Ex vivo Angiogenic Cell Expansion System Increases The Number and Vasculogenic Potential of Endothelial Progenitor Cells by Switching the Culture Gravity Condition
Hiroko Hagiwara, Ph.D.1, Akira Higashibata, Ph.D.2, Shiho Ogawa, Ph.D.2, Shigeyuki Kanazawa, MD, Ph.D.1, Hiroshi Mizuno, MD, Ph.D.1, Rica Tanaka, MD, Ph.D.1.
1Juntendo University School of Medicine, Tokyo, Japan, 2Japan Aerospace Exploration Agency, Ibaraki, Japan.
Purpose: A serum-free, ex vivo cell expansion system called the mononuclear cell quality and quantity control culture (MNC-QQc) system can increase the number of CD34-positive cells, which are an indicator for endothelial progenitor cells (EPCs). MNC-QQc cells have angiogenic potential that is 30 times higher than that of MNCs. Although MNC-QQc is already an effective therapy, we investigated whether microgravity (MG) can increase the potential of this culture system. MG was reported to increase the stem cell culture functionality. This study aimed to evaluate the effect of MG on MNC-QQc to increase the number and improve the function of angiogenic cells, such as EPCs.Methods: MNCs were isolated from peripheral blood of healthy volunteers (n = 8). MNC-QQc was performed under four different conditions: (1) normal MNC-QQc (Normal Control; NC), (2) earth gravity (EG) for 7 days in Disposable cell container (DCC), (3) MG for 7 days in DCC, and (4) MG for 3 days followed by EG for 4 days in DCC (Microgravity and Earth Gravity; ME). After 7 days of MNC-QQc, the total number and percentage of CD34-positive cells, an indicator of EPCs, were measured by FACS analysis. The vascular regeneration ability of MNC-QQc cells was evaluated by identifying definitive EPC colony-forming units (dEPC-CFU) and primitive EPC CFU (pEPC-CFU) in colony forming assays. EPC number was measured by EPC-culture assay, and gene expression was quantified by real-time PCR.Results: While none of the culture conditions changed the total cell number, the CD34-positive cell number was significantly higher in the MG and ME groups than in the NC group [MG vs. NC (4.90 ± 1.21 vs. 1.12 ± 0.3, p < 0.05) and ME vs. NC (5.5 ± 1.64 vs. 1.21 ± 0.3, p < 0.05)]. EPC number was significantly increased in the ME group compared to the NC and EG groups (ME: 233.4 ± 18.4 vs. NC: 104 ± 27.7 vs. EG: 182.1±15.3, p < 0.05). dEPC-CFU were significantly increased in the ME group compared to the NC and EG groups (dEPC-CFU/ME: 967.1 ± 197.8, NC: 594.8 ± 186.3, EG: 386.1 ± 77.2, p < 0.05). Furthermore, VEGF-A expression increased in the ME group compared to the NC group [ME vs. NC (3.84 ± 0.34 vs. 2.33 ± 0.34, p < 0.05)].Conclusion: Stimulation of MNC-QQc cells with MG increased the number of EPCs, such as CD34-positive cells, by enhancing their proliferation capacity. Furthermore, EG culture after MG stimulation induced vasculogenic differentiation of CD34 cells.This study indicated that MNC-QQc in combination with MG-EG conditions might be a more effective angiogenic cell expansion culture method and could be a valuable tool for therapeutic vasculogenesis and tissue regeneration.
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