Gibco™ PSC Definitive Endoderm Induction Kit

Catalog No.	Quantity
A3062601	1 kit

Catalog No. A3062601 Supplier Gibco™ Supplier No. A3062601

Description

Gibco™ PSC Definitive Endoderm Induction Kit consists of two xeno-free media that enable efficient induction of human pluripotent stem cells (PSCs) to definitive endoderm.

Gibco™ PSC Definitive Endoderm Induction Kit consists of two xeno-free media that enable efficient induction of human pluripotent stem cells (PSCs) to definitive endoderm. Unlike other methods that require multiple components and take five or more days, the PSC Definitive Endoderm Induction Kit can generate ≥90% CXCR4+/PDGFRα- definitive endoderm cells with only two components in just two days. Each medium is supplied as a 1X complete medium, requiring no mixing in of additional components, and the resultant definitive endoderm is capable of differentiating into midgut/hindgut, pancreatic endoderm, or liver bud progenitors that express relevant physiological markers.

Generation of high quality definitive endodermDefinitive endoderm generated using the PSC Definitive Endoderm Induction Kit is capable of differentiating into downstream lineages such as midgut/hindgut (expressing CDX2), pancreatic endoderm (expressing PDX2), and liver bud progenitors (expressing AFP).Rapid production of definitive endodermThe PSC Definitive Endoderm Induction Kit is capable of producing definitive endoderm in only two days.Generation of highly pure definitive endodermThe PSC Definitive Endoderm Induction Kit is capable of producing highly pure populations of definitive endoderm from pluripotent stem cells, showing ≥90% CXCR4+/PDGFRα- across multiple lines and high expression of key markers SOX17 and FOXA2.No mixing in of components requiredEach medium is supplied at a 1X concentration, requiring no mixing in of reagents. Simply thaw the media and add to PSC culture.

Specifications

• Cell Type: Stem Cells (Human)
• Content And Storage: • PSC Definitive Endoderm Induction Medium A
  • PSC Definitive Endoderm Induction Medium B
• Product Type: PSC Definitive Endoderm Induction Kit
• Quantity: 1 kit
• Shipping Condition: Dry Ice

Frequently Asked Questions (FAQs)

Can I cryopreserve definitive endoderm cells?

We don't recommend cryopreserving cells at the definitive endoderm stage. After definitive endoderm induction is complete, we recommend using the cells either for characterization or immediately starting downstream differentiation.

Can these cells be maintained in definitive endoderm medium?

No. Once definitive endoderm cells are induced, we recommend using the cells either for characterization or immediately starting downstream differentiation.

How can I characterize definitive endoderm cells?

Definitive endoderm cells generated using the PSC Definitive Endoderm Induction kit can be characterized by FACS or immunocytochemistry by looking at high expression of endoderm markers, including CXCR4, SOX17, and FOXA2.

I see floating cells after addition of medium B when using the PSC Definitive Endoderm Induction Kit. Is that normal?

It is normal to see floating cells after addition of medium B. Floating cells are not detrimental to definitive endoderm efficiency. Higher confluency (>15-30%) at the time of induction will generally lead to higher number of floating cells.

Do I have to incubate cells for exactly 24 hours in medium A or medium B when using the PSC Definitive Endoderm Induction Kit?

If necessary, cells can be incubated for 24 +/-3h.

Will the definitive endoderm cells divide in culture?

No. After the 2-day induction process, Definitive Endoderm cells will not divide further in culture.

Do I need to rinse cells between Definitive Endoderm Induction Medium A to Definitive Endoderm Induction Medium B switch?

No. It is not necessary to rinse cells when changing from Definitive Endoderm Induction Medium A to Definitive Endoderm Induction Medium B. However, Medium A should be completely removed before switching to Medium B.

At what confluency of PSCs should I begin induction?

We recommend starting with 15-30% confluent PSCs for induction. Below are examples showing optimum confluency before induction (24 h after seeding). Induction at higher starting confluency will lead to higher number of floating cells.

At what seeding density should I seed PSCs prior to differentiation?

The optimum range for seeding density is 0.01 × 10e6-0.04 x 10e6 cells/cm2. For example seed 0.09 x 10e6 - 0.38 x 10e6 cells per well of a 6-well plate.

