PGCLCs were induced from primed hESCs as described in Sasaki et al. (2015) (link), with some modifications (Chen et al., 2017b ). Day 7 hESCs were dissociated into single cells with 0.05% Trypsin-EDTA and plated onto Human Plasma Fibronectin (Invitrogen, 33016-015)-coated 12-well-plate at the density of 200,000 cells/well in 2 mL/well of iMeLC media, which is composed of 15% KSR, 1× NEAA, 0.1 mM 2-Mercaptoethanol, 1× PSG (Gibco, 10378-016), 1 mM sodium pyruvate (Gibco, 11360-070), 50 ng/mL Activin A (Peprotech, AF-120-14E), 3 μM CHIR99021 (Stemgent, 04-0004), 10 μM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50 ng/mL primocin in Glasgow’s MEM (GMEM) (Gibco, 11710-035). iMeLCs were dissociated into single cells with 0.05% Trypsin-EDTA after 24 h of incubation and plated into ultra-low cell attachment U-bottom 96-well plates (Corning, 7007) at the density of 3000 cells/well in 200 μL/well of PGCLC media, which is composed of 15% KSR, 1× NEAA, 0.1 mM 2-Mercaptoethanol, 1× PSG (Gibco, 10378-016), 1 mM sodium pyruvate (Gibco, 11360-070), 10 ng/mL human LIF (Millipore, LIF1005), 200 ng/mL human BMP4 (R&D systems, 314-BP), 50 ng/mL human EGF (R&D systems, 236-EG) 10 μM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50 ng/mL primocin in Glasgow’s MEM (GMEM) (Gibco, 11710-035).
Human plasma fibronectin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Human plasma fibronectin is a glycoprotein that is a major component of the extracellular matrix. It plays a role in cell adhesion, migration, and wound healing.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (6 mentions)
Human bmp4, by R&D Systems (5 mentions)
Activin a, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (5 mentions)
Primocin, by Thermo Fisher Scientific (3 mentions)
Ultra low cell attachment u bottom 96 well plates, by Corning (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Lif1005, by R&D Systems (2 mentions)
Human egf, by R&D Systems (2 mentions)
Human lif, by Merck Group (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
96 well u bottom plate, by Corning (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Hyaluronic acid, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
30 protocols using human plasma fibronectin
1
Differentiation of hESCs into PGCLCs
2
Differentiation of hESCs into PGCLCs
3
Differentiating hESCs into hPGCLC
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Primed hESCs were differentiated into hPGCLC as described in Sasaki et al. (2015) (link) with some modifications. Day-7 hESCs were dissociated into single cells with 0.05% trypsin-EDTA (Gibco) and plated onto a human plasma fibronectin (Invitrogen)-coated 12-well-plate at 200,000 cells/well cell density in 2 mL/well of incipient mesoderm-like cells (iMeLCs) medium, which is composed of 15% knockout serum replacement (KSR), 1× non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol, 1× penicillin-streptomycin-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 50 ng/mL Activin A (Peprotech), 3 μM CHIR99021 (Stemgent), 10 μM ROCKi (Y27632, Stemgent), and 50 ng/mL primocin in Glasgow's minimal essential medium (GMEM) (Gibco). Twenty-four hours later, iMeLCs were dissociated into single cells by 0.05% trypsin-EDTA, followed by plating onto ultra-low cell attachment U-bottom 96-well plates (Corning) at a density of 3,000 cells/well in 200 μL/well of hPGCLC medium, which is composed of 15% KSR, 1× NEAA, 0.1 mM 2-mercaptoethanol, 1× penicillin-streptomycin-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 10 ng/mL human leukemia inhibitory factor (Millipore), 200 ng/mL human BMP4 (R&D systems), 50 ng/mL human epidermal growth factor (R&D Systems), 10 μM of ROCKi (Y27632, Stemgent), and 50 ng/mL primocin in GMEM (Gibco). Day-4 hPGCLC aggregates were used for further analysis.
4
hESCs to human PGCLCs Differentiation
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hESCs were dissociated into single cells with 0.05% Trypsin-EDTA (GIBCO, 25300-054) and plated onto Human Plasma Fibronectin (Invitrogen, 33016-015)-coated 12-well-plates at the density of 200,000 cells/well in 2mL/well of iMeLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Strep-tomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 50ng/mL Activin A (Peprotech, AF-120-14E), 3mM CHIR99021 (Stemgent,04-0004), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035). After 24 hr, iMeLCs were dissociated into single cells with 0.05% Trypsin-EDTA and plated into ultralow cell attachment U-bottom 96-well plates (Corning, 7007) at the density of 3,000 cells/well in 200ml/well of hPGCLC media, which is composed of 15% KSR (GIBCO, 10828-028), 1x NEAA (GIBCO, 11140-050), 0.1mM 2-Mercaptoethanol (GIBCO, 21985-023), 1x Penicillin-Streptomycin-Glutamine (GIBCO, 10378-016), 1mM sodium pyruvate (GIBCO, 11360-070), 10ng/mL human LIF (Millipore, LIF1005), 200ng/mL human BMP4 (R&D systems, 314-BP), 50ng/mL human EGF (R&D systems, 236-EG), 10mM of ROCKi (Y27632, Stemgent, 04-0012-10), and 50ng/mL primocin in Glasgow’s MEM (GMEM) (GIBCO, 11710-035).
5
Optimizing Cell Culture Conditions
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Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), Hank’s buffered salt solution, Earle’s balanced salt solution, penicillin, streptomycin, human plasma fibronectin, and trypsin-ethylenediaminetetraacetic acid were purchased from Invitrogen Life Technologies, Carlsbad, CA. Type IV collagenase, HEPES (4-[2-hydroxyethyl] piperazine-1-ethanesulfonic acid), glucagon, calcium chloride, hydrocortisone, sodium dodecyl sulfate, hydrogen peroxide, glutaraldehyde, dicumarol, 3-methylcholanthrene, calf thymus DNA, chitosan, and hyaluronic acid (HA) was purchased from Sigma-Aldrich, St. Louis, MO. All other chemicals were purchased from Fisher Scientific, Waltham, MA, unless otherwise noted.
