Muse cells were plated at 1.55 × 104/cm2 and cultured in the adherent state for the entire period of induction on a laminin-coated surface. The cells were first incubated with DMEM low glucose containing 2% of FBS plus TGF-β1 (2.5 ng/mL, Wako), BMP-4 (5 ng/mL, Wako), BMP-2 (5 ng/mL, Sigma-Aldrich), activin A (10 ng/mL, Wako), Wnt-3a (50 ng/mL, R&D Systems, Minneapolis, MN, USA), and bFGF (10 ng/mL, Wako) for 7 d and then for 2 wk with DMEM low glucose containing 2% of FBS plus TGF-β1, insulin-like growth factor-1 (IGF-1, 5 ng/mL, Sigma-Aldrich), hepatocyte growth factor (HGF; 20 ng/mL, Wako), and cardiotrophin-1 (CT-1; 200 ng/mL, Sigma-Aldrich).
Activin a
Manufactured by Fujifilm
Sourced in United States, Japan
Activin A is a laboratory product manufactured by Fujifilm. It is a member of the transforming growth factor-beta (TGF-beta) superfamily of proteins. Activin A functions as a growth and differentiation factor.
Automatically generated - may contain errors
Lab products found in correlation
Bone morphogenetic protein 4 (bmp4), by Fujifilm (2 mentions)
Tgf β1, by Fujifilm (2 mentions)
Activin a, by R&D Systems (2 mentions)
Rankl, by Fujifilm (2 mentions)
M csf, by BioLegend (2 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
Insulin like growth factor 1 igf 1, by Merck Group (1 mentions)
Bmp 2, by Merck Group (1 mentions)
Dmem low glucose, by Fujifilm (1 mentions)
Tgf β1, by Merck Group (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Stem cell factor (scf), by Fujifilm (1 mentions)
Rock inhibitor, by Fujifilm (1 mentions)
Lif1005, by Merck Group (1 mentions)
Dorsomorphin, by Fujifilm (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
V bottom plate, by Thermo Fisher Scientific (1 mentions)
Alk5 inhibitor 2, by Santa Cruz Biotechnology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
8 protocols using activin a
1
Muse Cell Differentiation Protocol
2
Induction of Germline Cells from hAM-Muse Cells
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The previously described method of differentiation into germline cells was used44 (link) with minor modifications. hAM-Muse cells (10,000 cells/cm2) were cultured in alpha-MEM containing 10% FBS, 1% GlutaMaX, 0.1 mg/mL kanamycin sulfate, 50 ng/mL Activin A (014-23961, Wako, Osaka, Japan), 3 µM GSK3 inhibitor (039–20,831, Wako), and 10 µM ROCK inhibitor (030-24021, Wako) on adherent culture for 2 days (iMeLC induction). After 2 days of iMeLC induction, the cells were cultured in alpha-MEM containing 10% FBS, 1% GlutaMaX, 0.1 mg/mL kanamycin sulfate, 200 ng/mL BMP4 (022-17071, Wako), 1000 U/mL leukemia inhibitory factor (LIF1005, Milipore), 100 ng/mL stem cell factor (197-15511, Wako), 50 ng/mL (EGF; 059-07873, Wako), and 10 µM ROCK inhibitor on suspension culture using low-cell-binding V-bottom 96-well plates (3000 cells/well) and formed aggregations at 37 °C in 5% CO2. Aggregations of hAM-Muse cells after 2 or 4 days were subjected to qPCR. Aggregations after 4 days were fixed with 4% PFA in 0.01 M PBS, embedded in OCT compound, and cut into 8-µm-thick cryosections. These cryosections were subjected to immunocytochemistry as described above. Human iPS cells were induced by the same method for 6 days and used as a positive control.
3
Screening Chemical Effects on Pancreatic β-Cell Differentiation
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In vitro pancreatic β cell differentiation was performed as previously reported13 (link) with some modifications for the chemical screening assays. Briefly, to detect the effect of each chemical in the screening, we omitted the chemicals (10 μM forskolin, 5 μM Alk5i, 10 μM Dexamethasone, 10 mM nicotinamide) from the final stages of the original protocol. The hiPSCs were plated in a V-bottom plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4500 cells/well in hiPSC medium without bFGF for 3 days to form embryoid bodies and then induced for 1 day in 100 ng/mL activin A (R&D Systems, Minneapolis, MN, USA) and 3 μM CHIR (Axon Medchem, Groningen, the Netherlands). Next, the cells were induced with 100 ng/mL activin A for 2 days, then subjected to PP induction for 7 days with 1 μM dorsomorphin (Wako), 2 μM RA (Sigma-Aldrich), and 10 μM SB431542 (Cayman, Ann Arbor, MI, USA). For endocrine differentiation during the third stage of differentiation, we added the individual LOPAC chemicals (Sigma-Aldrich) at 5 μM or DMSO for control, or the cells were fully induced with 10 μM Fsk (Nacalai Tesque), 5 μM Alk5 inhibitor II (Santa Cruz, Dallas, TX, USA), 10 μM Dex (Sigma-Aldrich), and 10 mM Nico (Sigma-Aldrich) as a positive control.
