Protocol for differentiation of human pluripotent stem cells into definitive endoderm lineage in a chemically defined and growth factor-free system

Summary

Here, we present a protocol for the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) lineage. We describe steps for the resuscitation, passaging, and plating of hPSCs. We then detail procedures for culturing cells followed by immunostaining, imaging, and analysis. This chemically defined, small-molecule-based, recombinant protein-free system offers a cost-effective and scalable platform for generating endodermal derivatives, demonstrating efficiency for applications in drug screening, disease modeling, and regenerative medicine.

For complete details on the use and execution of this protocol, please refer to Zhao et al.¹

Graphical abstract

Highlights

• •Revive and passage human pluripotent stem cells (hPSCs)
• •Differentiate hPSCs into definitive endoderm (DE) via a chemically defined system
• •Validate DE differentiation by immunofluorescence staining

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) lineage. We describe steps for the resuscitation, passaging, and plating of hPSCs. We then detail procedures for culturing cells followed by immunostaining, imaging, and analysis. This chemically defined, small-molecule-based, recombinant protein-free system offers a cost-effective and scalable platform for generating endodermal derivatives, demonstrating efficiency for applications in drug screening, disease modeling, and regenerative medicine.

Before you begin

Institutional permissions (if applicable)

Human embryonic stem cells are commercially obtained and all relevant experiments were approved by the Ethical Committee of Sun Yat-sen University and comply with the 2021 Guidelines for Stem Cell Research and Clinical Translation (issued by the International Society for Stem Cell Research, ISSCR).

Preparations

Timing: 2–3 h

• 1.Culture all cells in a humidified incubator at 37°C with 5% CO₂. Prior to initiating each step, prepare and pre-warm all required medium to 37°C for 20 min. Please refer to key resources table for a comprehensive list of reagents and resources. Please refer to materials and equipment for detailed medium formulations.
• 2.
  Preparation of Matrigel:

  • a.
    Store undiluted Matrigel matrix at −20°C in a non-frost-free freezer.
  • b.
    Thaw Matrigel matrix vials overnight in ice at 2°C to 8°C. After thawing, gently swirl the solution on ice to ensure homogeneous distribution.
  • c.
    Aliquot 200 μL into pre-chilled polypropylene tubes and store at −70°C or −20°C to avoid repeated freeze-thaw cycles.
  • d.
    For immediate use, dilute one aliquot with 20 mL ice-cold DMEM/F12 medium.

Note: Maintain Matrigel on ice throughout handling, as it begins gelling above 10°C. Therefore, keep Matrigel matrix on ice always during handling.

Note: Pre-chill all pipette tips and labware contacting Matrigel. Use polypropylene or other freezer-compatible tubes and store at −70°C or −20°C. Diluted Matrigel can be stored in a refrigerator at 4°C for up to one month.

• 3.
  Preparation of Vitronectin:

  • a.
    Store undiluted Vitronectin at −80°C in a non-frost-free freezer.
  • b.
    Thaw Vitronectin at 25°C for 5–10 min, then place on ice.
  • c.
    Vitronectin can be divided into usage-size aliquots in polypropylene tubes and stored at −60°C to −80°C.
  • d.
    For immediate use, dilute one aliquot (200 μL) with 20 mL DMEM/F12 medium, diluted Vitronectin solution can be stored in a refrigerator at 4°C up to 2 weeks

Note: 25°C or shaking might result in an appearance of light turbidity. This does not impact product performance.

Note: After thawing, gently swirl the solution on ice to ensure homogeneous distribution.

• 4.
  Preparation of Synthemax:

  • a.
    Add 10 mL DMEM/F12 medium to the vial of Synthemax II-SC substrate.
  • b.
    Pipet up and down several times, washing the walls of the vial, to ensure the complete reconstitution of the powder in water.
  • c.
    This will result in 1 mg/mL Synthemax II-SC stock solution.
  • d.
    Dilute Synthemax II-SC stock solution 1:40 in DMEM/F12 medium to achieve a final concentration of 0.025 mg/mL.

