STEMdiff™ Definitive Endoderm Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Prior to differentiation using STEMdiff™ Definitive Endoderm, maintain the cells in their pluripotent state by culturing mTeSR™1 on Matrigel. -Matrix-coated plates should be prepared in advance and be brought to room temperature (15 - 25°C) for at least 30 minutes prior to use.	Differentiate the cells for 5 days using STEMdiff™ Definitive Endoderm media. -For optimal differentiation performance, cells must be exposed to complete TeSR™-E8™ Pre-Differentiation Medium for at least 24 hours before the next passaging step.

Upstream tips
Prior to differentiation using STEMdiff™ Definitive Endoderm, maintain the cells in their pluripotent state by culturing mTeSR™1 on Matrigel. -Matrix-coated plates should be prepared in advance and be brought to room temperature (15 - 25°C) for at least 30 minutes prior to use.
Protocol tips
Differentiate the cells for 5 days using STEMdiff™ Definitive Endoderm media. -For optimal differentiation performance, cells must be exposed to complete TeSR™-E8™ Pre-Differentiation Medium for at least 24 hours before the next passaging step.

Publication protocol

Ectoderm: hESCs were differentiated into neuroectoderm cells using the dual SMAD inhibition method. Briefly, hESCs were dissociated into single cells using Accutase and plated onto Matrigel-coated plates with ROCK inhibitor (Y-27632, final concentration 10 μM) to reach approximately 90% confluence on the next day. Differentiation was induced with SRM (DMEM/F12, 15% knockout serum replacement (KOSR), and 1% GlutaMax) supplemented with 10 μM SB431542 and 200nM Noggin for five to seven days.
Mesoderm: Differentiation of hESCs into mesoderm was performed as described in the literature (Lian et al., 2010) with some modifications. Briefly, hESCs were dissociated into single cells using Accutase and plated onto Matrigel-coated plates with ROCK inhibitor (Y-27632) to reach approximately 30% confluence on the next day. Differentiation was initiated with RPMI/B-27 (RPMI/1640, 2% B-27 (minus insulin), 0.5% GlutaMax, and 0.5% non-essential amino acid) supplemented with 10 μM CHIR-98014 for four days, with daily medium replenishment.

Endoderm: hESCs were dissociated into single cells using Accutase and plated onto Matrigel-coated plates with ROCK inhibitor (Y-27632) to reach approximately 90% confluence. The next day, differentiation was induced with RPMI/B-27 (RPMI/1640, 2% B-27, 0.5% GlutaMax, and 0.5% non-essential amino acid) supplemented with 100ng/ml Activin-A and 3 μM CHIR99021 for one day and then with 0.25ng/ml BMP4, 5ng/ml bFGF and 100ng/ml Activin-A for three more days. Alternatively, very consistent and reproducible endoderm could also be generated using the STEMdiff Definitive Endoderm Kit following the manufacturer’s instructions.

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Manufacturer protocol

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