6
Imaging Cell Adhesion on Nanofiber Scaffolds
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20 µm thick scaffolds with random or aligned 700 nm diameter electrospun polycaprolactone (PCL) fibers, mounted on 15 mm diameter plastic coverslips were purchased from Nanofiber Solutions (NanoAligned and NanoECM 24-well plate inserts, respectively). The scaffolds were placed in 12-well plates and adsorbed with 5 µg/ml human plasma fibronectin (Invitrogen and Corning, Fisher Scientific) in PBS for 30 min at room temperature or overnight at 5 °C. The scaffolds were rinsed once with PBS before seeding cells.
For timelapse experiments, cells were seeded on scaffolds in CCM1 (containing inhibitors as necessary) and then incubated for 10 min at 37 °C. The scaffolds were then inverted, placed in a glass-bottomed dish (produced in-house) filled with CCM1 (containing inhibitors as necessary), and quickly moved to the microscope where imaging began with 20 min of cell seeding. Z-stacks for high-resolution adhesion timelapse movies were acquired every 2 min for 20–60 min. Z-stacks for low-magnification, multi-cell movies were acquired every 1 min for 30–45 min.
For morphology experiments, cells were seeded on scaffolds in CCM1 (containing inhibitors as necessary), incubated for 30 min at 37 °C, fixed with 4% formaldehyde for 15 min, and then mounted on coverslips.
For timelapse experiments, cells were seeded on scaffolds in CCM1 (containing inhibitors as necessary) and then incubated for 10 min at 37 °C. The scaffolds were then inverted, placed in a glass-bottomed dish (produced in-house) filled with CCM1 (containing inhibitors as necessary), and quickly moved to the microscope where imaging began with 20 min of cell seeding. Z-stacks for high-resolution adhesion timelapse movies were acquired every 2 min for 20–60 min. Z-stacks for low-magnification, multi-cell movies were acquired every 1 min for 30–45 min.
For morphology experiments, cells were seeded on scaffolds in CCM1 (containing inhibitors as necessary), incubated for 30 min at 37 °C, fixed with 4% formaldehyde for 15 min, and then mounted on coverslips.
7
Cytotoxicity Assay using Cell Lines
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TNF-α was obtained from R & D (Heidelberg, Germany) and Calcein-AM from Invitrogen (Heidelberg, Germany). Ethanol, methanol and formic acid were supplied by Merck KGaA (Darmstadt, Germany). Cell culture media (MCDB131) and supplements (EGF, ECGF) for human umbilical vein endothelial cells (HUVECs) were obtained from Promocell (Heidelberg, Germany) and those for Caco-2 cells (Dulbecco’s Eagle’s Minimum Essential Medium (DMEM)) from Invitrogen (Heidelberg, Germany). Fetal calf serum (FCS) was supplied by Biochrome (Berlin, Germany), lysogeny broth (LB) for the culture of Gram-negative microorganisms by Life Technologies GmbH (Darmstadt, Germany) and tryptic soy broth (TSB) for the culture of Gram-positives by Merck KGaA (Darmstadt, Germany). Human plasma fibronectin was obtained from Invitrogen (Heidelberg, Germany). All chemicals were of analytical grade and cell culture tested.
8
hESCs to human PGCLCs Differentiation
9
Generation of Hematopoietic Stem Cells from iPSCs
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Patient-specific iPSCs were differentiated into hematopoietic stem cells (HSCs) under defined, serum-free and feeder-free conditions as previously reported.20 (link) Briefly, TrypLE (Invitrogen)-dissociated iPSCs were seeded onto 6-well plates that were precoated with 3 mg cm−2 human plasma fibronectin (Invitrogen) in mTeSR1 supplemented with an inhibitor of Rho-associated kinase (H1152, Sigma). After 1 day, mTeSR1 was replaced with a hematopoietic commitment medium containing Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 1 × HIT (Stem Cell Technologies), 450 μM monothioglycerol (Sigma), 50 ng ml−1 recombinant human BMP4 (R&D Systems), 50 ng ml−1 recombinant human vascular endothelial growth factor (Invitrogen), 0.1 mM NEAA (Invitrogen) and 2 mM L -glutamine (Invitrogen). After 6 days, the medium was changed to a hematopoietic maturation medium consisting of Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 5 U ml−1 heparin (Sigma), 25 ng ml−1 thrombopoietin (Invitrogen), 25 ng ml−1 human recombinant stem cell factor (Invitrogen), 25 ng ml−1 human recombinant FLT3L (Peprotech, Rocky Hill, NJ, USA), 10 ng ml−1 interleukin-3 (Invitrogen) and 10 ng ml−1 interleukin-6 (Invitrogen). The cells were incubated under hypoxic conditions (5% O2 balanced with nitrogen).
10
Endothelial and Intestinal Cell Culture
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M3G was purchased from Carl Roth GmbH (Karlsruhe, Germany), PCA from Sigma-Aldrich (Taufkirchen, Germany), TNF-α from R&D (Heidelberg, Germany), Calcein-AM and human plasma fibronectin from Invitrogen (Heidelberg, Germany). Cell culture media and supplements for human umbilical vein endothelial cells (HUVECs) were obtained from Promocell (Heidelberg, Germany), and those for Caco-2 cells and HT29-B6 cells from Invitrogen (Heidelberg, Germany). All chemicals were with analytical grade and cell culture tested.
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