Note: Store Corning Synthemax II-SC stock solution at 4°C for up to 6 months.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
FoxA2/HNF3β (D56D6) XP rabbit mAb, dilution 1:200	Cell Signaling Technology	Cat#:8186; RRID: AB_10891055
Human SOX17 antibody, dilution 1:200	R&D Systems	Cat#AF1924; RRID: AB_355060
Mouse monoclonal anti-GATA4 (clone: G-4), dilution 1: 400	Santa Cruz Biotechnology	Cat#SC25310; RRID: AB_627667
GATA-6 (D61E4) XP rabbit mAb, dilution 1:200	Cell Signaling Technology	Cat# 5851; RRID: AB_10705521
CD184 (CXCR4) monoclonal antibody (12G5), APC, eBioscience	Thermo Fisher Scientific	Cat#17-9999-42; RRID: AB_1724113
Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488, dilution 1:300	Thermo Fisher Scientific	Cat#A11001; RRID: AB_2534069
Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488, dilution 1:300	Thermo Fisher Scientific	Cat#A11008; RRID: AB_143165
Donkey anti-goat IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 647, dilution 1:300	Thermo Fisher Scientific	Cat#A21447; RRID: AB_2535864
Donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 555, dilution 1:300	Thermo Fisher Scientific	Cat#A31572; RRID: AB_162543
DAPI (10 mg)	Sigma	Cat#D9542
Chemicals, peptides, and recombinant proteins
TeSR-E8 kit for hPSCs maintenance	STEMCELL Technologies	05990
DMEM/F12	Gibco	11320-033
Vitamin C (Vc)	Sigma	A8960
CHIR99021 (10 mM)	Selleck	S2924; CAS:1797989-42-4
Accutase	STEMCELL Technologies	07920
Y-27632 2HCI	Selleck	S1049
Matrigel	BD Biosciences	354277
Vitronectin	Gibco	CTS279S3
Synthemax II-SC substrate	Corning	3535
UltraPure 0.5 M EDTA	Thermo Fisher Scientific	AM9260G
Dulbecco’s phosphate-buffered saline without Ca2+ or Mg2+	Gibco	14190136
LDN193189 (10 mM)	Selleck	S7507
Phosphate-buffered saline (PBS)	Gibco	10010-049
Triton X-100	Sigma	Cat#93443
Bovine serum albumin (BSA)	Solarbio	Cat#A8010
4% paraformaldehyde (PFA)	MeilunBio	MA0192
Experimental models: Cell lines
Human embryonic stem cell (hESC) line H1	WiCell	N/A
hESC line H9	WiCell	N/A
Human induced pluripotent stem cells (hIPSC) WTB	Conklin lab, Gladstone/UCSF	N/A
hIPSC WTC	Conklin lab, Gladstone/UCSF	N/A
Software and algorithms
ImageJ	ImageJ	https://imagej.net/ij/
Prism 9.0	GraphPad	https://www.graphpad.com/
FlowJo	FlowJo	https://www.flowjo.com/solutions/flowjo
ZEN2.3 (blue edition)	Zeiss	https://www.zeiss.com/microscopy/zh/products/software/zen-starter.html
BioRender	BioRender	www.biorender.com
Other
0.22-mm Millex-GP filter unit	Millipore	SLGP033RB
Zeiss LSM780 confocal microscope	Zeiss	LSM780
CytoFLEX-S	Beckman Coulter	B78557
MycoAlert mycoplasma detection kit	Lonza	LT-07-418
6-well plates	ExCell Bio	CS016-0092

Materials and equipment

All medium is prepared using sterile techniques. We recommend that cell culture is performed in a sterile environment without the use of antibiotics.

4C-DE induction basal medium

Reagent	Final concentration	Amount
DMEM/F12	N/A	500 mL
Vc	71 μg/mL	35.5 g
Total	N/A	500 mL

Note: Vc is chemically stable under standard ambient conditions (room temperature).