4
Endothelial Differentiation of Embryonic Stem Cells
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ESCs were differentiated into the endothelial cell lineages using the endothelial
differentiation protocol developed for human PSCs21 (link) with some modification. Our differentiation protocol consists of two
phases. First, ESCs were differentiated into epistem cell (EpiSC)-like cells22 (link) (phase I). Second, the EpiSC-like cells were differentiated into the
endothelial cell lineages21 (link) (phase II). At phase I, ESC colonies were entirely dissociated into
single cells and seeded on a well of a six-well plate coated with Fibronectin
(0.1%, Sigma) in ESC-cultured medium. One day after the culture, the medium was
changed to NDiff227 medium supplemented with Activin A (20 ng/ml; Wako, Japan),
basic FGF (12 ng/ml; Wako, Japan), and Rho kinase inhibitor (Y-27632, 10 μM).
The cells were continuously cultured for 2 days (phase I). Then, the cells were
cultured in DMEM with B27 without insulin (50×; Thermo Fisher, MA) supplemented
with GSK 3α-β inhibitor (CHIR-99021, 10 μM) for 2 days. Finally, they were
cultured in endothelial cell growth medium (EBM2; Lonza, Basel, Swiss) for 3
days (phase II).
differentiation protocol developed for human PSCs21 (link) with some modification. Our differentiation protocol consists of two
phases. First, ESCs were differentiated into epistem cell (EpiSC)-like cells22 (link) (phase I). Second, the EpiSC-like cells were differentiated into the
endothelial cell lineages21 (link) (phase II). At phase I, ESC colonies were entirely dissociated into
single cells and seeded on a well of a six-well plate coated with Fibronectin
(0.1%, Sigma) in ESC-cultured medium. One day after the culture, the medium was
changed to NDiff227 medium supplemented with Activin A (20 ng/ml; Wako, Japan),
basic FGF (12 ng/ml; Wako, Japan), and Rho kinase inhibitor (Y-27632, 10 μM).
The cells were continuously cultured for 2 days (phase I). Then, the cells were
cultured in DMEM with B27 without insulin (50×; Thermo Fisher, MA) supplemented
with GSK 3α-β inhibitor (CHIR-99021, 10 μM) for 2 days. Finally, they were
cultured in endothelial cell growth medium (EBM2; Lonza, Basel, Swiss) for 3
days (phase II).
5
Isolation and Culture of Rheumatoid Arthritis Synovial Cells
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A total of 6 patients with RA diagnosed based on the revised 1987 American College of Rheumatology criteria for RA were recruited. Synovial tissues were collected from 6 patients who underwent arthroplastic joint surgery and synovectomy.
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
6
Monocyte Differentiation into Osteoclasts
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Peripheral blood monocytes (PBMs) were cultured in a 96-well tissue culture plate at a concentration of 2 × 10 4 cells/well. Cultures were maintained in 200 μL of α-minimal essential medium containing 10% fetal bovine serum for 7-10 days with M-CSF (BioLegend) and RANKL (Wako Pure Chemical Industries) in the presence or absence of activin A (Wako Pure Chemical Industries). The cells were pretreated with M-CSF and/or activin A for 24 h, Pathobiology 2020;87:198-207 200 DOI: 10.1159/000506260 followed by the addition of RANKL to the culture medium. Every 3 days, half of the culture medium was replaced with fresh medium containing cytokines and chemicals.
7
Directed Differentiation of hPSCs into Pancreatic Progenitors
8
Conversion of Mouse ESCs to EpiSCs
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Mouse ESCs were seeded onto MEF feeders at a density of 1-3 x 10 5 cells per 3 cm dish and cultured in the ES medium over night at 37°C. For conversion of ES cells to EpiSC-like cells, ES cell medium was replaced with EpiSC medium (DMEM/F12 plus glutamax (Gibco), 1xNEAA (Life Technologies), 15% KSR (Life Technologies), 5 ng/mL of basic FGF (Reprocell), 10 ng/mL of Activin A (Wako) and 2 µM IWP-2 (Stemgent) and the cells were incubated at 37°C overnight. The day of the medium change was set as Day 0. On the next day (Day 1), cells were passaged using CTKCa dissociation buffer (phosphate buffered saline containing 0.25% trypsin (BD Diagnostic Systems), 1 mg/ml of collagenase (Life Technologies), 20% KSR (Life Technologies), 1 mM CaCl2) essentially as described by Sugimoto et al. [18] (link). The medium was changed every day and cells were passaged every other day. For harvesting primed PSC-like cells, cells were dissociated by 0.25% trypsin, 1 mM EDTA and the single cell suspension was used for single-cell capture or plate purification was done to remove feeder cells before harvesting.
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