Nicotinamide

Metformin hydrochloride, nicotinamide, and streptozotocin were purchased from Alfa Aesar. All other chemicals and reagents used were of analytical grade.

The cells were harvested and washed with ice-cold PBS (phosphate buffered saline, Caisson Labs), followed by lysis with Bullet Blender® (Next Advance, Inc.) in PLC lysis buffer [13 (link), 35 (link)]: 30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na₃VO₄ supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF, 10 μM TSA (Trichostatin A, Selleckchem), and 5 mM nicotinamide (Alfa Aesar). Total protein concentrations of the cell lysates were determined using the DC protein assay (Bio-Rad). Western blot and image analysis were conducted as described previously [13 (link)]. Antibody catalog numbers and vendors are as follows: C1 (A31857) and C3 (A21362) from Invitrogen; BNIP3 (ab10433) from Abcam; β-actin (MA5-15739), GAPDH (MA5-15738), and LC3 (PA1-16931) from Pierce; Mfn1 (sc-50330), Mfn2 (sc-50331), and Tfam (sc-28200) from Santa Cruz; PGC1α (Ab3242) from Millipore; VDAC (4661s) and Drp1 (8570) from Cell Signaling Technology; Beclin-1 3738s and Beclin-1 MABN16 from Cell Signaling Technology and Millipore, respectively; NRF1 (LS-B43) from LifeSpan BioSciences.

RPE cells were generated from iPSCs following the previously reported method [26 (link)]. Briefly, iPSCs were grown to 80% confluence in mTeSR^TMPlus medium and switched to RPE differentiation medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), 20% knockout serum replacement (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 1× non-essential amino acids (NEAA; Sigma, San Luis, MO, USA), 50 μM β-mercaptoethanol (Gibco), 1% penicillin–streptomycin (Gibco) and 10 mM nicotinamide (MedChemExpress, Princeton, NJ, USA) for 0–7 days. Then, nicotinamide was replaced with 100 ng/mL Activin A (Peprotech, Rocky Hill, NJ, USA) for another week. On day 14 of differentiation, Activin A was removed from the medium, and 3 μM CHIR99021 (MedChemExpress) was added. On day 42, RPE cells were collected by incubating in TrypLE Express (Gibco) for 30 min at 37 °C and then seeded on a Matrigel-coated 6-well plate with a density of approximately 1 × 10⁶ cells/well. Meanwhile, RPE differentiation medium was replaced with RPE maintenance medium consisting of DMEM, 4% KSR, 1× NEAA, 1% penicillin–streptomycin and 50 μM β-mercaptoethanol without molecule compound. At this point, the cells were defined as passage 1 (P1). Only RPE cells at P2 and P3 were used in this study.

The protocol for differentiation of pluripotent hiPSCs into RPE was adapted from Buchholz et al., 2013 [14 (link)]. For differentiation, iPSCs were plated at 70–80% confluence on 9.6cm² wells coated with 1X Matrigel (Corning) or Synthemax (3535XX1 Corning, Corning NY) or vitronectin (PeproTech) in basal neural induction medium (DMEM/F12+ 1X N2+ 1X B27 + 1X Non-essential amino acids, Thermo Fisher Scientific) supplemented for 2 days with Nicotinamide (10mM) (Sigma-Aldrich, St. Louis, MO), Noggin (50ng/ml) (PeproTech), Dkk-1 (10ng/ml) (R&D Systems, Minneapolis, MN.) and IGF-1 (10ng/ml) (R&D Systems) with a medium change on day 1. On days 3 and day 4, the Noggin concentration was reduced to 10ng/ml and bFGF was added at 5ng/ml (R&D Systems). The Nicotinamide, bFGF and Noggin were removed from the media on day 5 and on days 5 and 6 the basal medium was supplemented with only Dkk-1 (10ng/ml), IGF1 (10ng/ml) and Activin A (100ng/ml) (PeproTech). The DKK and IGF 1 were then removed and from day 7 to day 14 the basal medium was supplemented with Activin A (100ng/ml), and SU5402 (10μm) (Sigma-Aldrich). CHIR 99021(3μM) (TOCRIS, Bristol, UK.) was also added from day 8 to day14. (See Fig 1). All media supplements were removed for the expansion of iPSC-derived RPE.

ADHLSCs at passage 5 or 6 were seeded at a density of 1 × 10⁴ cells/cm² onto collagen I-coated 175 cm² flasks in complete DMEM medium. Twenty-four hours later, the culture medium was switched to IMDM (Thermo Fisher Scientific). Cells were then subjected to a four-step differentiation protocol as previously described [39 (link)]. First, cells were incubated for 2 days with IMDM containing 20 ng/ml epidermal growth factor (EGF) (PeproTech) and 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech). Then, the cells were incubated for 10 days with IMDM containing 20 ng/ml hepatocyte growth factor (HGF) (PeproTech), 10 ng/ml bFGF, nicotinamide 0.61 g/l (Sigma Aldrich), and 1% insulin-transferrin-selenium (ITS) (Invitrogen) premix. Next, the cells were incubated for 10 days with IMDM containing 20 ng/ml HGF, 20 ng/ml oncostatin M (OSM) (PeproTech), 0.61 g/l nicotinamide, and 1% ITS premix. Finally, the cells were treated with IMDM containing 20 ng/ml OSM, 1 μM dexamethasone (Sigma Aldrich), and 1% ITS premix for 10 days. For each step, the medium was changed every three days. Negative controls were performed using IMDM supplemented with 1% FCS and 1% of Penicillin/Streptomycin. Cells were harvested either after each step or at the end of the differentiation protocol and used for MLR, RT-PCR, fluorescence microscopy, or flow cytometry analysis.

To differentiate pancreatic ductal cells into beta-like pancreatic islet cells, the method from previously reported was used in our study (11 (link)), 5 × 10⁵ cells were seeded into 6-well cell culture plates and cultured in DMEM/F12 containing 10 nM exendin-4, 100 pM recombinant human hepatocyte growth factor (HGF), 2 nM activin A, and 10 mM nicotinamide (Peprotech, USA) thus culture medium was changed every 2 days for 14 days. Insulin and glucagon levels were detected using immunofluorescence. Randomly selected 10 non-overlapping visual fields were observed and photographed to calculated positive ratios.