PSC Definitive Endoderm Induction Kit - FAQs

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.

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

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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.

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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.

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

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

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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.

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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.

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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.

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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.

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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.

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

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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.

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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.

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No other components are required.

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

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No. The formulation is animal origin-free.

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

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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.

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

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

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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.

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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.

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

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

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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.

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No. These media are not available separately.

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

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The formulation (components and concentrations) is proprietary.

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

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

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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.

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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.

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

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

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

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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.

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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.

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

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).

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

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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.

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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.

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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).

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