4C-DE induction basal medium can be stored at 4°C for up to 1 month and pre-warmed at 37°C for at least 20 min prior to use.

4C-DE induction medium Ⅰ (day 0-1)

Reagent	Final concentration	Amount
DMEM/F12	N/A	500 mL
CHIR99021 (10 mM)	3 μM	150 μL
Vc	71 μg/mL	35.5 g
Total	N/A	500 mL

Note: CHIR99021 stock solution can be stored at −80°C for up to 6 months. For convenience, prepare 0.1 mL diluted aliquots and store them at −20°C for routine use. Avoid repeated freezing and thawing. Prepare fresh medium and filter-sterilize through a 0.22 μm filter before use.

To address potential inaccuracies in dispensing volumes of CHIR99021, an initial 10- to 100-fold dilution of the 10 mM stock solutions with DMSO is recommended to enhance pipetting precision.

4C-DE induction medium Ⅰ can be stored at 4°C for up to 1 month and pre-warmed at 37°C for at least 20 min prior to use.

4C-DE induction medium Ⅱ (day 1-3)

Reagent	Final concentration	Amount
DMEM/F12	N/A	500 mL
Vc	71 μg/mL	35.5 g
LDN193189 (10 mM)	0.1 μM	5 μL
Total	N/A	500 mL

Note: LDN193189 stock solution can be stored at −80°C for up to 6 months. For convenience, prepare 0.1 mL diluted aliquots and store them at −20°C for routine use. Avoid repeated freezing and thawing. Prepare fresh medium and filter-sterilize through a 0.22 μm filter before use.

To address potential inaccuracies in dispensing volumes of LDN193189, an initial 10- to 100-fold dilution of the 10 mM stock solutions with DMSO is recommended to enhance pipetting precision.

4C-DE induction medium Ⅱ can be stored at 4°C for up to 1 month and pre-warmed at 37°C for at least 20 min prior to use.

Step-by-step method details

Resuscitate human pluripotent stem cells

Timing: 4 days

This section details how to resuscitate and culture hPSCs followed by daily medium changes until they are ready for passaging.

• 1.Add 800 μL diluted Matrigel to each well of a 6-well plate and incubate at 37°C for 30 min, or add 1 mL of diluted Vitronectin and incubate at 25°C for 1 h (troubleshooting 4).

CRITICAL: Matrigel tends to solidify at low temperatures (−20°C – 4°C) and should be transferred from −20°C to 4°C in advance for slow thawing, avoiding repeated freeze-thaw cycles. Aspirate the diluted Vitronectin/Matrigel solution and discard immediately prior to use. Do not allow the culture surface to dry.

• 2.Rapidly thaw the frozen hPSCs at 37°C water bath (troubleshooting 5).

CRITICAL: Minimize freeze-thaw cycles to prevent cellular damage. Transfer thawed cells immediately to pre-warmed medium.

Note: The H1 cell line of human embryonic stem cells (hESCs) were thawed and revived in this protocol.

• 3.Resuspend thawed cells in a 15 mL centrifuge tube with equal volume of E8 medium, centrifuge at 200 × g at 25°C for 5 min. Aspirate supernatant and gently resuspend the cell pellet in 1 mL of E8 medium.

CRITICAL: Handle with care during resuspension to reduce mechanical damage that could lead to cell death.

• 4.
  Aspirate the coating solution from the culture plate:

  • a.
    Add 1 mL of E8 medium to each well, followed by 1 mL the cellular suspension.
  • b.
    Subsequently, add Y-27632 to achieve a final concentration of 5 μM.
  • c.
    Gently mix the contents to ensure uniform distribution of the components.
  • d.
    Incubate the plate at 37°C with 5% CO₂ (troubleshooting 4).
• 5.On the subsequent day, aspirate the medium and add 2 mL of fresh E8 medium per well. Refresh the medium daily thereafter, when cells reach 80–90% confluence, passage them.

