Stemdiff definitive endoderm kit

iPSCs were differentiated to iHeps using previously established protocols.¹⁷ (link)^,23 (link) Differentiating iHeps were maintained in a 5% CO₂, 5% O₂, 90% N₂ environment. iPSCs were dissociated to a single-cell suspension using Gentle Cell and replated at a density of 1 × 10⁶ cells. Twenty-four hours later, cells were differentiated to definitive endoderm for 4 days using the StemDiff Definitive Endoderm Kit (StemCell Technologies, catalog #05110), as per manufacturer’s instructions. On D5 of the protocol, definitive endoderm cells were dissociated with Gentle Cell to determine endoderm efficiency by flow cytometry and replated in hepatocyte differentiation media using stage-specific growth factors for directed differentiation to hepatocytes.¹⁷ (link) A detailed protocol for the hepatocyte differentiation is available for free download at: http://www.bu.edu/dbin/stemcells/protocols.php.

iPSC directed differentiation to the hepatic lineage was performed using our previously published protocol.^{21 (link),25 (link)} Briefly, undifferentiated iPSCs were cultured until confluent then passaged at Day 0 using GCDR, replated at 1 × 10⁶ cells per well of a Matrigel coated 6 well plate, and placed into hypoxic conditions (5% O2, 5%CO₂, 90%N₂) for the remainder of the differentiation. They were patterned into definitive endoderm using the STEMdiff Definitive Endoderm Kit per manufacturer’s instructions over 5 days (StemCell Technologies). Cells were passaged on day 5 of differentiation using GCDR, with endoderm efficiency confirmed via cell surface staining for CXCR4 and cKit, and endoderm subsequently grown in serum free base media supplemented with stage specific growth factors to specify the hepatic lineage and induce maturation (Figure 1B). Detailed protocols for derivation of iPSC-hepatocytes are available for free download at: https://crem.bu.edu/cores-protocols/.

Differentiation of hiPSCs in 2D monolayer cultures (Fig. 1A) was performed according to Szkolnicka et al (11 (link)) with minor changes. In detail, hiPSCs were plated onto Matrigel-coated [1:20 diluted in Dulbecco's modified Eagle's medium (DMEM)/F12 medium] 24-well plates and differentiated into definitive endodermal (DE) cells using the STEMdiff™ Definitive Endoderm kit (Stemcell Technologies, Inc.) according to the manufacturer's instructions until day 5. From day 5 to 8 cultures were maintained in SR-DMSO-Medium [knockout DMEM supplemented with 20% knockout serum replacement medium, 0.5% GlutaMAX, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, DMSO (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin]. From day 9 on, hepatocyte maturation medium [HepatoZYME-SFM, 1% GlutaMAX, 1% penicillin/streptomycin, 10 µM hydrocortisone 21-hemisuccinate sodium salt (Sigma-Aldrich; Merck KGaA), 10 ng/ml human HGF and 20 ng/ml human OSM (both from PeproTech EC Ltd., London, UK)] was used and renewed every other day. All reagents were purchased from Thermo Fisher Scientific, Inc., if not stated otherwise.

DE induction was performed using the STEMdiff Definitive Endoderm Kit from STEMCELL Technologies according to the manufacturer's protocol with small modifications. On the day before DE induction (day-1), culture of the OARSAi002-A iPSC line was dissociated into single cells using TrypLE Select Enzyme (1X). Collected cells were resuspended into Cellartis DEF-CS medium supplemented with GF-1, GF-2, and GF-3 and seeded onto a 6-well tissue culture plate pre-coated with LDEV-free, hESC-qualified Matrigel (Corning Inc.) at a cell density of 2.1x10⁵ cells/cm². The plate was incubated at 37˚C and supplied with 5% CO₂, and the cells reached ~90-100% confluence by the following day. On day 0, cells were washed once with DMEM/F12 (Thermo Fisher Scientific, Inc.) and replaced with STEMdiff Endoderm basal medium with 1X supplement MR and 1X supplement CJ. Cells were then incubated with STEMdiff Endoderm basal medium with 1X supplement CJ on days 1-3, with medium replenished daily. On day 4, the protein expression of key DE markers, SOX17 and forkhead box protein A2 (FOXA2), were evaluated by immunocytochemistry staining on 4% paraformaldehyde (PFA)-fixed cells using SOX17 mouse anti-human monoclonal antibody (Abcam) and FOXA2 rabbit anti-human monoclonal antibody (Abcam), as described below.