Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Materials
2.2. Synthetic Surface Preparation on TCP Dishes of PMEDSAH-g Dishes
2.3. Preparation and Use of 10% Human Serum (HS) Treated (HSt)-PMEDSAH-g Dishes
2.4. Preparation of Matrigel-Coated Dishes
2.5. Human Pluripotent Stem Cells (hPSC) Culture
2.6. Quantitative Analysis of the Total Cell Number
2.7. Immunofluorescence Staining
2.8. Flow Cytometry Analysis
2.9. RNA Isolation and Quantitative Real-Time PCR
2.10. Embryoid Body (EB) Formation and Analysis of hPSC Self-Renewal and Pluripotency
2.11. Cytogenetic Evaluation
2.12. Reprogramming of Human Somatic Cells into iPSC on 10% HSt-PMEDSAH-g Dishes and Quantification of Reprogramming Efficiency
2.13. Alkaline Phosphatase (AP) Staining
2.14. Data Analysis
3. Results
3.1. Optimization and Standardization of PMEDSAH Grafting (PMEDSAH-g) and hPSC Culture
3.2. Culture and Expansion of hPSC on 10% HSt-PMEDSAH-g Dishes
3.3. Characterization of hPSC Cultured on W10% HSt-PMEDSAH-g Dishes
3.4. Reprogramming of Human Somatic Cells on W10% HSt-PMEDSAH-g Dishes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Supplements Systematically Tested to Promote Cell Attachment | Concentration | Observations | |||
---|---|---|---|---|---|
Integrin Activation | Divalent Cations | Cytokines | Serum | ||
Commercial peptide (QHREDGS) [20,21] | 10 nM | No cell attachment | |||
Integrin activating antibody [22,23] | 2 µg/mL | Low and unstable cell attachment on W0 | |||
Altering the pH of media used [24,25] | 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0 | Low cell attachment, which was lost by W1 | |||
Ca2+, Mg2+, Mn2+ [26,27] | 3 mM | Cell attachment sustained until W3 | |||
Phorbol-myristate acetate (PMA) [29] | 100 ng/mL | No cell attachments | |||
Retinoic acid (RA) [30] | 1 mM | Low and unstable cell attachment on W0 | |||
Fetal Bovine serum (FBS) [31] | 10% | Cell attachments and differentiation | |||
Knockout™ Serum Replacement (KOSR) [7] | 10% | No cell attachments | |||
Human serum (HS) [3,33] | 1% | Low cell attachment on W0, lost all cells by W4 | |||
HS | 10% | Consistently stable, and long-term cell attachment | |||
HS | 100% | Highest cell attachment, but high degree of differentiation during each passage |
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Timilsina, S.; McCandliss, K.F.; Trivedi, E.; Villa-Diaz, L.G. Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions. Bioengineering 2023, 10, 999. https://doi.org/10.3390/bioengineering10090999
Timilsina S, McCandliss KF, Trivedi E, Villa-Diaz LG. Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions. Bioengineering. 2023; 10(9):999. https://doi.org/10.3390/bioengineering10090999
Chicago/Turabian StyleTimilsina, Suraj, Kaitlyn Faith McCandliss, Evan Trivedi, and Luis G. Villa-Diaz. 2023. "Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions" Bioengineering 10, no. 9: 999. https://doi.org/10.3390/bioengineering10090999