StemXVivo Endoderm Kit
StemXVivo Endoderm Kit Summary
A kit optimized to efficiently differentiate pluripotent cells into definitive endodermal cells under serum-free conditions.
Key Benefits
- Definitive endoderm differentiation in 4 days
- Pre-optimized to reduce experimental variation
- Endodermal cells are 70–90% positive for SOX17
- Endodermal cells express minimal to non-detectable levels of SOX7
Why Use Pre-optimized Reagents for Endoderm Differentiation?
Pluripotent stem cells can differentiate into all three germ layers including the definitive endoderm which gives rise to cells of the liver, lung, pancreas, and gut lining. Inefficient or inconsistent differentiation represents a costly and time-consuming obstacle in stem cell differentiation.
High quality media and defined differentiation supplements can reduce experimental variability and increase differentiation efficiency.
The StemXVivo® Endoderm Kit:
- Includes premium quality reagents that are optimized to induce endoderm differentiation in 4 days.
- Includes components with low lot-to-lot variation to reduce experimental variability.
- Yields definitive endodermal cells expected to be at 70–90% positive for SOX17.
- Yields an endodermal cell population with little to non-detectable SOX7 expression.
The StemXVivo® Endoderm Kit includes a serum-free, defined media supplement and premium quality recombinant proteins that have been optimized to efficiently differentiate pluripotent stem cells into definitive endodermal cells.
Cells are differentiated using two differentiation media that are made by combining the media supplement and the recombinant proteins included in this kit with RPMI medium. Differentiation Media I contains Activin A, FGF basic, and Wnt-3a whereas Differentiation Media II contains Activin A and FGF basic.
- Endoderm Base Media Supplement (50x)
- Recombinant Human Activin A
- Recombinant Human FGF Basic
- Recombinant Human Wnt-3a
- Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
Specifications
Product Datasheets
Scientific Data
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Definitive Endoderm Differentiation of BG01V Human Embryonic Stem Cells. BG01V cells were differentiated into definitive endoderm using the media supplements included in this kit (Catalog # SC019B). To evaluate lineage commitment, the cells were stained with the Anti-Human SOX17 antibody included in this kit. The cells were stained with NorthernLights™557-Conjugated Donkey Anti-Goat IgG Secondary Antibody (R&D Systems, Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue).
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The Differentiation of BG01V Human Embryonic Stem Cells into Definitive Endoderm Confirmed by Cell Markers (SOX17+/CXCR4+/FOX2A+/SOX7low). BG01V human embryonic stem cells were differentiated using the StemXVivo®Endoderm Kit (Catalog # SC019) and stained withA) a Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924),B) a Goat Anti-Human HNF-3 beta/FoxA2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2400), orC) a Goat Anti-Human SOX7 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2766). In all images, cells were stained with the NorthernLights™557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001, red) and the nuclei were counterstained with DAPI (blue). Endoderm-differentiated cells were also stained withD) a PE-conjugated Mouse Anti-Human CXCR4 Monoclonal Antibody (Catalog # FAB173P; filled histogram) or a PE-conjugated Mouse IgG2BIsotype Control (Catalog # IC0041P; open histogram). High levels of SOX17, CXCR4, and HNF-3 beta/FoxA2 combined with low levels of SOX7 indicate definitive endoderm differentiation.
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Differentiation of Human Amniotic Fluid Stem Cells into Definitive Endoderm.
The StemXVivo®Endoderm Kit (Catalog # SC019) was used to differentiate first-trimester human amniotic fluid stem cells (AFSCs) into definitive endoderm by culturing AFSCs in media containing recombinant human FGF basic, recombinant human Wnt-3a, and recombinant human Activin A for one day. On day 2, the media was removed and replaced with fresh media containing recombinant human FGF basic and recombinant human Activin A. The media was replaced twice daily until day 5 when cells were analyzed for expression of definitive endoderm markers.A.Before differentiation (day 0) AFSCs express the extra-embryonic endoderm marker SOX7, but not the definitive endoderm markers SOX17, HNF-3 beta, CXCR4, and GSC, as determined by quantitative real-time RT-PCR. After differentiation (day 5) into definitive endoderm, expression of SOX17, HNF-3 beta, CXCR4, and GSC was detected, but not expression of SOX7.***P< 0.001.B.Before differentiation (day 0) AFSCs fail to express the definitive endoderm marker CXCR4 as assessed by flow cytometry. However, after differentiation (day 5) the cells express CXCR4 (isotype control in black).***P< 0.001.
Adapted from: Moschidou, D. et al. (2012) Mol. Therapy 20: 1953-1967
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Expression of SOX17 and HNF-3 beta in Human Amniotic Fluid Stem Cells Differentiated into Definitive Endoderm.
