T6397

Phase 1 of this study applied 100 ng/mL PTH 1-34 (Sigma-Aldrich, P3671), T3 (Sigma-Aldrich, T6397), or T4 (Sigma-Aldrich, T2376) during week 1 (that is, daily for days 1 to 7) or week 3 (that is, daily for days 15 to 21) of a 4-week culture period. A concentration of 100 ng/mL was chosen to maximize the potential of a cellular response [11 (link),22 (link),23 (link)]. Phase 2 evaluated the use of 25 ng/mL PTH during week 2 (that is, daily for days 8 to 14), week 3 (that is, daily for days 15 to 21), or weeks 2 to 4 (that is, daily for days 8 to 28) sequentially after 0 or 100 ng/mL T3 application during week 1. A dosing study using PTH (0, 5, 10, 25, 50, and 100 ng/mL) revealed no differences in any measured parameters, with the exception of 25 ng/mL PTH being the highest concentration to result in the highest cellular content (Additional file 1). A concentration of 25 ng/mL PTH was thus selected for use in Phase 2. All controls received no hormone treatment.

Human immortalized PTECs (RPTEC/TERT1; ATCC CRL4031) were cultured using the supplier's specified protocol with some modifications. The cell media was modified to contain DMEM F-12 without glucose (pH 7.3 ± 0.05) (ATCC, 30-2006), NaHCO₃ (1.2 mg mL⁻¹) (Sigma-Aldrich, S5761), d-glucose (100 mg dL⁻¹) (Sigma-Aldrich, G7021), ITS (1× concentration, 13146-5ML; Sigma), triiodothyronine (5 pM) (Sigma-Aldrich, T6397), sodium selenite (3.65 ng mL⁻¹) (Sigma-Aldrich, S5261), PGE1 (25 ng mL⁻¹) (Cayman, 13010), hydrocortisone (25 ng mL⁻¹) (Sigma-Aldrich, H0888), ascorbic acid (3.5 μg mL⁻¹) (Sigma-Aldrich, A92902), and EGF (10 ng mL⁻¹) (R&D systems, 236-EG). The PTECs were cultured up to passage 20.
Glomerular microvascular endothelial cells (HGECs, human primary; Cell Systems) from decapsulated glomeruli isolated from normal human kidney cortical tissue, were cultured utilizing complete classic medium with 10% serum and CultureBoost (Cell systems, 4Z0-500) according to the supplier's specified protocol. The HGECs were cultured up to passage 8 and then plated onto laminin 521 coating (10 μg ml⁻¹).

The SGBS pre-adipocyte cell strain was obtained from the laboratory of Prof. Martin Wabitsch et al.^{76 (link)}. SGBS pre-adipocytes were grown to near confluence and then incubated (d0) in a serum-free differentiation medium [2 μmol/l rosiglitazone (Cayman #714740), 25 nmol/l dexamethasone (Sigma Aldrich #D-1756), 0.5 mmol/l methylisobuthylxantine (Sigma Aldrich #I-5879), 0.1 μmol/l cortisol (Sigma Aldrich #H-0888), 0.01 mg/ml transferrin (Sigma Aldrich #T-2252), 0.2 nmol/l triiodothyronine (Sigma Aldrich #T-6397), and 20 nmol/l human insulin (Sigma Aldrich #19278)]. After 4 days the medium was changed, and the cells were further cultured in medium supplemented with 0.1 μmol/l cortisol, 0.01 mg/ml transferrin, 0.2 nmol/l triiodothyronine, and 20 nmol/l human insulin. The SGBS pre-adipocytes were exposed to DEHP [50 µg/ml] (Sigma Aldrich) from d0-d4. DEHP had been solved in DMSO (Sigma Aldrich) in a 1000-fold stock solution. The maximum concentration of DMSO in the culture media was 0.1%. Controls were run as vehicle controls with 0.1% DMSO in the media. The dose used is comparable to DEHP doses found in neonates undergoing clinical procedures, such as transfusion or extracorporeal membrane oxygenation as well as in whole blood and blood components^{77 –79 (link)}. All experiments have been finalized at d8 of differentiation.