Subculture and expand human pluripotent stem cells

Timing: 4 days

This section details how to subculture cells on Matrigel or Vitronectin-coated plates using EDTA or Accutase, centrifuge and reseed in E8 medium at the desired ratio.

• 6.Add 800 μL diluted Matrigel to each well of a 6-well plate and incubate at 37°C for 30 min, or add 1 mL of diluted Vitronectin and incubate at 25°C for 1 h (troubleshooting 4).
• 7.
  Cell passage:

  • a.
    Aspirate the medium from the culture wells.
  • b.
    Add 600 μL of 0.5 mM EDTA per well and incubate at 25°C for 4 min.
  • c.
    Aspirate the dissociation solution and add 1 mL E8 medium.
  • d.
    Gently pipette to resuspend cells and transfer the cellular suspension to a 15 mL centrifuge tube (Alternatively, 800 μL of Accutase per well can be used, with incubation at 37°C for 8 min. Subsequently, add 800 μL of DMEM/F12 to resuspend cells.) (troubleshooting 3).

CRITICAL: EDTA: ensuring precise timing to prevent incomplete detachment or excessive damage to the cells.

Accutase: Temperature-sensitive, requires precise control and is suitable for experiments with single-cell suspensions.

• 8.Centrifuge the cellular suspension at 200 × g at 25°C for 5 min. Carefully aspirate the supernatant and resuspend the cell pellet in 1 mL E8 medium. Seed cells at a splitting ratio of 1:3 to 1:10, depending on the experimental requirements.

CRITICAL: All cell lines should be confirmed to be mycoplasma-free by using the MycoAlert Mycoplasma Detection Kit.

Note: High ratio (1:10): Recommended for rapid expansion (e.g., scaling for cryopreservation). Low ratio (1:3): Preferred for maintaining > 90% viability or in differentiation protocols.

Differentiate human pluripotent stem cells into endoderm by 4C induction

Timing: 3 days

This section details a 4C induction system (CHIR99021/LDN193189/Vc/Synthemax II-SC Substrate) for DE differentiation.

• 9.Add 1 mL diluted Synthemax to each well of a 6-well plate and incubate at 25°C for 2 h. Aspirate all remaining solution (vessels will appear to be dry). The vessel is ready to use, or can be stored at 4°C for up to 3 months.

CRITICAL: It is critical to use the exact dispense volumes to ensure a concentration of 5 μg/cm² on the surface.

Note: Be sure to review the SDS and dispose of all Synthemax II-SC liquid waste as chemical waste in appropriate lab disposal site. In addition, use all appropriate cell culture personal protective equipment (lab coat and gloves) when handling Synthemax II-SC solution.

• 10.Maintain undifferentiated hPSCs in E8 medium. For digestion, treat cells with Accutase and incubate them at 25°C for 5 min to generate single-cell suspensions (troubleshooting 3).

CRITICAL: Accurately control the digestion time and temperature to ensure that cells are digested into a single-cell suspension.

• 11.Seed cells at a density of 2 × 10⁵ cells per well on Synthemax-coated plates in E8 medium containing 5 μM Rho kinase inhibitor Y-27632 and grown to 80%–90% confluence (troubleshooting 4).

CRITICAL: Cell density: Accurately control the initial density (2×10⁵ cells/well). Too high a density may lead to uneven differentiation, while too low a density may affect cell-to-cell signaling.

The addition of 5 μM Y-27632 can inhibit apoptosis and increase single-cell survival rate. It is important to note that its duration of action should not be too long (usually 24–48 h).

• 12.Culture cells in DMEM/F12 medium supplemented with 71 μg/mL Vc for 3 days, with the addition of 3 μM CHIR99021 from days 0-1 and 0.1 μM LDN193189 from days 2-3 (troubleshooting 2 and 5) (Figure 1A).