I use TesR Medium as a PSC culture medium. Is PSC Definitive Endoderm Induction Medium compatible with this medium?

Optimal results are obtained by using Essential 8 Medium as a pluripotent stem cell medium, however mTesR1 or TeSR-E8 can be used as substitute, if desired.

What is the recommended method to seed PSC to start Definitive Endoderm Induction?

When seeding PSCs for Definitive Endoderm induction, cells should be plated as very small clumps using Accutase Reagent. They can also be seeded as singularized cells using TrypLE Reagent. To promote cell survival, you can treat the cells overnight with ROCK inhibitors including RevitaCell Supplement , Y27632, or Thiazovivin.

Can I use PSCs directly from thaw or do I need to allow them to recover when using the PSC Definitive Endoderm Induction Kit?

It is recommended to expand cells through at least one passage before seeding cells for definitive endoderm induction.

What dissociation reagent should I use with the PSC Definitive Endoderm Induction Kit?

For passaging PSCs we recommend EDTA, and for seeding cells for definitive endoderm induction we recommend Accutase Enzyme for small clumps and TrypLE Reagent for singularized cells.

What are the critical parameters in my starting PSCs that influence the differentiation outcome?

It is critical that human PSCs are high quality, karyotypically normal, exhibit pluripotency markers, and kept under passage 100. Upon thaw, culture cells for at least one passage before seeding for definitive endoderm induction.

Are there any additional components that need to be added to the media of the PSC Definitive Endoderm Induction Kit?

No other components are required.

What plate formats have been tested for PSC Definitive Endoderm Induction Medium?

We typically use 6-well plate format; however, other standard formats can also be used.

Are there any animal origin components in the PSC Definitive Endoderm Induction media?

No. The formulation is animal origin-free.

Do the PSC Definitive Endoderm Induction media need to be filtered prior to use?

The media are aseptically prepared and sterile filtered so they do not need to be filtered prior to use.

What substrate should I use with the PSC Definitive Endoderm Induction Kit?

We recommend using Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700), but Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301 or Cat. No. A1413302) can also be used.

Which specific lineages can be obtained from definitive endoderm cells generated using PSC Definitive Endoderm Induction Kit?

PSC Definitive Endoderm Induction kit can be used to generate a wide range of definitive endoderm-derived cells including mid/hindgut, liver, and pancreas.

Can the PSC Definitive Endoderm Induction Kit be used with species other than human?

Preliminary data have shown 82% Sox17 expression when the kit is used with mouse ESCs.

How does this media compare to published protocols?

PSC Definitive Endoderm Induction Medium is highly efficient, simpler (less steps and less components), and quicker (less time to definitive endoderm) than many published methods.

Which stem cell lines has the PSC Definitive Endoderm Induction Kit been tested with?

This product has been tested across multiple hESC and iPSC cell lines. hESC cell lines tested include H9, HES2 and HES3; and iPSC cell lines tested include those derived from either CD34+ cord blood or from fibroblasts reprogrammed by CytoTune-iPS Sendai or CytoTune-iPS 2.0 Sendai or by mRNA transfection.

What markers can I expect my definitive endoderm cells to express?

Definitive endoderm cells generated using PSC Definitive Endoderm Induction kit are positive for CXCR4, Sox 17, FoxA2, and are negative for PDGFRalpha.

What quality controls are used to ensure product performance for the PSC Definitive Endoderm Induction Kit?

Each lot is tested for % efficiency of definitive endoderm induction using flow cytometry analysis. Additionally, it is tested for sterility, endotoxin, pH, and osmolality.

How many definitive endoderm cells can I produce from my starting population of PSCs?

Approximately 8-fold increase in cell number can be expected. For example, if you seed 0.09 x 10e6 H9-ESC cells per well of a 6-well plate, you may expect 0.8 x 10e6 cells by day 3. Yield may vary depending upon the starting PSC cell line.

Can I purchase either Definitive Endoderm Induction Medium A or Definitive Endoderm Induction Medium B separately?

No. These media are not available separately.

What is the purpose of each individual medium in Definitive Endoderm Induction Kit?

Definitive Endoderm Induction Medium A: Pushes PSCs toward Anterior Primitive Streak (APS) fate
Definitive Endoderm Induction Medium B: Induces definitive endoderm formation.

