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Theranostics 2016; 6(11):1899-1917. doi:10.7150/thno.15412 This issue Cite

Research Paper

Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion

Yi Shuai^1,2*, Li Liao^1,2*, Xiaoxia Su³, Yang Yu^1,4, Bingyi Shao^1,4, Huan Jing^1,2, Xinjing Zhang^1,4, Zhihong Deng^5✉, Yan Jin^1,2✉

1. State Key Laboratory of Military Stomatology, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China
2. Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China
3. Department of Orthodontics, Stomatology Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi 710032, People's Republic of China
4. College of Stomatology, Chongqing Medical University, Chongqing, People's Republic of China
5. Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, People's Republic of China.
* These authors contributed equally to the study.

✉ Corresponding authors: Yan Jin, MD. PhD. Tel: +86-29-84776147, Fax: +86-29-83218039, E-mail: yanjinedu.cn; Zhihong Deng, MD. PhD. Telephone: +86-29-84776472, Fax: +86-29-83218039, E-mail: dengzhedu.cn.

Abstract

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSCs therapy. Here, we report a melatonin-based strategy to improve cell therapy of in vitro cultured MSCs. Among four small molecules with anti-aging and stem cell-protection properties (rapamycin, resveratrol, quercetin and melatonin), colony forming, proliferation, and osteogenic differentiation assay showed that melatonin was the most efficient to preserve self-renewal and differentiation properties of rat bone marrow MSCs (BMMSCs) after long-term passaging. Functional assays confirmed melatonin treatment did not affect the colony forming, proliferation and osteogenic differentiation of BMMSCs cultured for 1 or 4 passages, but largely prevented the decline of self-renew and differentiation capacity of BMMSCs cultured for 15 passages in vitro. Furthermore, heterotopic osteogenesis assay, critical size calvarial defects repair assay, osteoporosis treatment and experimental colitis therapy assay strongly certified that melatonin preserved the therapeutic effect of long-term passaged BMMSCs on bone regeneration and immunotherapy in vivo. Mechanistically, melatonin functioned by activating antioxidant defense system, inhibiting the pathway of cell senescence, and preserving the expression of gene governing the stemness. Taken together, our findings showed that melatonin treatment efficiently prevented the dysfunction and therapeutic failure of BMMSCs after long-term passaging, providing a practical strategy to improve the application of BMMSCs in tissue engineering and cytotherapy.

Keywords: cell culture, mesenchymal stem cells, melatonin, osteogenesis, small molecule.

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