CRITICAL: CHIR99021 (3 μM): Activates the Wnt/β-catenin pathway to promote endodermal specification. It is essential to strictly control the concentration, as excessive amounts may induce other lineages (such as mesoderm).

Figure 1.

Representative microscopy images of DE cells induced by 4C: Morphology and differentiation efficiency

(A) A schematic of the protocol used to differentiate of human pluripotent stem cells into DE by 4C.

(B) Immunofluorescence analysis of DE markers SOX17, FOXA2, GATA4, and GATA6 on Day 3 DE induced by Activin A and 4C illustrated by the upper schematic (n = 3 biologically independent experiments). Scale bars, 50 μm.

(C) Percentage of SOX17⁺/FOXA2⁺ and GATA4⁺/GATA6⁺ DE of cells under Activin A (AA) or 4C condition at D3 (n = 3 biologically in dependent experiments). Data are represented as mean ± SE. ∗∗p<0.01; ∗∗∗p<0.001; n.s., no significant.

LDN193189 (0.1 μM): it is necessary to synchronize precisely with CHIR99021 to avoid signal interference.

Note: Our differentiation protocol employs CHIR99021 to indirectly activate endogenous Nodal signaling via temporal modulation of canonical Wnt/β-catenin pathways.² This approach eliminates reliance on exogenous growth factors (e.g., Activin A) while achieving high-purity lineage specification, thereby offering a simplified, scalable, and chemically defined strategy for deriving endodermal and cardiac lineages through coordinated activation of intrinsic developmental cues.

Assessment for the efficiency of human pluripotent stem cell differentiation into endoderm by immunofluorescence analysis of endodermal marker genes

Timing: 2 days

This section details the procedure for evaluating the differentiation efficiency of hPSCs into DE using immunofluorescence staining of endoderm markers (SOX17/FOXA2/GATA4/GATA6).

Note: SOX17, a member of the Sry-related HMG-box (SOX) transcription factor family and is directly involved in the specification of the endodermal lineage. During embryonic development, the expression of SOX17 initiates at the gastrulation stage, specifically marking DE cells and inhibiting the expression of mesodermal or ectodermal genes.^3,4,5

FOXA2, a pioneer transcription factor of the Forkhead box family, orchestrates endoderm patterning and subsequent organogenesis (e.g., hepatic and pancreatic development). In embryonic development, it is localized to the intestinal endoderm and the ventral midline of the central nervous system, and continues to be expressed in subsequent hepatocyte differentiation.^4,6

Therefore, both FOXA2 and SOX17 can serve as marker genes for the endoderm. These steps describe the detailed procedures for immunofluorescence staining analysis of endoderm-specific genes.

• 13.After 24 h of differentiation, aspirate the medium and gently wash cells three times with PBS, allowing each wash to stand for 3 min.
• 14.Fix cells with 4% PFA for 15–30 min at 25°C.

Pause point: The fixed cells can be kept in PBS at 4°C up to 1 week.

• 15.Wash cells three times with PBS for 5 min each time and then permeabilized with 0.3% Triton X-100 for 15 min at 25°C.
• 16.Block cells with 2% BSA buffer for 1 h at 25°C.

Note: To prepare a 2% (weight/volume) BSA solution, dissolve 2 g of BSA in 100 mL of phosphate-buffered saline (PBS), with subsequent storage at 4°C for up to 72 h.

• 17.Dilute primary antibodies SOX17 (1:200), FOXA2 (1:200), GATA4 (1:400), and GATA6 (1:200) in 2% BSA solution according to the manufacturer’s technical datasheet guidelines.

