Abstract
BackgroundMesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs.MethodshUCB-MSCs were maintained under hypoxic conditions (3% O2) with 1.8 mM Ca2+ during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs.ResultsThe proliferation of Ca2+/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca2+/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca2+/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs.ConclusionsThese findings suggest that a Ca2+/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.
Highlights
Mesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and have many clinical applications
Optimization of culture conditions for increasing the proliferation capacity of hUCB‐MSCs To determine whether the modification of culture conditions can stimulate cell proliferation, hUCB-MSCs were treated with C a2+, hypoxia, or combined C a2+/hypoxia
After isolation of mononuclear cells (MNCs) from the UCB, the cells were cultured under normal or Ca2+/hypoxia culture conditions (Fig. 2c). hUCB-MSCs isolated with Ca2+/hypoxia exhibited 2.7 × 105-fold greater total growth at passage 12 than hUCB-MSCs isolated under normal culture conditions
Summary
Mesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and have many clinical applications. We report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). We determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs. Human mesenchymal stem cells (hMSCs), which are capable of self-renewal and differentiation into various. It is difficult to obtain the large numbers of cells required for MSC clinical applications (i.e., up to 5 × 106 MSCs/kg body weight) with conventional culture methods [12]. Calcium (Ca2+) is a ubiquitous intracellular signal responsible for regulating numerous cellular processes such as cell differentiation, proliferation, apoptosis, and secretion. The effects of the combination of Ca2+ and hypoxia on the expansion of hMSCs have not been elucidated to date
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