Taurine

To analyse neutrophils, monocytes, macrophages, and dendritic cells, a single cell suspension was prepared from hearts before or at various time points after MI (days 1, 3, 5, 7, and 28 after operation). The hearts were mechanically dissociated using surgical scissors and subsequently treated with a 1x Hank's Balanced Salt Solution (HBSS) (Invitrogen, USA), enzymatic dissociation buffer containing 0.1 mg/mL Liberase TH Research Grade (Roche Diagnostics, UK), 50 μg/mL of DNaseI (Roche Diagnostics, UK), 10 mM HEPES (Invitrogen, USA), and 30 mM Taurine (Sigma, UK) for 4 cycles of 10 min at 37°C. After each 10 min incubation cycle, the cells were collected and filtered using a 70 μm cell strainer (BD Pharmingen, USA) and an equal volume of ice cold 1x HBSS containing 10 mM HEPES, 30 mM Taurine (Sigma, UK), and 20% Fetal Bovine Serum (FBS) (GE Healthcare, USA) was added to the enzyme dissociation buffer. The cells were pelleted at 320 g for 7 min at 4°C and washed with the 1x HBSS media solution containing 20% FBS as described above. Under these isolation conditions, adult cardiomyocytes are predominately lysed, as the enzymatic dissociation buffer is toxic to these large, fragile cells [22 (link)]. An aliquot of the cell suspension was used to quantify the cell concentration/mL using a Beckman Coulter Vi-Cell XR cell counter (Beckman Coulter, High Wycombe, UK).

To evaluate the effects of taurine on synapse formation, presynaptic visceral dorsal 4 (VD4) and postsynaptic left pedal dorsal 1 (LPeD1) cells in L. stagnalis were paired and cultured in the absence or presence of 1 mM taurine (Sigma-Aldrich) in DM (no trophic factors) or CM (with trophic factors) overnight. Intracellular recordings were made the next day to monitor the development of synapses by recording postsynaptic potentials (PSPs) in the LPeD1 cell following the presynaptic stimulus. Current injection-induced action potentials in the presynaptic VD4 neuron triggered 1:1 PSPs in the postsynaptic LPeD1 neuron, indicating the formation of functional synapses. The current injection was made using a built-in current injector (Dual Channel Intracellular Recording Amplifier IR-283; Cygnus Technology, Delaware Water Gap, PA, USA). Averages of four successive PSPs measured from treated and untreated control groups were compared to determine the effects of taurine on the incidence and strength of synapse formation. The percentage of synapse formation was determined as the number of pairs that exhibited quantifiable transmission of stimuli between cells out of the total number of pairs that were treated.

Male C57BL/6 mice (8–10 weeks, 25–30 g) from Shanghai SLAC Laboratory Animal Co. Ltd were housed in a specific pathogen-free environment (22.5 ± 5.0°C, 43 ± 5% humidity, and 12/12-h light/dark).
Thirty milligrams per kilogram MPTP (Cat# M0896; Sigma-Aldrich, St Louis, MO, USA), prepared using sterile saline, was injected intraperitoneally once daily for 5 consecutive days to establish the PD model, whereas controls were injected with sterile saline solution (0.9% NaCl, wt/vol). Mice were sacrificed 2 days after the last injection.
For the taurine supplementation experiment, 150-mg/kg taurine (Cat# T0625, Sigma-Aldrich), prepared using sterile saline, was injected intraperitoneally every day for 4 weeks (55 (link)). Two weeks after the taurine administration, MPTPs were given to construct the PD mice model.

Animals were housed with a 12-hour dark/12-hour light cycle, or kept in complete darkness, depending on the experimental design, with food and water available ad libitum. Animals were fed the RM1 diet, which has a low protein content (12.91% digestible protein; Special Diet Service (SDS) – 801010; http://www.sdsdiets.com/pdfs/RM1-A-P.pdf), except during the dietary intake experiment, in which animals were fed the RM3 diet containing 19.94% digestible protein (Special Diet Service (SDS) – 801030 http://www.sdsdiets.com/pdfs/RM3-A-P.pdf). The Ush1g^−/− and Ush1c^−/− albino mice used in the study were backcrossed with BALB/cJ mice seven and six times, respectively. During taurine supplementation experiments, taurine (Sigma-Aldrich) was dissolved in the drinking water of the treated group, at a concentration of 0.1 M, and administered for four months. Six or more animals were randomly assigned in the different group to be able to perform statistical analysis. All experiments were carried out in accordance with European Community Council Directives (86/609/EEC) and the ARVO (Association for Research in Vision and Ophthalmology) statement for the use of animals in ophthalmic and visual research. The present study was ethically approved by the French « Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche » under the number 02892.02.

IPC differentiation was performed by using 10 days 3-step differentiation protocol as performed by Sawangmake et al. (2014) (13). Briefly, 1 × 10⁶ of hDPSCs were seeded in 60 mm non-treated culture dish (Eppendorf, Hamburg, Germany) as a single cell suspension. The cells were maintained in serum-free medium (SFM)-A for 3 days, SFM-B for 2 days and SFM-C for 5 days and the medium were changed every 48 h. The medium used in this experiment were SFM-DMEM (Thermo Fisher Scientific Corporation, USA) with different supplementation. The supplementation of each medium were respectively as follows; SFM-A: 1% BSA (Cohn fraction V, fatty acid free; Sigma, Missouri, USA), 1X insulin-transferrin-selenium (ITS) (Invitrogen, USA), 4 nM activin A (Sigma, Sigma, Missouri, USA), 1 nM sodium butyrate (Sigma, Missouri, USA), and 50 μM beta-mercaptoethanol (Sigma, Missouri, USA); SFM-B: 1% BSA, 1X ITS, and 0.3 mM taurine (Sigma, Missouri, USA); and SFM C: 1.5% BSA, 1X ITS, 3 mM taurine, 100 nM glucagon-like peptide (GLP)-1 (Sigma, Missouri, USA), 1 mM nicotinamide (Sigma, Missouri, USA), and 1x non-essential amino acids (NEAAs) (Sigma, Missouri, USA).

To produce the definitive endoderm layer as a starting point of beta cell differentiation, both SCAP and BMSCs cells were plated in 6-well plates until reaching 50–60% confluence, then the cell culture media was replaced by PSC Definitive Endoderm (DE) Induction media (Thermofisher, Waltham, MA, USA). At day 1, PSC Definitive Endoderm (DE) Induction medium A was added to the cells. At day 2, PSC Definitive Endoderm (DE) Induction media A was replaced with PSC Definitive Endoderm (DE) Induction medium B. At day 3, for the differentiation of the cells into a specific line of endoderm (Pancreatic differentiation), cells were induced by adding Advanced DMEM-F12 (Thermofisher, Waltham, MA, USA), supplemented with 1% BSA (Bovine serum albumin, Biowest, Nuaillé, France), 1% ITS (Insulin Transferrin selenium, Sigma, Burlington, MA, USA), 0.3 mM Taurine (Sigma, Burlington, MA, USA) and without serum for 2 days. At day 5, differentiating functional pancreatic cells were induced by Advanced DMEM-F12 (Thermofisher, Waltham, MA, USA), supplemented with 1.5% BSA (Biowest, Nuaillé, France), 1.5% ITS (Sigma, Burlington, MA, USA), 3 mM Taurine (Sigma, Burlington, MA, USA), 100 nM GLP (glucagon-like peptide Sigma, Burlington, MA, USA) and 1 mM nicotinamide (Sigma, Burlington, MA, USA) without serum for 10 days. The medium was exchanged every 2 days.