StemXVivo Endoderm Kit

Name: StemXVivo Endoderm Kit
Brand: R&D Systems
SKU: SC019B

R&D Systems, part of Bio-Techne | Catalog # SC019B

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Product Datasheet / COA / SDS

For endoderm differentiation of human pluripotent stem cells

Key Product Details

Summary for StemXVivo Endoderm Kit

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.

Kit Contents

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

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Product Specifications for StemXVivo Endoderm Kit

Species	Human
Source	N/A

Scientific Data Examples for StemXVivo Endoderm Kit

	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).
	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 IgG_2BIsotype 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.
	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
	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

Preparation & Storage

Shipping Conditions	The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage	Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

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

View Graphic Protocol

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.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 1

Plate BG01V human embryonic stem cells onto the coated plates at 3.3 x 10³ cells/cm² in MEF Media containing FGF basic.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 2

Culture the cells overnight at 37 °C and 5% CO₂. The next day each well should contain a tightly packed monolayer of cells

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 3

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.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 4

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.

Protocol for Human Monocyte-derived Dendritic Cell Differentiation Step 5

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.

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StemXVivo Endoderm Kit