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

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.

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“…Furthermore, PL has been shown to provide an increase to the growth rates of MSC populations grown outside of fetal bovine serum, while still ensuring the chromosomal stability of the cell line over multiple generation. Moreover, additional trophic factors which improve the proliferation and differentiation of BM-MSCs into neurons and neuron-like cells in vitro have been identified, including melatonin, ATP, neurotrophin-3 (NT-3), rolipram, β-mercaptoethanol (BME), CHIR99021 (CHIR), lithium chloride, and others (Tu et al, 2014; Abdullah et al, 2016; Joe and Cho, 2016; Narcisi et al, 2016; Shuai et al, 2016; Yan et al, 2016). Co-culturing BM-MSCs with active neurons or culturing BM-MSCs on media conditioned by mature neurons also appears to promote their differentiation towards neural cell types (Tu et al, 2014; Kil et al, 2016).…”