Monothioglycerol

Manufactured by Merck Group

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Monothioglycerol is a chemical compound used in various laboratory applications. It is a colorless, viscous liquid with the chemical formula HOCH2CH(SH)CH2OH. Monothioglycerol serves as a reducing agent and can be used in the synthesis of other chemical compounds.

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Monothioglycerol (MTG, (12 citations) α-monothioglycerol (9 citations) α-monothioglycerol (MTG), (2 citations)

101 protocols using monothioglycerol

Culturing G1E and Fetal Liver Erythroid Cells

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G1E-ER-GATA-1 (RRID:CVCL_D047) cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (ThermoFisher, Waltham, MA) containing 15% FBS (Gemini, West Sacramento, CA), 1% antibiotic/antimycotic (Corning, Tewksbury, MA), 2 U/ml erythropoietin (Amgen, Thousand Oaks, CA), 120 nM monothioglycerol (Sigma, St Louis, MO), 0.6% conditioned medium from an SCF producing CHO cell line, and 1 μg/ml puromycin (Gemini) (Fujiwara et al., 2009 (link)). G1E (RRID:CVCL_D046 cells were cultured without puromycin.
G1E cells were derived from GATA-1-null murine ES cells. ES cells were cultured under conditions that promoted the development of definitive erythroid cells (Weiss et al., 1997 (link)). G1E-ER-GATA-1 cells are G1E cells stably expressing GATA-1 fused to the ligand-binding domain of human estrogen receptor (Gregory et al., 1999 (link)). G1E and G1E-ER-GATA-1 cells were a kind gift from Dr. Mitchell J. Weiss (St. Judes).
Fetal liver erythroid precursors cells were cultured in StemPro-34 (ThermoFisher) with 1x nutrient supplement (ThermoFisher), 2 mM glutamax (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 100 μM monothioglycerol (Sigma), 1 μM dexamethasone (Sigma), 0.5 U/ml of erythropoietin, and 1% conditioned medium from a kit ligand producing CHO cell line. Cells were cultured in a humidified incubator at 37°C (5% carbon dioxide).

McIver S.C., Katsumura K.R., Davids E., Liu P., Kang Y.A., Yang D, & Bresnick E.H. (2016). Exosome complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis. eLife, 5, e17877.

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Inducible GFP Expression in Mouse Embryonic Stem Cells

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Mouse embryonic stem cells (mESCs) with a Doxycycline inducible GFP transgene ^{32 (link)} were maintained on gelatin coated tissue culture treated plates in serum-free conditions. Media (2i plus LIF) was composed of 50% Neurobasal media (Gibco) and 50% DMEM/F12 (Corning cellgro) supplemented with 0.5X N2 and 0.5X B27 (Gibco), 100U/mL Penicillin, 0.1 mg/ml Streptomycin (Corning), 0.05% BSA (Gibco), 2mM Glutamine (Corning), 0.15 mM monothioglycerol (Sigma), 1000U/ml mouse LIF (ESGRO, Sigma), 3uM CHIR99021 (Stem Cell Technologies), and 1uM PD0325901 (Stem Cell Technologies). To produce embryoid bodies (EBs) mESCs were differentiated in serum free differentiation media (SFD). SFD consisted of 75% IMDM (Gibco), 25% Ham’s F12 (Corning cellgro), 0.5X N2 (Gibco), 0.5X B27 (Gibco), 0.05% BSA (Gibco), 0.5mM Ascorbic Acid (Sigma), 2mM Glutamine (Corning), 0.45 mM monothioglycerol (Sigma), 100U/mL Penicillin and 0.1 mg/ml Streptomycin (Corning). On the first day of differentiation, mESCs were dissociated with Accutase (Sigma) and plated at 40,000 cells/ml in 12.5ml of SFD in 100mm petri dishes. After 48 hours, EBs were dissociated with Accutase and plated again at 80,000 cells/ml in SFD supplemented with 75ng/ml Activin A (R&D Systems). After 24 hours, expression of GFP was induced by adding Doxycycline (Sigma) for 24 hours before EBs collection.

de Silva N., Lacko L.A., Jamies E.A., Evans T, & Hurtado R. (2024). Atacama Clear for Complex 3D Imaging of Organs. bioRxiv.

