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Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low physiological oxygen (O 2 ) environment. Investigators have become increasingly aware of the effects of O 2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O 2 environment for cell culture. The aim of this review was to discuss an "hMSC checklist" as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation and growth, attachment and migration, genomic stability, and paracrine activity. Evidence drawn from the current literature demonstrates that low O 2 environments could improve most "hMSC checklist" attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O 2 studies. Therefore, "hypoxia" as a term and as a culture condition is discussed. The biology of short-term (acute) versus long-term (chronic) hypoxia is considered, and a nascent hypothesis to explain the behavior of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse and driving toward a regulatory recognizable "hMSC checklist," we may be better able to provide the patient population with safe and efficacious regenerative treatments.

The Role of Dissolved Oxygen Levels on Human Mesenchymal Stem Cell Culture Success, Regulatory Compliance, and Therapeutic Potential