Note: GATA4⁷ and GATA6, members of the zinc finger transcription factor family, play pivotal roles in the development of endodermal organs including the pancreas, liver, and intestines. These transcription factors maintain the lineage of the endoderm by suppressing alternative signaling pathways. In murine blastocysts, GATA6 serves as a marker of the primitive endoderm and regulates cellular fate determination. Embryos deficient in GATA6 fail to establish proper endoderm formation, whereas ectopic GATA6 expression drives the differentiation of embryonic stem cells toward endodermal lineages.⁸ The complementary regulatory functions of GATA4 and GATA6 provide comprehensive characterization of endodermal cellular identity, establishing these factors as robust biomarkers for endoderm specification in developmental studies. The complementary regulatory functions of GATA4 and GATA6 provide comprehensive characterization of endodermal cellular identity, establishing these factors as robust biomarkers for endoderm specification in developmental studies.

• 18.Add diluted primary antibodies to cover the cell monolayer, incubate cells with the primary antibody at 4°C for 12–16 h.
• 19.Aspirate antibodies and wash cells three times with PBS for 5 min each time.

CRITICAL: Keep cells away from light in the followed steps.

• 20.Dilute isotype-matched secondary antibodies with 2% BSA according to the technical datasheet guidelines.
• 21.Add diluted secondary antibodies to cover the cell monolayer, and incubate at 25°C for 2 h.
• 22.Aspirate secondary antibodies and wash cells three times with PBS for 5 min each time.
• 23.Add diluted DAPI solution to cover the cell monolayer and incubate at 25°C for 5 min.

Note: Prepare a 1 mg/mL DAPI stock solution by dissolving the compound in sterile distilled water. Aliquot the solution into sterile 1 mL microcentrifuge tubes and store at −20°C for up to three months. Working solutions are prepared by diluting the stock solution with PBS at a 1:1000 ratio.

• 24.Aspirate diluted DAPI solution and wash cells three times with PBS for 5 min each time.
• 25.Observe and analyze stained cells using a Zeiss LSM 780 confocal microscope (Figure 1B) (troubleshooting 2, 5, and 7).

Assessment for the efficiency of human pluripotent stem cell differentiation into endoderm by flow cytometry analysis

Timing: 1 day

This section details the procedure for evaluating the differentiation efficiency of hPSCs into DE using flow cytometry analysis of endoderm markers (CXCR4).

Note: CXCR4, a chemokine receptor highly expressed in DE cells,⁹ serves as a lineage marker in conjunction with FOXA2, GATA4, and others. Notably, under simulated microgravity conditions, the upregulation of CXCR4 expression has been linked to Wnt pathway activation,¹⁰ thereby promoting endoderm differentiation. Furthermore, CXCR4 mediates the directed migration of endodermal progenitor cells by binding with its ligand SDF-1 (CXCL12),¹¹ facilitating the spatial organization of endodermal tissues during early embryogenesis.

• 26.On differentiation day 3, gently aspirate the culture medium, and wash cells 2-3 times with preheated 37°C PBS buffer to aspirate residual culture medium and impurities.
• 27.Add Accutase digestion solution (just enough to cover the cell monolayer), and incubate the culture dish in a 37°C cell incubator for 10 min.

Note: Observe the cell digestion under a microscope. When most of the cells (80%–95%) become round and detached, gently tap the bottom of the culture dish to completely detach the remaining cells.

• 28.Neutralize the digestion reaction with an equal volume of DMEM/F12 medium (preheated to 37°C in advance). Gently pipette the cells to disperse them into a single-cell suspension.
• 29.Transfer the cellular suspension to a 15 mL centrifuge tube and centrifuge at 200 × g for 5 min at 25°C. Then aspirate the supernatant.

CRITICAL: Aspirate the supernatant thoroughly without disturbing the pelleted cells to eliminate residual medium and digestion enzymes, minimizing interference with downstream processes.

• 30.Resuspend the cell pellet in PBS supplemented with 2% BSA using gentle trituration to achieve monodisperse suspension.

CRITICAL: Prevent cell aggregation that could compromise subsequent cell counting accuracy.

• 31.
  Load 10 μL cellular suspension onto a hemocytometer under a coverslip using bubble-free technique and calculate cellular density.

