Recombinant human insulin

Human recombinant insulin (Sigma-Aldrich, Poznan, Poland), essentially fatty acid free human serum albumin (HSA) (Sigma-Aldrich), 4-(1-pyrenyl)butyric acid (Merck, Warsaw, Poland), Human recombinant insulin (Sigma-Aldrich), Congo Red, Thioflavin T (Sigma-Aldrich, Poznan, Poland), and all necessary amino acid derivatives (Merck) were used as received. NaH₂PO₄∙2H₂O, Na₂HPO₄∙12H₂O, TX-100, EtOH, MeOH, and NaCl (Sigma-Aldrich) were used for the buffer preparation. Water was purified with the use of a Millipore Milli-Q Plus system.
Analytical HPLC: UltiMate 3000 UHPLC System Thermo Scientific™; column parameters: Kinetex 2.6 u C18 100A, 100 × 4.6 mm, 20 °C; diode array UV/Vis detector (DAD); eluent ACN/H₂O; gradient 0–2 min 3/97, 2–31 min 95/5, 31–32 min 0/100, 32–33 min 0/100, 33–35 min 3/97, 35–37.5 min 3/97.
Preparative HPLC: CombiFlash, EZPrep, Teledyne ISCO, Supelco Discovery BIO Wide Pore C18 column (25 cm × 21.2 mm, 10 mm; Sigma Aldrich); flow rate, 5 mL/min; detection wavelengths, 220 and 254 nm); gradient ratio A (0.1% TFA in ACN)/ B (0.1% TFA in H₂O) 0:100 to 18:82 in 30 min, followed by an isocratic run for 5 min.
ESI/MS: micrOTOF-Q III spectrometer Bruker Daltonics equipped with electrospray source (ESI) and time of flight detector (TOF).

Osteogenic differentiation was conducted. In brief, ADSCs at passage 2 were induced by 2.5 weeks of feeding (twice a week) with osteogenic induction medium consisting of 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM ascorbate (all from Sigma-Aldrich Chemical Company, St Louis, MO, USA), and 10% fetal calf serum (FCS) in DMEM/F12 basal medium. Osteogenic differentiation was subsequently confirmed by mineralized matrix deposition by 0.2% alizarin red-S staining.
Adipogenic differentiation was then performed. In short, the cells were induced by 3 cycles of induction/maintenance using adipogenic induction medium consisting of 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 10 μg/ml recombinant human insulin, 100 mM indomethacin (all from Sigma-Aldrich Chemical Company, St Louis, MO, USA), and 10% FCS, and using adipogenic maintenance medium comprising of only 10 μg/ml recombinant human insulin and 10% FCS. After that, the induced cells were subjected to incubation for another 7 days in adipogenic maintenance medium. Adipogenic differentiation was then confirmed by the formation of neutral lipid-vacuoles stainable with 0.18% oil Red-O for 5 min (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Primary normal human dermal fibroblasts served as negative control (NC). Each experiment was run in triplicate.

For osteogenic differentiation, the MenSCs were plated at 3 × 10³ cells/cm² and treated with the human MSC osteogenic differentiation medium (Cyagen, China). The osteogenic medium consisted of 100 nM dexamethasone (DEX), 0.2 mM ascorbate, 1 mM glutamine, 10 mM b-glycerophosphate, 1% penicillin–streptomycin, and 10% FBS and was completely changed every 3 days for up to 21 days. Cells were then fixed by 4% formaldehyde and stained with Alizarin red for 5 min.
For adipogenic differentiation, MenSCs were cultured in DMEM/F12 supplemented with 10% FBS, 1 μM DEX, 10 μg/ml recombinant human insulin, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX) and 1 μM rosiglitazone (Sigma-Aldrich) for 6 days. Then, culture medium was replaced with DMEM/F12 supplemented with 10% FBS and cells were cultured for the next 3 days. Then they were treated with DMEM supplemented with 10% FBS, 1 μM DEX, 10 μg/ml recombinant human insulin, and 60 μM indomethacin (Sigma-Aldrich) up to 12 days^{44 (link)}. For Oil Red O stain analysis, cells were fixed in 4% formaldehyde and then stained with Oil Red O for 30 min.