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Differentiation of hESCs to Hematopoietic Lineage

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Nearly confluent hESCs, maintained in feeder free conditions, were treated with 50 mM EDTA/PBS solution for 1 min at 37 °C and upon addition of defined medium, the cells were manually cut with StemPro EZ Passaging Tool (Life Technologies) to obtain similar size pieces of colonies to generate homogenous sizes of self-aggregated EBs. Cell clumps collected with serological pipette were left to settle down at room temperature and supernatant was removed. Aggregates were resuspended in serum free StemPro^®-34 SFM media (Thermo Fisher) supplemented with 0.5% penicillin/streptomycin, L-glutamine (2 mM), ascorbic acid (1 mM), monothioglycerol (4 × 10 − 4 M; Sigma-Aldrich), transferrin (150 mg/ml) and BMP-4 (10 ng/ml), seeded into low attachment dishes (Corning) to generate embryoid bodies under hypoxic conditions for 4 days. Human haemangioblasts were FACS sorted based on KDR expression and further cultured on fibronectin or GO coated cover slips in serum free StemPro^®-34 SFM media (Thermo Fisher) supplemented with 0.5% penicillin/streptomycin, L-glutamine (2 mM), ascorbic acid (1 mM), monothioglycerol (4 × 10 − 4 M; Sigma-Aldrich), 150 mg/ml transferrin, 10 ng/ml VEGF, 5ng/ml bFGF, 50 ng/ml hSCF, and hIL6 (10 ng/ml) (PropsecBio).

Garcia-Alegria E., Iluit M., Stefanska M., Silva C., Heeg S., Kimber S.J., Kouskoff V., Lacaud G., Vijayaraghavan A, & Batta K. (2016). Graphene Oxide promotes embryonic stem cell differentiation to haematopoietic lineage. Scientific Reports, 6, 25917.

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Directed Differentiation of iPSCs into Endoderm

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Induced pluripotent stem cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 10⁴ cells per cm² in primed hiPS cell medium (KSR/FGF2). After 48 h, the medium was changed daily with RPMI-based medium with B27 supplement (Gibco, ThermoFisher Scientific), 100 ng ml⁻¹ activin A (Peprotech), 1 µM CHIR99021, 1% penicillin and streptomycin for 3 days. On days 4–8, the medium was changed daily with DMEM/F12-based medium with N2 (Gibco, ThermoFisher Scientific) and B27 supplements, 0.05 mg ml⁻¹ ascorbic acid (Sigma-Aldrich), 0.4 mM monothioglycerol (Sigma-Aldrich), 2 µM dorsomorphin (Peprotech), 10 µM SB-431542 (Miltenyi Biotec), 1% penicillin and streptomycin. On days 9–12, the medium was changed daily with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 20 ng ml⁻¹ BMP4 (Peprotech), 0.5 µM all-trans retinoic acid (Sigma-Aldrich), 3 µM CHIR99021, 1% penicillin and streptomycin. On days 12–20, the medium was changed every other day with DMEM/F12-based medium with B27 supplement, 0.05 mg ml⁻¹ ascorbic acid, 0.4 mM monothioglycerol, 10 ng ml⁻¹ FGF10 (Stemcell Technologies), 10 ng ml⁻¹ FGF7 (Peprotech), 3 µM CHIR99021, 50 nM dexamethasone (Sigma-Aldrich), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1% penicillin and streptomycin.

Buckberry S., Liu X., Poppe D., Tan J.P., Sun G., Chen J., Nguyen T.V., de Mendoza A., Pflueger J., Frazer T., Vargas-Landín D.B., Paynter J.M., Smits N., Liu N., Ouyang J.F., Rossello F.J., Chy H.S., Rackham O.J., Laslett A.L., Breen J., Faulkner G.J., Nefzger C.M., Polo J.M, & Lister R. (2023). Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature, 620(7975), 863-872.

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Directed Differentiation of iPSCs to Hepatocyte-like Cells

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Normal donor iPSCs grown on passage 3 irradiated CF1 MEFs were
differentiated into DECs for 5–7 days as previously described^{3 (link)} using insulin-free B27
(Invitrogen) instead of FBS. DECs were differentiated directly (without
splitting) into iPSC-Heps by culture in IMDM (Invitrogen) containing B27 with
insulin (Invitrogen), 1% Glutamax, 0.3 mM monothioglycerol
(Sigma-Aldrich), 1% Antibiotic-Antimycotic (Invitrogen), 0.126 U/mL
human insulin (Sigma-Aldrich), 10 ng/mL bFGF, 20 ng/mL BMP4, and 100 nM Dex for
5 days. Maturation was continued using the same medium additionally supplemented
with 20 ng/mL HGF for 15–20 days before switching to HCM supplemented
with 20 ng/mL HGF and 20 ng/mL OSM for 5–7 days similar to previously
reported protocols^{1 (link),3 (link)}. All growth factors were
purchased from R&D. Differentiation was performed entirely at
37°C in 5% O2/5% CO2 with daily media changes.