  • a.
    Place the hemocytometer under a microscope and count the number of cells according to the counting rules.
  • b.
    Calculate cellular density of the suspension and adjust cell concentration according to experimental needs.
• 32.Transfer 5 × 10⁵ cells suspension to a new centrifuge tube and add 1 μL of APC-conjugated anti-CXCR4 antibody, mixing gently. Take another 5 × 10⁵ cells suspension without adding antibody as a negative control group.

CRITICAL: Be accurate with the amount of antibody added to avoid affecting experimental results due to excessive or insufficient antibody.

• 33.Place the cells labeled with antibody and the negative control group on ice and incubate in the dark for 30 min.

CRITICAL: During the incubation process, keep the cell suspension in a dark environment to prevent fluorescence quenching. The centrifuge tube can be wrapped with tin foil or placed in a dark box.

• 34.Harvest cells from ice and centrifuge at 200 × g for 5 min at 25°C. Carefully aspirate and discard the supernatant, then wash the cells twice with PBS buffer.

CRITICAL: After each wash, ensure that as much of the supernatant is removed as possible to eliminate unbound antibodies.

• 35.Gently pipette the cell suspension to ensure a uniform single-cell suspension. Transfer the cell suspension to a flow cytometry tube, taking care to avoid the formation of bubbles.
• 36.Open the CytoFLEX-S flow cytometer and configure detection parameters. Insert the flow cytometry tube into the sample slot and initiate the detection program. Record cell fluorescence intensity and cell count.
• 37.After detection, use FlowJo software to analyze the data (troubleshooting 6).

Expected outcomes

hPSC-derived DE exhibits multilineage differentiation potential, generating respiratory epithelium, hepatocytes, pancreatic cells, and intestinal lineages. These derivatives demonstrate significant translational potential for applications including drug screening, disease modeling (e.g., type Ⅰ diabetes), toxicity assessment, and cell replacement therapies targeting conditions such as acute liver failure.

In this protocol, we established a xeno-free, chemically defined culture system that efficiently directs hPSC differentiation into homogeneous DE populations. Treatment with a small molecule cocktail induced robust endodermal specification, as evidenced by co-expression of characteristic markers FOXA2⁺, SOX17⁺, GATA4⁺, and GATA6⁺. The system achieves differentiation efficiencies exceeding 98%, generating endodermal progenitors competent for downstream lineage specification into diverse endodermal derivatives (Figure 1).

Collectively, this optimized protocol enables cost-effective and reproducible differentiation of hPSCs into DE cells through well-defined chemical cues.

Quantification and statistical analysis

• 1.Images are captured by Zeiss LSM 780 confocal microscope, analyzed by ZEN blue software. After opening the relevant image files, adjust the brightness and contrast of each channel or change channel color as needed, and export the images in TIFF format.
• 2.Perform image quantification using ImageJ software. For endodermal markers, calculate separately the ratio of FOXA2⁺/SOX17⁺ and GATA4⁺/GATA6⁺ cells to the total number of cells within each field of view. The ratio of endodermal cells should be calculated based on 3 biologically independent experiments conducted for each group, comparing the treatment group to the control group (Figure 1C).
• 3.Data are expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism 9.5.0 software. Differences between the two groups were evaluated using an unpaired, two-tail Student’s t test. Differences were considered statistically significant if p-value < 0.05.

Limitations

The effectiveness and applicability of the protocol may vary across different hPSCs lines, and may requiring adjustments of treating duration and reagent concentration individually.

The solubility and storage conditions (e.g., repeated freeze-thaw cycles) of CHIR99021 and LDN193189 may adversely affect their biological activity, necessitating the establishment of standardized batch quality control protocols.

The passage number of thawed cells may adversely affect differentiation efficiency and cellular viability, necessitating the use of cells maintained below 55 cumulative population doublings in order to gain high differentiational efficiency.

