Definitive Endoderm Differentiation

With the Gibco PSC Definitive Endoderm Induction Kit, you can expect:

• Fast, efficient differentiation: Generate definitive endoderm cells from PSCs in just 48 hours with a streamlined workflow.
• Ready-to-use formulation: Two fully optimized media help eliminate the need for component mixing or additional supplements.
• Accelerated workflows: Produce high-quality, reproducible cells up to 50% faster than many published protocols.
• High-purity output: Achieve ≥90% expression of key endoderm markers SOX17 and FOXA2 across multiple PSC lines.
• Downstream functionality: Obtain functional cells capable of differentiating into hepatic, pancreatic, and other endoderm-derived lineages.
• Validated data: See the proof

Generating definitive endoderm cells from PSCs is a crucial step before cells can be differentiated into downstream endoderm lineages. Why not get through that step faster? Compared to other differentiation protocols*, the PSC Definitive Endoderm Induction Kit produces cells in up to 50% less time and requires no predifferentiation or mixing of media.

Cells generated using the PSC Definitive Endoderm Induction Kit show ≥90% CXCR4⁺/PDGFRα^– across multiple cell lines and express the key markers Sox17 and FoxA2.

Figure 1. Definitive endoderm CXCR4⁺/PDGFRα^– cells derived from stem cells. The PSC Definitive Endoderm Induction Kit media produce definitive endoderm populations with high efficiency (>90%) across hESC and iPSC cell lines, including episomal and CytoTune reprogrammed lines. Representative dot plots show CXCR4⁺/PDGFRα^– cell populations. For each experiment, unstained cells were used to set quadrant gates.

Figure 2. Immunocytochemistry of hESCs treated with PSC Definitive Endoderm Induction Kit media. At day 3, induced cells were immunostained for the endodermal transcription factors Sox17 and FoxA2, and the pluripotent marker Oct4. Nuclei were counterstained with DAPI (blue) to assess total cell numbers.

Functional cells generated using this kit are capable of differentiating into midgut/hindgut, pancreatic endoderm, or liver bud progenitors that express relevant physiological markers. Data credited to L.T. Ang, K.M. Loh, and B. Lim of the Genome Institute of Singapore.

Immunocytochemistry (10x) of H1 ESCs treated with PSC Definitive Endoderm Induction Kit media and differentiated into liver bud progenitors. Cells were stained for (A) nuclei, (B) AFP, and (C) merged.

• H9 ESCs were seeded at 20% density on Matrigel coated 6-well plates in Essential 8 (E8) Medium containing RevitaCell according to Gibco PSC Definitive Endoderm Induction Kit (Gibco DE kit) product insert.
• To induce definitive endoderm, medium was changed to DE kit Medium A on Day 1 and DE kit Medium B on Day 2 after seeding.
• On Day 3, the hepatoblast specification protocol was followed according to each protocol and continued through the end of hepatocyte specification.
• Morphology images were captured daily. Protein and RNA was collected from H9 cells cultured in E8 for 3 days, DE induced cells on Day 3, and at the end of each protocol’s hepatocyte specification protocol.

Hepatic Differentiation Protocol 1: Szkolnicka D, Farnworth SL, Lucendo-Villarin B, et al. (2014) Deriving functional hepatocytes from pluripotent stem cells. Curr Protoc Stem Cell Biol 30:1G.5.1–1G.5.12 
Hepatic Differentiation Protocol 2: Hannan NR, Segeritz CP, Touboul T, et al. (2013) Production of hepatocyte-like cells from human pluripotent stem cells. Nat Protoc 8(2):430–437. 

Representative data showing utility of Gibco DE upstream of hESC differentiation to hepatocytes using two different published methods.

A. Representative images of hESC H9 cells cultured for 3 days in E8 medium, H9 cells cultured and treated with Gibco DE kit at day 3, and hepatocyte-like cells generated downstream of DE at the end of differentiation protocol for each method tested. B. qRT-PCR analysis of the above cells to determine their stem-ness (Nanog, Oct3/4), induction to definitive endoderm (CXCR4, FOXA2) and finally expression of hepatic markers (AFP and Albumin).

1. Efficiently reprogram somatic cells to pluripotent stem cells (PSCs) using the CytoTune-iPS 2.0 Sendai Reprogramming Kit.
2. Culture and expand PSCs feeder-free with Essential 8 Medium.
3. Induce definitive endoderm in 2 days using the PSC Definitive Endoderm Induction Kit.
4. Differentiate to the desired terminal lineage.
   
   

* Protocol provided for the STEMdiff™ Definitive Endoderm Kit is an estimate based on the published web protocol.