The StemXVivo®Endoderm Kit (Catalog # SC019) was used to differentiate first-trimester human amniotic fluid stem cells (AFSCs) into definitive endoderm by culturing AFSCs in media containing recombinant human FGF basic, recombinant human Wnt-3a, and recombinant human Activin A for one day. On day 2, the media was removed and replaced with fresh media containing recombinant human FGF basic and recombinant human Activin A. The media was replaced twice daily until day 5 when cells were analyzed for expression of definitive endoderm markers. Prior to differentiation (day 0), AFSCs show little to no expression of the definitive endoderm markers SOX17 and HNF-3 beta as assessed by confocal microscopy. However, after differentiation (Day 5), cells express both SOX17 and HNF-3 beta. The nuclei were stained with DAPI (blue). Scale Bar, 50 µm.
Adapted from: Moschidou, D. et al. (2012) Mol. Therapy 20: 1953-1967
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human pluripotent stem cells are differentiated into endoderm cells using the following endoderm differentiation procedure:
- Plate cells on coated plates
- Replace MEF Conditioned Media with Differentiation Media
- Evaluate differentiation status using the included SOX17 antibody
- Cells are ready for downstream applications on Day 4
Reagents Provided
Reagents supplied in the StemXVivo® Endoderm Kit (Catalog # SC019B):
- Endoderm Base Media Supplement (50x)
- Recombinant Human Activin A
- Recombinant Human FGF Basic
- Recombinant Human Wnt-3a
- Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
Other Supplies Required
Reagents
- Human pluripotent stem cells
- RPMI Medium 1640
- DMEM/F-12
- GlutaMAX™ (Invitrogen), or equivalent
- Penicillin-Streptomycin
- Phosphate-Buffered Saline (PBS)
- Cultrex® Basement Membrane Extract Reduced Growth Factor, PathClear®(Catalog # 3433-005-01) or StemXVivo® Culture Matrix (Catalog # CCM013)
- MEF Conditioned Media (Catalog # AR005)
- Trypan Blue Stain
- Accutase® (Innovative Cell Technologies), or equivalent
- BSA, very low endotoxin
- Sterile, deionized water
Materials
- 60 mm tissue culture dishes
- 24-well culture plates
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 0.2 mm syringe filter
- 0.2 mm, 500 mL filter units
- 10 mL syringes
- Serological pipettes
- Pipettes and pipette tips
Equipment
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- Centrifuge
- Hemocytometer
- Inverted microscope
Procedure Overview
This protocol is designed for BG01V human embryonic stem cells grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005). If using different cell lines or growth media, this protocol may need to be optimized.
Coat wells with Cultrex® Basement Membrane Extract (Catalog # 3432-005-01) or StemXVivo® Culture Matrix (Catalog # CCM013).
Incubate at room temperature for 1-2 hours.
Plate BG01V human embryonic stem cells onto the coated plates at 3.3 x 103 cells/cm2 in MEF Media containing FGF basic.
Culture the cells overnight at 37 °C and 5% CO2. The next day each well should contain a tightly packed monolayer of cells
Day 1 of Differentiation
Remove the MEF Conditioned Media from the wells 12-24 hours after initial plating.
Add fresh MEF Conditioned Media containing FGF basic.
Incubate at 37 °C and 5% CO2 for a minimum of 2-4 hours prior to adding Differentiation Media I.
Remove the MEF Conditioned Media from each well.
Wash each well once with once with 1X PBS.
Add Differentiation Media I to each well and incubate overnight at 37 °C and 5% CO2.
Day 2 and 3 of Differentiation
Approximately 12-16 hours after adding Differentiation Media I, replace the media with Differentiation Media II.
Incubate Continue to replace Differentiation Media II every 8-12 hours.
Day 4 of Differentiation
On Day 4, the cells are ready for further differentiation to downstream cell types or analysis by immunocytochemistry and/or flow cytometry.
Citations for StemXVivo Endoderm Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 5
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A Mutant Methionyl-tRNA Synthetase-Based toolkit to assess induced-Mesenchymal Stromal Cell secretome in mixed-culture disease models
Authors: Burgess, JD;Amerna, D;Norton, ES;Parsons, TM;Iii, RBP;Faroqi, AH;Wszolek, ZK;Cazares, HG;Kanekiyo, T;Delenclos, M;McLean, PJ;
Research square 2023-05-03
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Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties
Authors: MA Fayaz, GDS Rosa, A Honaramooz
Cells, 2021-10-20;10(11):. 2021-10-20
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Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function
Authors: MJ Munro, L Peng, SK Wickremese, ST Tan
PLoS ONE, 2020-05-19;15(5):e0232934. 2020-05-19
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Generation of an induced pluripotent stem cell cohort suitable to investigate sporadic Alzheimer's Disease
Authors: DC Schöndorf, M Elschami, M Schieck, E Ercan-Herb, C Weber, Y Riesinger, S Kalman, D Steinemann, DE Ehrnhoefer
Stem Cell Res, 2018-12-05;34(0):101351. 2018-12-05
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Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach.
Authors: Moschidou D, Mukherjee S
Mol. Ther., 2012-07-03;0(0):. 2012-07-03
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