3 mM glucose (Sigma Aldrich) + 0.5% w/v Albumax + 2 mM L-Carnitine), (4) FA only (RPMI/B27–glucose + 0.5% w/v Albumax + 2 mM L-Carnitine), (5) Maturation media (MM) [13] (DMEM without glucose (Thermo Fisher Scientific) supplemented with 3 mM glucose (Sigma Aldrich), 10 mM L-lactate (Sigma Aldrich), 5 μg/mL Vitamin B12 (Sigma Aldrich), 0.82 μM Biotin (Sigma Aldrich), 5 mM Creatine monohydrate (Sigma Aldrich), 2 mM Taurine (Sigma Aldrich), 2 mM L-carnitine (Sigma Aldrich), 0.5 mM Ascorbic acid (Sigma Aldrich), 1× NEAA (Thermo Fisher Scientific), 0.5% (w/v) Albumax (Thermo Fisher Scientific), 1× B27 and 1% KOSR (Thermo Fisher Scientific)). |
Wnt signaling | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After 24 h, hiPSC-CMs were treated with 4 μM dimethyl sulfoxide (DMSO, control; Fisher Scientific, Waltham, MA, USA), 2 μM Chiron (Wnt activation), or 4 μM C59 (Wnt inhibition, Tocris) overnight (n = 8 wells per condition for LIVE/DEAD; n = 5 wells per condition for MTT). |
Mechanics (PDMS) | A thin layer (<0.5 mm) of polydimethylsiloxane (PDMS) (Sylgard 184 and Sylgard 527 in a 2:1 ratio, Dow, Midland, MI, USA) was glued into the bottom of a 12-well tissue culture plate using Dow sealant mixed with ethanol. The PDMS was treated with sterile-filtered polydopamine (PDA, MilliporeSigma) solution (0.01% (w/v) in 1 M Tris-HCl buffer (pH 8.5) overnight at 4 °C [92]. The PDA was then aspirated, the PDMS was washed with sterile-filtered mH2O twice, and finally coated with Geltrex (0.1 mg/mL) overnight at 4 °C. HiPSC-CMs were seeded onto PDMS substrate or standard Geltrex-coated tissue culture plastic (110,000 cells/well) and maintained for one week prior to hypoxia (n = 3 wells per condition). |
Alignment (PCL) | Nano-aligned PCL scaffolds (Nanofiber Solutions, Dublin, OH, USA) were glued into the bottom of a 12-well tissue culture plate using Dow sealant mixed with ethanol and then coated with Geltrex (0.1 mg/mL) overnight at 4 °C. HiPSC-CMs were seeded onto PCL substrate or standard Geltrex-coated (0.1 mg/mL) tissue culture plastic (110,000 cells/well) and maintained for one week prior to hypoxia (n = 3 wells per condition). |
Electrical pacing (EP) | HiPSC-CMs were seeded into a Geltrex-coated (0.1 mg/mL) 6-well cell culture plate (300,000 cells/well). After 24 h, hiPSC-CMs were subjected to electrical pacing or no electrical pacing (n = 3–4 wells per condition). Electrical paced hiPSC-CMs were stimulated with a 4 ms biphasic field pulse stimulus at 1 Hz and 4 V/cm using a 6-well electrode insert (C-Dish, IonOptix, Westwood, MA, USA) connected to the IonOptix culture pacing system (C-Pace EP, IonOptix, Westwood, MA, USA). |
Heat shock (HS) | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After 24 h, hiPSC-CMs were subjected to one hour of heat shock at 42 °C. Four groups (n = 5 wells per condition) were tested in hypoxia: (1) Control (37 °C, normoxia), (2) heat shock only (42 °C, normoxia), (3) hypoxia only (37 °C, hypoxia), and (4) heat shock + hypoxia (42 °C, hypoxia). |
Hypoxia preconditioning (HPC) | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After an overnight recovery period, hiPSC-CMs were subjected to 30 min of hypoxia (1% O2, 5% CO2) 24 h prior to damage induction. Four groups (n = 5 wells per condition) were tested in hypoxia: (1) Control (no pre-exposure, no hypoxia), (2) pre-exposure only, (3) hypoxia only, and (4) pre-exposure and hypoxia. |
Apoptosis inhibition (cyclosporine) | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After 24 h, hiPSC-CMs were treated with 0.1 μM Cyclosporine A overnight prior to hypoxia or left as untreated controls (n = 5 wells per condition) [20]. |
Mitochondrial stabilization (Mito-TEMPO) | HiPSC-CMs were seeded into Geltrex-coated (0.1 mg/mL) 96-well plates (10,000–12,000 cells/well). After 24 h, hiPSC-CMs were treated with 25 μM Mito-TEMPO (MilliporeSigma) overnight prior to hypoxia or left as untreated controls (n = 5 wells per condition) [19]. |
RPMI/B27 (high glucose media) | ECTs were cultured for seven days, with RPMI/B27 with media being replaced on alternate days. |
Maturation medium (MM) | ECTs were cultured for seven days, with MM (Table 1) [13] being replaced on alternate days. |
FA + high glucose media | ECTs were cultured for seven days, with FA + high glucose media (RPMI/B27 + 0.5% w/v Albumax + 2 mM L-Carnitine) being replaced on alternate days. |
RPMI/B27 + HPC | ECTs were cultured for seven days, with RPMI/B27 (Thermo Fisher Scientific) media being replaced on alternate days. On day 6, ECTS were exposed to 30 min of hypoxia (1% O2, 5% CO2) for 30 min approximately 24 h before hypoxia damage induction, and then returned to the incubator. |
MM + HPC | ECTs were cultured for seven days, with MM (Table 1) [13] being replaced on alternate days. On day 6, ECTS were exposed to 30 min of hypoxia (1% O2, 5% CO2) for 30 min approximately 24 h before hypoxia damage induction, and then returned to the incubator (normoxia). |
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Snyder, C.A.; Dwyer, K.D.; Coulombe, K.L.K. Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning. Int. J. Mol. Sci. 2024, 25, 9627. https://doi.org/10.3390/ijms25179627
Snyder CA, Dwyer KD, Coulombe KLK. Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning. International Journal of Molecular Sciences. 2024; 25(17):9627. https://doi.org/10.3390/ijms25179627
Chicago/Turabian StyleSnyder, Caroline A., Kiera D. Dwyer, and Kareen L. K. Coulombe. 2024. "Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning" International Journal of Molecular Sciences 25, no. 17: 9627. https://doi.org/10.3390/ijms25179627