The human lung cancer cell lines PC-9, HCC827, H3255, H1650, NCI-H322M, and NCI-H226 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). PC-9, HCC827, H1650, NCI-H322M and NCI-H226 were cultured in RPMI 1640 medium (SH3080901b, Hyclone) supplemented with 10% FBS (10099141, Gibco), L-glutamine (25030-081, Invitrogen), and 100 U/ml penicillin and streptomycin (SV3001001, Hyclone). H3255 was cultured in ACL-4 medium (serum-free) supplemented with 0.02 mg/ml insulin (I-2797, Sigma-Aldrich), 0.01 mg/ml transferrin (T-5391, Sigma-Aldrich), 25 nM sodium selenite (S-9133, Sigma-Aldrich), 50 nM hydrocortisone (H-6909, Sigma-Aldrich), 1 ng/ml epidermal growth factor (E-9644, Sigma-Aldrich), 0.01 mM ethanolamine (E-0135, Sigma-Aldrich), 0.01 mM phosphorylethanolamine (P-0503, Sigma-Aldrich), 100 pM triiodothyronine (T-6397, Sigma-Aldrich), 0.5% (w/v) bovine serum albumin (A-9418, Sigma-Aldrich), 10 mM HEPES (15630-080, Gibco), 0.5 mM sodium pyruvate (11360-070, Gibco), and 2 mM L-glutamine (G-5763, Sigma-Aldrich).

After PBS rinse, cells are exposed to DMEM with B27 (100×), EGF (100 ng/ml, Peprotech) + NICO (Sigma, N0636, 10 mM) + Noggin (100 ng/ml) + Vc (0.25 mM). Cells are fed with fresh medium every other day.
V-bottom plate-based aggregation: stage-4 cells were rinsed with PBS and then incubated with Accutase (Millipore) for 10–15 min at 37 °C. Dissociated single cells were rinsed twice with DMEM/F12 and spun at 300 × g for 3 min. The resulting cells were re-suspended in aggregation medium (5a-Medium supplied with 10 μM Y27632). Cell solution with 0.1–0.4 (according to experimental design) million cells was added into each well of V-bottom 96-well plate, followed by a spun at 300 × g for 3 min. The plate was put into 37 °C incubator for 8–12 h to form clusters. 5a-Medium is made of V4b-Medium + heparin (Sigma, H3149, 10 µg/ml) + ZnSO₄ (10 µM, Sigma, Z0251) + LDN (Selleck Chemical, S2618, 100 nM) + T3 (1 µM, Sigma, T6397) + RA (0.05 µM) + SANT1 (0.25 µM; Tocris) + GABA (1 mM, Sigma) + human EGF (100 ng/ml, Peprotech) + NICO (10 mM) + Vc (0.25 mM). V4b-Medium (800 ml) is made of 340 ml MCDB 131 medium + 170 ml F12 medium + 170 ml KO-DMEM medium + 3.9 ml Glucose (45%, Sigma) + 80 ml 20% FF-BSA + 16 ml Sodium Bicarbonate (7.5%) + 4 ml ITX + 8 ml Pen/Strep + 8 ml Glutamax; all items are from Thermo Fisher Scientific unless indicated.

To minimize ‘edge-effects’ due to the increased rate of evaporation or warming of media, wells on the outer edges of the plate were filled with HBSS, while cells were grown in the wells of the middle part of the plate. The effect of NMDA (100 µM; solubilized in water, Tocris Bioscience, Cat. No. 0114), dimethylsulfoxid (DMSO, 0.01% v/v, Sigma-Aldrich, D8418), triiodothyronine (T3, 1 µM, solubilized in DMSO, Sigma-Aldrich, T6397), SSM5 (10 µg/mL) and 12D7 (10 µg/mL) in myelinating cultures was investigated between DIV 21–28, DIV 28–35 and DIV 35–42. In order to avoid potential effects related to the location of the wells on the microplates, substances were added to the wells following a pre-generated random pattern that varied from one experimental repeat to the next. Four technical replicates per condition were used for each of four independent culture preparations. Treatments were performed three times a week by replacing half the medium.

Murine brown preadipocytes were kindly provided by Associate Professor Patrick Seale (Harms et al., 2015 (link)). Preadipocytes immortalized with SV40 large T antigen were propagated in basal DMEM (Sigma-Aldrich, D6429) containing 10% FBS (Sigma-Aldrich, F7524) and 1% penicillin/streptomycin. Cells were passaged when they reached 70-80% of confluence. Media was changed every 2^nd day. Two days post 100% confluency, cells were induced to differentiate with DMEM supplemented 10% FBS, 1% penicillin/streptomycin, insulin (20 nM) (Sigma-Aldrich, I9278), dexamethasone (0.5 μM) (Sigma-Aldrich, D4902), T3 (1 nM) (Sigma-Aldrich, T6397), and 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) (Sigma-Aldrich, I5879). On day 2 of differentiation, dexamethasone and IBMX were omitted from the media. From day 4 of differentiation and onwards, cells were cultured in propagation medium. Cells were harvested or assayed on day 7 of differentiation. The cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.