Stem cells are currently used in clinical applications to augment the healing of orthopedic tissue defects. Their applicability to multiple other therapeutic situations has also been investigated. One approach to eliciting the therapeutic benefits of stem cells is to inject them into the defect site in suspension or in a delivery gel.1 However, the therapeutic potential of stem cells may best be realized via tissue-engineering approaches to develop biological tissue substitutes which, through in vitro cultivation, can be functional at the time of implantation. The use of stem cell-based approaches in combination with scaffolds and bioreactor systems has resulted in engineered tissues of various mesenchymal lineages including bone,2, 3 cartilage,4 fat,5 and ligament,6 among others.
Stem cells produce all multicellular tissues in the body through proliferation and differentiation in tightly controlled in vivo environments. Because of their plasticity, they are particularly sensitive to their immediate environments. In vivo they are thought to reside in specific “niches,” which maintain their pluripotent or multipotent capabilities. Consequently, differentiation along specific lineages is thought to coincide with their migration out of their specific niche into an environment that provides appropriate differentiation cues.7 The in vitro conversion of unspecialized cells to immature, but functional tissues depends on establishing the cellular microenvironment, regulated so that key in vivo stimuli, which guide cellular organization and development, are recapitulated.8 As the field of tissue engineering has matured, new technology has been employed to regulate the application of mechanical as well as biological factors to developing tissue constructs. However, one developmentally important stimulus that is still rarely accounted for during in vitro culture is the oxygen tension. The role of oxygen as a metabolic substrate for cells in three-dimensional (3D) organization has now been investigated extensively9–12 and others have reviewed techniques utilized to mitigate mass transport limitations and avoidance of anoxic regions and steep oxygen gradients in thick 3D tissue constructs.13, 14 However, much less studied (but gaining increasing attention) has been of the utilization of oxygen in its role as a signaling molecule that influences stem cell survival, proliferation, and differentiation in culture.
The effect of oxygen tension on stem cell physiology has been studied for over 30 years beginning with the haematopoietic system.15–17 For haematopoietic stem cells (HSCs), it has been found that cultivation under low oxygen tensions maintained a significantly higher number of long-term colony initiating cells (LTC-ICs) relative to cultures under ambient (20%, v/v) oxygen concentrations.18–20 Recently, it has also been seen for several other stem and progenitor cell populations that cultivation under hypoxic conditions resulted in enhanced proliferation and maintenance of their naïve states.21 In vivo studies have shown that mesenchymal stem and progenitor cells home specifically to hypoxic events and function as therapeutic agents, enabling limited regeneration to damaged tissues.22–24 In particular, the stem cells have demonstrated the potential to organize themselves into vascular structures as well as secrete angiogenic growth-factors in response to hypoxic challenges. Several in vitro studies have also shown that both bone marrow- and adipose-derived stem cells upregulate VEGF expression during hypoxia,25–28 which along with other factors, may be responsible for the “cyto-protective” effects on neighboring cells.26–29 Although the identity of cellular oxygen sensor is being debated,30, 31 emerging evidence indicate that some of the effects of hypoxia on stem cell function are directly regulated by hypoxia-inducible factor (HIF) proteins. The role of HIFs in regulating stem cells' response to hypoxia has been recently reviewed by Keith and Simon32 and is not included in this review.
This review focuses on the role of oxygen tension on the stem cells' development into mesenchymal tissues in vitro. As a result, oxygen's influence as a signaling molecule (rather than metabolic substrate) on the proliferation, differentiation, and tissue development is discussed. Although there have been studies on numerous types of stem and progenitor cells, we discuss only embryonic stem cells (ESCs) and adult mesenchymal stem cells (MSCs) derived either from the bone marrow or adipose tissues in terms of their expansion and terminal differentiation into tissues of mesenchymal lineage. Cells from various mammalian species are included as they share many properties. The physiological relevance of low oxygen tension as an environmental parameter that uniquely benefits stem cells' expansion and maintenance is described. This provides a context for reviewing the results of in vitro studies of stem cells cultivated under hypoxic conditions. Finally, we discuss the impact of these findings on tissue engineering approaches and the need to specifically regulate the oxygen content of the cellular microenvironment in order to optimize in vitro tissue development is discussed in the last section. It is noted that hypoxia refers to the condition when oxygen tension is below physiological level but is used in this review to describe O2 lower than 21% for consistency with conventional terminology.