Human Pluripotent Stem Cell Functional Identification Kit
R&D Systems, part of Bio-Techne | Catalog # SC027B
Key Product Details
Summary for Human Pluripotent Stem Cell Functional Identification Kit
Kit Summary
To verify pluripotency in human stem cells by in vitro functional differentiation.
Key Benefits
- Verifies the pluripotency of your starting population
- Only takes 5 days
- Reduces experimental variation
Why functionally verify human stem cell pluripotency in vitro?
To determine if a cell is truly a pluripotent stem cell, it is important to verify its ability to differentiate into each of the three germ layers.
The teratoma assay is a standard way to assess pluripotency, but this method requires expensive animal models and is time-consuming, often requiring 6 to 10 weeks before pluripotency status can be determined. Similarly, embryoid body assays are time-consuming and rely on random differentiation.
- Uses fully defined supplements to drive reproducible differentiation.
- Provides results in 5 days to save time and reagents.
- Avoids tissue embedding and sectioning procedures.
- Can verify the ability to differentiate into 3 germ layers without the use of an animal model.
- Verifies a healthy, pluripotent starting stem cell population to increase consistency between studies and reduce unwanted experimental variability.
Kit Contents
This kit contains the following specially formulated media supplements and growth factors to drive pluripotent stem cell differentiation and a marker to characterize each of the three germ layer cell types.
- Differentiation Base Media Supplement (50X)
- Ectoderm, Mesoderm, and Endoderm Differentiation Supplements
- Ectoderm Marker: Goat Anti-Human Otx2 Antigen Affinity-purified Polyclonal Antibody
- Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal AntibodyV
- Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
The quantity of each component in this kit is sufficient to make 200 mL of media for differentiation. This is enough media for the differentiation of one 24-well plate of each cell type.
The acute and chronic effects of over-exposure to the reagents in this kit are unknown. Safe laboratory handling procedures should be followed and protective clothing should be worn when handling kit reagents.
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.
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Verification of Induced Pluripotent Stem Cell Pluripotency. iPS2 human induced pluripotent stem cells were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). The kit also contains Goat Anti-Human Otx2 (ectoderm), Goat Anti-Human Brachyury (mesoderm), and Goat Anti-Human SOX17 (endoderm) Antigen Affinity-purified Polyclonal Antibodies for the confirmation of differentiation status. To further evaluate lineage commitment, cells were stained with a Goat Anti-Human SOX1 Antigen Affinity-purified Antibody (Catalog # AF3369), a Goat Anti-Human HAND1 Antigen Affinity-purified Antibody (Catalog # AF3168), and a Goat Anti-Human HNF-3 beta/FoxA2 Antigen Affinity-purified Antibody (Catalog # AF2400). The cells were stained using the NorthernLights™557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips. |
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Analysis of Functional Tri-lineage Differentiation using Flow Cytometry. JOY6 human induced pluripotent stem cells were differentiated to ectoderm, mesoderm, and endoderm using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC027B). Differentiation was analyzed by flow cytometry using the protocol provided with this kit. Differentiated cells (orange) show increased expression of their respective lineage-specific markers (ectoderm - Otx2, mesoderm - Brachyury, endoderm - SOX17) compared to undifferentiated JOY6 pluripotent stem cells (blue). |
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Verification of Human Embryonic Stem Cell Pluripotency.
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Verification of Human Induced Pluripotent Stem Cell Pluripotency. Two human induced pluripotent stem cells lines (ADLF1 and FAB2) were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). The kit also contains Goat Anti-Human Otx2 (ectoderm), Goat Anti-Human Brachyury (mesoderm), and Goat Anti-Human SOX17 (endoderm) Antigen Affinity-purified Polyclonal Antibodies for the confirmation of differentiation status. The cells were stained using the NorthernLights™ 557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (blue). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, the pluripotency status of human stem cells is verified using the following in vitro differentiation procedure:
- Culture pluripotent cells of interest
- Induce endoderm, mesoderm, and ectoderm differentiation with media supplements
- Evaluate differentiation using germ layer markers and fluorescent ICC
Kit Components
Reagents supplied in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B):
- Differentiation Base Media Supplement (50X)
- Ectoderm Differentiation Supplement
- Mesoderm Differentiation Supplement
- Endoderm Differentiation Supplement I
- Endoderm Differentiation Supplement II
- Ectoderm Marker: Goat Anti-Human Otx2 Antigen Affinity-purified Polyclonal Antibody
- Mesoderm Marker: Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody
- Endoderm Marker: Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody
Note: The quantity of each component in this kit is sufficient to make 200 mL of medium for differentiation. This is enough medium for the differentiation of one 24-well plate for each lineage.
The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into hepatocyte-like cells.
Other Supplies Required
Reagents
- RPMI
- BSA, very low endotoxin
- D-MEM/F-12 (1X)
- GlutaMAX™ (Invitrogen or equivalent)
- Penicillin-Streptomycin
- Phosphate Buffered Saline (PBS)
- Trypan Blue Solution
- MEF Conditioned Media (Catalog # AR005 or equivalent)
- StemXVivo™ Culture Matrix (100X) (Catalog # CCM013), Cultrex® PathClear® BME Reduced Growth Factor Basement Membrane Extract (Catalog # 3433-005-01), or equivalent
- Recombinant Human FGF basic (Tissue culture grade; Catalog # 4114-TC or equivalent)
- Accutase® (Innovative Cell Technologies or equivalent)
Materials
- Human pluripotent stem cells
- 24-well culture plates
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 0.2 μm syringe filter
- 10 mL syringe
- Pipettes and pipette tips
- Serological pipettes
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Inverted microscope
- 37 °C water bath
Procedure Overview
This protocol is designed for BG01V human embryonic stem (hES) cells and iPS2 human induced pluripotent stem (iPS) 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.
Note: The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.
Undifferentiated Cell Preparation
Add 1:100 Culture Matrix or Cultrex BME in sterile PBS.
Add 1.1x105 cells/cm2 in MEF Conditioned Media containing 4 ng/mL FGF Basic.
Differentiation Procedures
| Ectoderm | Mesoderm | Endoderm | |
| Day 1 | Replace media with Ectoderm Differentiation Media. | Replace media with Mesoderm Differentiation Media. | Replace media with Endoderm Differentiation Media. |
| Day 2 | Repeat | Repeat media change 12-16 hours later. ICC detection of Brachyury (24-36 hours after initial differentiation) | 16-24 hours later, Replace media with Endoderm Differentiation Media II. |
| Day 3 | Repeat | Replace media with Endoderm Differentiation Media II. | |
| Day 4 | ICC detection of Otx2. | ICC detection of SOX17. |
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