Zhu S., Rezvani M., Harbell J., Mattis A.N., Wolfe A.R., Benet L.Z., Willenbring H, & Ding S. (2014). Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature, 508(7494), 93-97.

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SFEBq Differentiation Protocol for Hypothalamic Neurons

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Each cell was cultured according to the SFEBq protocol [6 (link)]. In brief, mESCs were enzymatically dissociated to single cells in 0.25% Trypsin-EDTA (Wako; Cat# 201–16945) and quickly reaggregated in differentiation medium (3,000 cells per well) using 96-well U-bottom low cell-adhesion plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan; Cat# MS-9096U). The differentiation medium was growth factor-free CDM (gfCDM), which contains Iscove’s modified Dulbecco’s medium (Gibco; Cat# 31980030) /Ham’s F-12 (Gibco; Cat# 31765035) 1:1, 1×chemically defined lipid concentrate (Gibco; Cat# 11905031), monothioglycerol (450 μM; Sigma-Aldrich; Cat# M6145, CAS# 96-27-5) and 5 mg/ml purified bovine serum albumin (BSA, Sigma-Aldrich; Cat# A9418, CAS# 9048-46-8). The differentiation medium was used from days 0–7. To regulate Sonic hedgehog (Shh) signal transduction, 10 nM SAG (Cayman Chemicals, Ann Arbor, MI, USA; Cat# 11914, CAS# 912545-86-9) was added to the culture from day 4 for induction of ventral hypothalamic neurons, such as POMC, AgRP and NPY.

Kawata M., Kodani Y., Ohkuma M., Miyachi E.I., Kaneko Y.S., Nakashima A., Suga H., Kameyama T., Saito K, & Nagasaki H. (2022). Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain. PLOS ONE, 17(11), e0276694.

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Differentiation of ESCs into Cardiomyocytes

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ESCs were directed to differentiate into cardiomyocytes as described previously^{15 (link)}. Briefly, cells were depleted from the feeder layer with a standard technique and aggregated into EBs using the hanging drop method. Next, EBs were dissociated and cultured at a density of 100,000 cells/ml for two days in serum-free media (3 parts Iscove’s Modification of DMEM (IMDM) (Cellgro): 1 part Ham’s F12 (Gibco), 0.05% bovine serum albumin (BSA), 2 mM GlutaMax (Gibco), B27 supplement (Gibco), N2 supplement (Gibco) supplemented with 50 mg/ml ascorbic acid, and 4.5×10⁻⁴ M monothioglycerol (Sigma)). Around 48 hours later, EBs were dissociated and re-aggregated in the presence of hVEGF (5 ng/mL), Activin A (5 ng/ml), and hBMP4 (0.25 ng/ml) (all from R&D Systems). EBs were further dissociated and replated at 500,000 cells/well in a 24 well plate in StemPro-34 (Gibco) supplemented with 5 ng/mL hVEGF, 10 ng/mL human basic FGF, and 25 ng/mL FGF10 (all from R&D Systems).

Hazra R., Brine L., Garcia L., Benz B., Chirathivat N., Shen M.M., Wilkinson J.E., Lyons S.K, & Spector D.L. (2022). Platr4 is an early embryonic lncRNA that exerts its function downstream on cardiogenic mesodermal lineage commitment. Developmental cell, 57(21), 2450-2468.e7.

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Directed Differentiation of iPSCs into ECs

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Regarding endothelial differentiation, on day 0, iPSC colonies were dissociated into single cells with Accutase (Sigma). Cells were resuspended in basic medium containing StemPro34 (Invitrogen), L-glutamine (Invitrogen), transferrin (Roche), monothioglycerol (Sigma), and ascorbic acid (Sigma), and Y-27632 (naclai tesque), BMP-4 (Peprotech), and Matrigel (BD) were added to form EBs. On day 1, the medium was supplemented with Activin A (R&D Systems) and BMP4. On day 4, EBs were seeded in Matrigel-coated dishes, and the medium was supplemented with VEGF (R&D Systems) for EC expansion. On day 14, the EBs were harvested using Accutase (Sigma-Aldrich), and CD31 positive cells were positively sorted using magnetic-activated cell sorting using anti-CD31 (Miltenyi) (Fig. 7a).

