Optimization of culture conditions for rapid clinical‐scale expansion of human umbilical cord blood‐derived mesenchymal stem cells

Abstract

Background Mesenchymal 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 (Ca 2+ ) 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. Methods hUCB-MSCs were maintained under hypoxic conditions (3% O 2 ) with 1.8 mM Ca 2+ 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. Results The proliferation of Ca 2+ /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 Ca 2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca 2+ /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, Ca 2+ /hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs. Conclusions These findings suggest that a Ca 2+ /hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes. Electronic supplementary material The online version of this article (doi:10.1186/s40169-017-0168-z) contains supplementary material, which is available to authorized users.

Intracellular Calcium Determines the Adipogenic Differentiation Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells via the Wnt5a/β-Catenin Signaling Pathway

“…Treatment of extracellular calcium has also been shown to increase the extent of calcium influx through the calcium channel [ 37 ]. Therefore, to examine the causative role of Ca 2+ in hUCB-MSCs adipogenic differentiation, the cells were treated with calcium chloride (1.8 mM) to elevate the intracellular Ca 2+ level during 5 days of culture (i.e., one passage culture of MSCs-H or MSCs-L, Supplementary Figure 5 ) [ 38 ]. Furthermore, the cells were pretreated with 5 μ M BAPTA-AM, which is a cell-permeant intracellular Ca 2+ chelator, for 2 h. To analyze whether Ca 2+ or BAPTA-AM treatment altered the MSC characteristics of the cells, the expressions of MSC-specific antigens and multilineage differentiation factors were compared in the different treatment groups (see Supplementary Figure 6 ).…”

Mesenchymal stem cells: Cell therapy and regeneration potential

“…Therefore, it is imperative to develop methods that can maintain MSCs without losing their self-renewal and differentiation potential. A number of studies have reported large-scale production of MSCs for clinical and commercial usage by devising newer methods such as three-dimensional (3-D) culture, bioreactors, hypoxic conditions, and the addition of growth factors (Choi et al, 2017;Dayment et al, 2014;Gottipamula, Muttigi, Kolkundkar, & Seetharam, 2013;Sart & Agathos, 2016;Swamynathan et al, 2014). Using a microcarrier-based suspension system, MSCs were amplified to a 10 3 -fold increase, which was significantly higher as compared with the 200-fold increase in monolayer culture (Yuan, Kallos, Hunter, & Sen, 2014).…”

“…Maintenance and self-renewal (K. A. Jackson et al, 2001;Law & Chaudhuri, 2013) Large-scale production for clinical and commercial usage (Choi et al, 2017;Gottipamula et al, 2013;Sart & Agathos, 2016;Swamynathan et al, 2014) Standardization of criteria for characterization (Robey, 2017;Sacchetti et al, 2016) Differentiation of MSCs in vitro and in vivo (Alimperti, You, George, Agarwal, & Andreadis, 2014;K. A. Jackson et al, 2001) Homing and localization to the sight of injury (Fiumana et al, 2013;Young, Flynn, & Amsden, 2018) Cell survival and integration in vivo (J.…”

Protective effects of human umbilical cord blood‑derived mesenchymal stem cells against dexamethasone‑induced apoptotic cell death in hair follicles

“…Preparation of hUCB-MSCs. This study was approved by the Institutional Review Board of MEdIPOST co., Ltd. collection of hUcB and isolation and culture of hUcB-MScs were performed as previously described (22). Mononuclear cells were isolated from hUcBs by centrifugation on a Ficoll-Hypaque gradient (density, 1.077 g/cm 3 ; Sigma-Aldrich; Merck KGaA).…”