What is the formulation of the medium in the PSC Definitive Endoderm Induction Kit?

The formulation (components and concentrations) is proprietary.

Once thawed, can the media be refrozen?

Each medium can be thawed, aliquoted, and refrozen once without affecting the performance.

What is the shelf life of thawed media in the PSC Definitive Endoderm Induction Kit?

Thawed medium can be stored at 2-8 degrees C for up to two weeks.

How should the media be thawed from the PSC Definitive Endoderm Induction Kit?

Each 50 mL media bottle can be thawed at 4 degrees C overnight, at room temperature (15-25 degrees C) for approximately 2 hours, or at 37 degrees C for approximiately 20 minutes.

What is the shelf life of this medium in the PSC Definitive Endoderm Induction Kit?

Shelf life for this product has not yet been established. However, in preliminary experiments, the product is shown to be stable for at least 6 months from time of manufacture when properly stored at -20 degrees C.

How is the PSC Definitive Endoderm Induction Kit stored?

PSC Definitive Endoderm Induction Kit is supplied as two bottles of frozen media. Each should be stored at -20 degrees C, protected from light.

How is PSC Definitive Endoderm Induction Kit shipped?

PSC Definitive Endoderm Induction Kit is shipped on dry ice. Upon receipt, store at -20 degrees C.

What is the format of the PSC Definitive Endoderm Induction Kit?

The kit is supplied as two 1X complete media (ready-to-use): PSC Definitive Endoderm Medium A and PSC Definitive Endoderm Medium B.

What are the benefits of using PSC Definitive Endoderm Induction Kit?

Unlike other methods that require multiple components and take five or more days, the PSC Definitive Endoderm Induction Kit can generate ?90% CXCR4+/PDGFRalpha- definitive endoderm cells with only two ready-to-use media in just two days. Each medium is supplied as a 1X complete medium, requiring no mixing of additional components, and resultant definitive endoderm is capable of differentiating to a wide range of definitive endoderm derived cells including mid/hindgut, liver, and pancreas.

What is PSC Definitive Endoderm Induction Kit?

PSC Definitive Endoderm Induction Kit consists of two animal origin-free media that enable efficient induction of human pluripotent stem cells (PSCs) to definitive endoderm.

What substrate should I use with the PSC Definitive Endoderm Induction Kit?

These two proteins are the same but have different protein concentrations due to the different methods used to measure the protein concentrations.

Is StemFlex Medium compatible with downstream differentiation kits available from Thermo Fisher Scientific?

Yes. We have seen compatibility with the following differentiation kits provided by Thermo Fisher Scientific: PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), PSC Definitive Endoderm Induction Kit (Cat. No. A3062601), PSC Neural Induction Medium (Cat. No. A1647801), and PSC Dopaminergic Neuron Differentiation Kit (Cat. No. A3147701).

I am using the Gibco PSC Definitive Endoderm Induction kit. Is it necessary to wash the cells between Medium A and Medium B?

No, it is not necessary to wash between Medium A and Medium B.

What is the purpose of each individual medium in the Gibco Definitive Endoderm Induction Kit?

The PSC Definitive Endoderm Induction Medium A pushes PSCs towards anterior primitive streak (APS) fate and the PSC Definitive Endoderm Induction Medium B induces definitive endoderm formation.

Which dissociation reagent should I use with the Gibco PSC Definitive Endoderm Induction Kit?

We recommend using EDTA for passaging PSCs. For seeding cells for definitive endoderm induction, we recommend using StemPro Accutase Cell Dissociation Reagent for small clumps and Gibco TrypLE Enzyme for singularized cells.

Can I use PSCs that are long-term cultured in Gibco Essential 8 Flex Medium for downstream differentiation?

We have seen no effect on downstream differentiation potential for cells cultured in Essential 8 Flex Medium for up to 15 passages. Tri-lineage potential has been demonstrated from embryoid bodies as well as using Gibco PSC Neural Induction Medium (Cat. No. A1647801), Gibco PSC Cardiomyocyte Differentiation Kit (Cat. No. A25042SA) and Gibco PSC Definitive Endoderm Induction Kit (Cat. No. A27654SA).