Sekine S.I., Kaneko M., Tanaka M., Ninomiya Y., Kurita H., Inden M., Yamada M., Hayashi Y., Inuzuka T., Mitsui J., Ishiura H., Iwata A., Fujigasaki H., Tamaki H., Tamaki R., Kito S., Taguchi Y., Tanaka K., Atsuta N., Sobue G., Kondo T., Inoue H., Tsuji S, & Hozumi I. (2019). Functional evaluation of PDGFB-variants in idiopathic basal ganglia calcification, using patient-derived iPS cells. Scientific Reports, 9, 5698.

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Chemically Defined hPSC Hematopoietic Differentiation

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hPSC hematopoietic differentiation was carried out under chemically defined conditions as previously described with some modification (Pang et al., 2013 (link), Wang et al., 2012 (link)). hPSCs were dissociated into a single-cell suspension using 1 mg/mL Accutase (Gibco) and plated on Matrigel-coated dishes at a density of 3.5 × 10⁴ cells/well (12-well plate) in mTeSR1 medium with 10 μM Y27632 (Calbiochem). After 24 hr, hPSCs were induced for stepwise differentiation. First, cells were cultured in Custom mTeSR1 medium supplemented with 40 ng/mL activinA (Peprotech) and 50 ng/mL BMP4 (Peprotech) for 2 days. Second, cells were incubated with Custom mTeSR1 medium supplemented with 40 ng/mL VEGF (Peprotech) and 50 ng/mL bFGF (Peprotech) for 2 days. Third, cells were incubated with Custom mTeSR1 medium supplemented with 40 ng/mL VEGF, 50 ng/mL bFGF, and 20 μM SB 431542 (STEMGENT) for 3 days. Finally, differentiated cells were transferred to low-attachment plates and cultured for 6 days in mTeSR1 medium containing 50 ng/mL stem cell factor (SCF) (Peprotech), 50 ng/mL TPO (Peprotech) and 50 ng/mL interleukin-3 (IL-3) (Peprotech), 1 mM GlutaMAX (Gibco), 2% B27 (Gibco), 0.1 mM monothioglycerol (Sigma-Aldrich), 1% insulin-transferrin-selenium (Gibco), 1% N-acetylaspartate (Gibco), 1% penicillin/streptomycin.

Wang H., Liu C., Liu X., Wang M., Wu D., Gao J., Su P., Nakahata T., Zhou W., Xu Y., Shi L., Ma F, & Zhou J. (2018). MEIS1 Regulates Hemogenic Endothelial Generation, Megakaryopoiesis, and Thrombopoiesis in Human Pluripotent Stem Cells by Targeting TAL1 and FLI1. Stem Cell Reports, 10(2), 447-460.

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Efficient Generation of hEROs from hESCs

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Human embryonic stem cell line (H9) was kindly provided by Stem Cell Bank, Chinese Academy of Sciences. After being tested without mycoplasma contamination, this hESC line was used in present study and maintained in mTeSR1 (Stem Cell Technologies) without feeders. hEROs were generated following Kuwahara’s protocol^{10 (link)}. In brief, hESCs were dissociated into single-cell suspensions in TrypLE Express (Gibco) containing 0.05 mg/ml DNaseI (Roche) and 20 μM Y-27632 (Merck). The retinal differentiation medium consisted of 45% IMDM (Gibco), 45% F12-Glutamax (Gibco), 450 μM monothioglycerol (Sigma-Aldrich), and 1% Chemically Defined Lipid Concentrate (Gibco) supplemented with 10% knockout serum replacement (KSR, Gibco) and 20 μM Y-27632. On day 0, 100 μl of cell suspension (1.2 × 10⁴ cells) was placed in each well of low-cell-adhesion V-bottom 96-well plates (Sumitomo Bakelite). On day 6, the medium was exchanged for fresh retinal differentiation medium supplemented with 1.5 nM bone morphogenetic protein 4 (BMP4, Peprotech). Thereafter, half of the medium was replaced every three days. On day 18, hERO was transferred to low-cell-adhesion dishes (Greiner) in long-term culture medium containing Dulbecco’s modified Eagle’s medium (DMEM)/F12-Glutamax (Gibco) with 1% N2 supplement (Gibco), 10% fetal bovine serum (FBS, Gibco), 0.5 μM RA (Sigma) and 0.1 mM taurine (Sigma).

Zou T., Gao L., Zeng Y., Li Q., Li Y., Chen S., Hu X., Chen X., Fu C., Xu H, & Yin Z.Q. (2019). Organoid-derived C-Kit+/SSEA4− human retinal progenitor cells promote a protective retinal microenvironment during transplantation in rodents. Nature Communications, 10, 1205.

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