ESCs were directed to differentiate into cardiomyocytes as described previously15 (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).
Hbmp4
Manufactured by R&D Systems
HBMP4 is a recombinant human bone morphogenetic protein 4 (BMP4) produced in HEK293 cells. BMP4 is a member of the transforming growth factor-beta (TGF-β) superfamily and plays a role in the regulation of cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hvegf, by R&D Systems (1 mentions)
Stempro 34, by Thermo Fisher Scientific (1 mentions)
Human basic fgf, by R&D Systems (1 mentions)
Fgf10, by R&D Systems (1 mentions)
Monothioglycerol, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Activin a, by R&D Systems (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Accutase, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Heparin, by STEMCELL (1 mentions)
Relesr, by STEMCELL (1 mentions)
5 protocols using hbmp4
1
Differentiation of ESCs into Cardiomyocytes
2
Neurosphere Formation and Quiescence
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Sorted NSCs or total SVZ cell suspensions were grown at 37°C in 5% CO2 in neurosphere medium composed of DMEM/F12 (Life Technologies) supplemented with 0.6% glucose (Sigma), 2 μg/mL heparin (STEMCELL Technologies), 1× insulin-selenium-transferrin (Life Technologies), N-2 supplement (Life Technologies), and B-27 without vitamin A supplement (Life Technologies), and in the presence of 20 ng/mL EGF (Millipore) and 10 ng/mL FGF2 (Millipore).
After 7 days, neurospheres were counted under an inverted microscope. Neurospheres were centrifuged and incubated for 5 min in the presence of Accutase (Sigma) then were mechanically dissociated. Dissociated cells were plated in neurosphere medium at a density not exceeding 1.4 cells/μL in 12- or 24-well plates.
Quiescence was induced in vitro by removing growth factors and by adding 25 ng/mL hBMP4 (R&D Systems).
For RNA measurement, cells were counted on day 3 and lysed in RLT buffer (Qiagen) for RNA isolation and qRT-PCR experiments.
After 7 days, neurospheres were counted under an inverted microscope. Neurospheres were centrifuged and incubated for 5 min in the presence of Accutase (Sigma) then were mechanically dissociated. Dissociated cells were plated in neurosphere medium at a density not exceeding 1.4 cells/μL in 12- or 24-well plates.
Quiescence was induced in vitro by removing growth factors and by adding 25 ng/mL hBMP4 (R&D Systems).
For RNA measurement, cells were counted on day 3 and lysed in RLT buffer (Qiagen) for RNA isolation and qRT-PCR experiments.
3
Differentiation of hESCs to NPCs and MES
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H9 hESCs (WiCell) were maintained in mTeSR1 medium (Stem Cell Technologies) on Matrigel (BD Biosciences)-coated tissue culture plates. H9 cells were passaged every 5 days by ReLeSR (Stem Cell Technologies). For NPC differentiation, hESCs were passaged as single cells using Accutase (Life Technologies) on Matrigel with ROCK inhibitor (Y-27632, Millipore) and induced to differentiate with SB431542 (10 µM, Tocris Bioscience) and NOGGIN (200 ng/ml, PeproTech)52 (link). To derive MES, hESCs were plated on Matrigel-coated plates as single cells and induced to differentiate with mTeSR1 containing 5 ng/ml hBMP4 (R&D)59 (link).
4
Quantitative Analysis of BMP4-Induced mES Genes
5
Directed Differentiation of hESCs to hPGCLCs
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Naive H9 and naive WIBR3 OCT4-dPE-GFP hESCs were propagated in serum-free N2B27 medium (N2 & B27; Life Technologies) supplemented with 20 ng/mL hLIF (Cambridge Stem Cell Institute [SCI]), 1 μM MEK inhibitor PD0325901 (SCI), 3 μM GSK3 inhibitor CHIR99021 (SCI), and 2 μM protein kinase C inhibitor Gö6983 (Sigma-Aldrich), as described previously (Guo et al., 2016 , Takashima et al., 2014 (link)). The medium was refreshed every day and cells were passaged every 4–5 days. hEpiLC were induced by plating 2 × 105 naive hESCs on a well of a 6-well plate coated with growth factor reduced Matrigel (Corning) in N2B27 medium supplemented with 1 ng/mL TGF-β1 (Peprotech), 12 ng/mL bFGF (SCI), and 1% KSR (Gibco). The medium was changed every day. hPGCLCs were induced by plating 3–4 × 103 day-4 hEpiLCs in a well of an Ultra-Low attachment U-bottom 96-well plate (Corning) in GK15 medium (Glasgow’s minimal essential medium [Life Technologies] with 15% KSR [Life Technologies], 0.1 mM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM β-mercaptoethanol) supplemented with 500 ng/mL hBMP4 (R&D Systems), 20 ng/mL hLIF (SCI), 100 ng/mL hSCF (R&D Systems), and 50 ng/mL hEGF (R&D Systems). Cells were cultured in 5% O2 and 5% CO2 in a humidified incubator at 37°C.
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