Glutamax supplement

2-Hydroxymelatonin (2(OH)Mel), 6-hydroxymelatonin (6(OH)Mel), bovine serum albumin (BSA), ethanol (EtOH), glucose, melatonin, and sodium pyruvate were purchased from Sigma (St. Louis, MO, USA). AFMK and GlutaMAX™ Supplements were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Other reagents were supplied as follows: EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany); RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA), high capacity cDNA Reverse Transcription Kit (Applied Biosystems), DyNamo Flash SYBR Green qPCR Kit (Thermo Scientific, Waltham, MA, USA), KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA), the Cell Mito Stress Test media was supplemented with 2 mM GlutaMAX™ Supplement (L-alanyl-L-glutamine dipeptide in NaCl), Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA), lactate colorimetric assay kit (Cell Biolabs, Inc. San Diego, CA, USA), TeloTAGGG Telomerase Elisa assay (Roche, Basel, Switzerland), and a set of human primers for real-time PCR (Eurofins Genomics, Ebersberg, Germany).

Osteogenesis and adipogenesis were assessed in monolayer by priming hMSCs with protein–surfactant complex or PBS, culturing the cells at 37 °C for 24 h and then inducing differentiation using minimum essential medium containing NaHCO₃ with 100 units ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 2 mM GlutaMAX supplement, 10% (v/v) FBS and 50 μl ml⁻¹ of freshly-added osteogenic or adipogenic supplement (R&D Systems, UK). The hMSCs were cultured under these conditions for 3 weeks, with media changes twice a week. The hMSCs were then fixed and stained with either Alazarin Red (osteogenesis) or Oil Red (adipogenesis). Chondrogenesis was assessed by priming hMSCs with protein–surfactant complex or PBS, culturing the cells at 37 °C for 24 h, and then engineering cartilage tissue. For full differentiation studies see Supplementary Methods.