Stem Cell Culture Requires Low Oxygen
Technical Note
Hypoxia-Enabled Stem Cell Culture
Stem cell research has revolutionized regenerative medicine and tissue engineering. A critical factor in advancing stem cell culture is the optimization of the microenvironment, particularly oxygen levels. Hypoxia, or reduced oxygen availability, has emerged as a pivotal condition for maintaining stem cell potency and promoting specific differentiation pathways. This technical note explores the importance of hypoxia in stem cell culture, the molecular pathways involved, and its implications for optimizing stem cell applications.
Importance of Hypoxia in Stem Cell Culture
In vivo, stem cells often reside in niches characterized by low oxygen levels (3-13%), such as the bone marrow or the embryonic inner cell mass. These hypoxic conditions play a crucial role in regulating stem cell self-renewal, proliferation, and differentiation. Mimicking this natural environment in vitro has been shown to optimize stem cell culture, while culture at atmospheric oxygen levels produces suboptimal and unwanted results.
Molecular Pathways Involved in Hypoxia versus Hyperoxia Responses
Cellular responses to extremes in oxygen levels are mediated by a complex network of molecular pathways, the most notable being the Hypoxia-Inducible Factor (HIF) signaling pathway and the Nuclear factor erythroid 2-Related Factor 2 (NRF2) pathway.
- HIF Stabilization and Activation:
- Under normoxic conditions, HIF-α subunits are hydroxylated by prolyl hydroxylase domain (PHD) enzymes, marking them for degradation via the ubiquitin-proteasome pathway.
- In hypoxic conditions, the lack of oxygen inhibits PHD activity, allowing HIF-1α to accumulate and translocate to the nucleus, where it dimerizes with HIF-1β.
- The HIF complex binds to hypoxia-responsive elements (HREs) in hundreds of target genes, upregulating pathways involved in glycolysis, angiogenesis, and cell survival.
- Metabolic Reprogramming:
- Hypoxia shifts cellular metabolism from oxidative phosphorylation to glycolysis, reducing reactive oxygen species (ROS) production and protecting cells from oxidative damage.
- This metabolic reprogramming supports the maintenance of pluripotency and enhances cell survival under low oxygen conditions.
- Epigenetic Regulation:
- Hypoxia influences the expression of chromatin-modifying enzymes, such as histone demethylases, which further regulate genes associated with stemness and differentiation.
- Wnt/β-Catenin Pathway Interaction:
- Hypoxia has been shown to enhance the activity of the Wnt/β-catenin pathway, a critical regulator of stem cell fate.
- NRF2 Activation:
- The transcription factor NRF2, encoded by the NFE2L2 gene, is a crucial master regulator for maintaining cellular redox homeostasis in response to oxidative stress, which is often a result of excess O2.
- Under normal conditions, NRF2 is bound by two molecules of KEAP1, an adapter protein for E3 ubiquitin ligases, resulting in consistent degradation by the 26S proteasome.
- Under oxidative stress conditions, ROS or electrophiles interact with or modify KEAP1, resulting in conformational changes in the KEAP1 protein that disrupt the NRF2-KEAP1 complex. Free NRF2 translocates to the nucleus, initiating the binding of the NRF2–sMAF complex to antioxidant response elements (AREs), resulting in the transactivation of target genes.
- NRF2 activation triggers the expression of hundreds of genes involved in various cellular processes, including antioxidant and xenobiotic responses, cell proliferation and survival, and metabolism, among others.
HIF-1 and NRF2 share a complex network of interactions both positively and negatively influencing the other’s expression and activity depending on the cellular context. Both are integral for stem cell function and survival.
Implications for Optimizing Stem Cell Culture
Leveraging hypoxia in stem cell culture requires careful control of oxygen levels and an understanding of its effects on cell behavior. Here are some strategies and considerations for optimizing culture conditions:
- Oxygen Tension Control:
- Specialized hypoxia chambers or incubators can maintain oxygen levels between 1-5%, mimicking physiological hypoxia. Embrient’s Modular Incubator Chamber (MIC-101) is ideal for low or custom oxygen cell culture. It is simple-to-use and easy to clean, while its sealed environment minimizes consumption of expensive gas mixes.
- Real-time oxygen monitoring ensures consistency and reproducibility.
- Supplementation with Hypoxia-Mimetic Agents:
- Compounds like cobalt chloride (CoCl2) or dimethyloxalylglycine (DMOG) can stabilize HIF-α, simulating hypoxic effects without altering atmospheric oxygen levels.
- Lineage-Specific Applications:
- Hypoxia can be tailored to promote specific differentiation pathways for targeted therapeutic applications. Examples are in the illustration below:
Challenges and Future Directions
Despite its benefits, hypoxia-enabled culture presents challenges, including the risk of variability in oxygen delivery and the need for standardization across labs. Future research should focus on:
- Developing more robust hypoxia-mimetic systems, capable of large-scale cultures.
- Investigating the interplay between hypoxia and other niche factors, such as extracellular matrix and mechanical cues.
- Exploring long-term effects of hypoxia on genomic stability and therapeutic potential.
- Overcoming the common practice of culturing cells at normoxic conditions.
Conclusion
Hypoxia plays a fundamental role in optimizing stem cell culture by mimicking the natural microenvironment, activating critical molecular pathways, and directing cell fate decisions. By refining our understanding and control of hypoxic conditions, researchers can improve the efficiency and reliability of stem cell-based therapies, paving the way for advances in regenerative medicine and beyond.
References
- Di Mattia M, Mauro A, Citeroni MR, Dufrusine B, Peserico A, Russo V, Berardinelli P, Dainese E, Cimini A, Barboni B. Insight into Hypoxia Stemness Control. Cells. 2021 Aug 22;10(8):2161. doi: 10.3390/cells10082161. PMID: 34440930; PMCID: PMC8394199.
- Dai X, Yan X, Wintergerst KA, Cai L, Keller BB, Tan Y. Nrf2: Redox and Metabolic Regulator of Stem Cell State and Function. Trends Mol Med. 2020 Feb;26(2):185-200. doi: 10.1016/j.molmed.2019.09.007. Epub 2019 Nov 1. PMID: 31679988.
- Bae T, Hallis SP, Kwak MK. Hypoxia, oxidative stress, and the interplay of HIFs and NRF2 signaling in cancer. Exp Mol Med. 2024 Mar;56(3):501-514. doi: 10.1038/s12276-024-01180-8. Epub 2024 Mar 1. PMID: 38424190; PMCID: PMC10985007.
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