Troubleshooting

Problem 1

Cell death or microbial contamination during culture and differentiation (relate to each step).

Potential solution

Strict aseptic techniques; prepare fresh medium and sterilize it through 0.22 μm filters; regularly test for mycoplasma contamination.

Problem 2

Insufficient expression of endodermal markers (SOX17 and FOXA2) after differentiation (relate to steps 12 and 25).

Potential solution

The low differentiation efficiency may be due to the excessive duration or concentration of CHIR99021, leading to a shift towards mesoderm, or the delayed addition of LDN193189. The expression of markers can be monitored by quantitative real time polymerase chain reaction or immunofluorescence, and the treatment window of small molecules can be dynamically adjusted.

Problem 3

Over-digestion or under-digestion with Accutase affects the preparation of single-cell suspensions (relate to steps 7 and 10).

Potential solution

Accutase digestion must be strictly controlled at 25°C for 5 min to avoid cell damage from over-digestion or cell cluster residues from under-digestion. If digestion is not complete, a cell sieve can be used to obtain a single-cell suspension of stem cells.

Problem 4

Poor attachment of cells in the coated plate (relate to steps 1, 4, 6, and 11).

Potential solution

The possible reasons are uneven coating of Matrigel, Vitronectin, Synthemax or insufficient concentration of Y-27632. Solutions: Check the coating process (use centrifugation to ensure even coverage, replace with a new cell culture plate for coating); verify the activity of the Y-27632 batch.

Problem 5

The low differentiation efficiency of human pluripotent stem cells into endodermal cells is indicated by a low positive ratio of SOX17⁺ and FOXA2⁺ (relate to steps 2, 12 and 25).

Potential solution

Before differentiation, it is necessary to confirm the pluripotency (such as OCT4⁺ expression) and karyotype stability of hPSCs (Figure 2). Additionally, variations in different batches of Synthemax or small molecule reagents may affect the results, and it is recommended to perform preliminary experiments to verify new batches.

Figure 2.

Representative microscopy images of hPSCs: Morphology and pluripotent marker genes

(A) After 48 h in culture, check the cells under bright-field for shape and adhesion. Scale bars, 250 μm.

(B) After 48 h in culture, immunofluorescence analysis of the pluripotent markers SOX2, OCT4, NANOG of hPSCs. Scale bars, 100 μm.

Problem 6

Flow cytometry indicates that the proportion of cells with CXCR4⁺ is on the low side (relate to step 38).

Potential solution

The quality and specificity of antibodies are crucial for experimental results. Select high-quality antibodies and conduct appropriate validation and optimization to ensure that the antibodies can specifically recognize the target antigen. Secondly, strictly ensure the light-protection process during antibody incubation to prevent fluorescence quenching.

Problem 7

The intrinsic cellular state heterogeneity of cryopreserved hPSCs post-thawing significantly compromises differentiation efficiency.

Potential solution

Parallel differentiation of isogenic cell batches using conventional Activin A protocol (Figures 1B and 1C) versus 4C method was analyzed through ImageJ-based morphometric quantification to evaluate differentiation efficiency.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jia Wang (jiawang@uhrs.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Zhiju Zhao (zhijuzhao@cuhk.edu.hk).

Materials availability

This study did not generate any unique reagents.

Data and code availability

This study did not generate/analyze any datasets or code.

Acknowledgments

This work was supported by the National Key R&D Program of China (2023YFA1801200), the National Natural Science Foundation of China (92268105, 32430031, 32200685, and 32471160), the Natural Science Foundation of Guangdong Province (2022A1515011819), the Taishan Scholar Foundation of Shandong Province (tsqn202306271), and the Qingdao Municipal Science and Technology Bureau (23-2-8-smjk-11-nsh).

Author contributions

J.W. and N.C. conceived and led the project. H.E. and Y.G. carried out the experiments and performed data analysis. H.E., Z.Z., X.H., and Y.G. wrote and edited the manuscript. All authors critically reviewed and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.