StemXVivo Hepatocyte Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # SC033
Key Product Details
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Human Induced Pluripotent Stem Cells Express TRA-1-81. JOY1 human induced pluripotent stem cells were stained with the NL493-conjugated TRA-1-81 (green) antibody included in the GloLIVE™Human Induced Pluripotent Stem Cell Live Cell Imaging Kit. Cells were counterstained with Hoechst 33342 (blue). |
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Hepatocyte Differentiation Efficiency Quantified by Flow Cytometry. BG01V human embryonic stem cells and induced pluripotent (iPS2) stem cells were differentiated using the StemXVivo®Hepatocyte Differentiation Kit. Differentiation efficiency was quantified by flow cytometric detection of the cell surface hepatocyte protein, Asialoglycoprotein Receptor 1 (ASGPR1; blue histogram). Differentiation of ESCs using our kit resulted in ~78% ASGPR1+ hepatocyte-like cells. Differentiation of iPSCs using the kit resulted in ~70% ASGPR1+ hepatocyte-like cells. Staining levels were compared to an isotype control antibody (gray histogram). |
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Pluripotent Stem Cell-Derived Hepatocyte-Like Cells Express Functional Cytochrome P450. Pluripotent Stem Cell-Derived Hepatocyte-Like Cells Express Functional Cytochrome P450.BG01V human embryonic stem cells were differentiated into hepatocyte-like cells using the StemXVivo®Hepatocyte Differentiation Kit. Hepatocyte-like cells were either left untreated or incubated for 24 hours in beta-naphthoflavone (BNP; 20 μM) to induce the expression of the cytochrome p450 enzyme, Cyp1A1. All wells were then incubated in ethoxyresorufin (29 mM), which is metabolically processed by Cyp1A1 to produce the fluorescent metabolite, resorufin. Cell culture medium (50 μl) was removed from each well every 15 minutes for 75 minutes. Resorufin absorbance was read at 570nm on a plate reader. BNP-treated hepatocyte-like cells had increased Cyp1A1 activity as shown by an increase in absorbance (A.V.) over time. |
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Induction and Activity of Cyp3A4 Expression in Hepatocytes Derived from Human Embryonic Stem Cells. Hepatocyte-like cells differentiated from BG01V Human Embryonic Stem Cells using the StemXVivo®Hepatocyte Differentiation Kit were treated for 48 hours with either 50 µM Dexamethasone (Tocris; Catalog # 1126) or 25 µM Rifampicin (Tocris; Catalog # 4121) to induce Cyp3A4 expression. Dexamethasone treatment resulted in a 3.25-fold increase in Cyp3A4 activity compared to untreated controls. Rifampicin treatment induced a 2.69-fold increase in Cyp3A4 activity relative to untreated controls. Cyp3A4 activity was measured using P450-Glo CYP3A4 cell-based assay (Promega; Cat # V8901). |
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Acetaminophen is Toxic to Pluripotent Stem Cell-Derived Hepatocyte-like Cells. Acetaminophen is Toxic to Pluripotent Stem Cell-Derived Hepatocyte-like Cells.Human induced pluripotent (iPS2) stem cells were differentiated into hepatocyte-like cells using the differentiation kit. Hepatocyte-like cells were treated with acetaminophen (1 mM or 10 mM) dissolved in EtOH (Catalog # 1706) for 22 hours. Hepatocyte-like cells incubated in EtOH only were used as a control. Treated cells were assessed by the Resazurin metabolism assay (Catalog # AR002). Cells treated with EtOH alone efficiently metabolized Resazurin into resorufin, a fluorescent biproduct, resulting in a cumulative increase in fluorescence over time. Acetaminophen was dose-dependently toxic to hepatocyte-like cells. Acetaminophen-treated cells had reduced metabolic activity, as quantified by a decreased accumulation of resorufin fluorescence over time. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human stem cell pluripotency can be verified in live cells prior to colony selection or experimentation using this 30 minute procedure:
- Pluripotent stem cell marker antibodies are added directly to the cells of interest
- After 30 minutes, the cells are washed and analyzed for the expression of pluripotency markers
- Positive colonies can be selected for expansion in culture
Reagents supplied in the StemXVivo® Hepatocyte Differentiation Kit (Catalog # SC033)
- Hepatocyte Differentiation Cocktail I
- Hepatocyte Differentiation Cocktail II
- Hepatocyte Differentiation Cocktail III
- Hepatocyte Differentiation Cocktail IV
- Hepatocyte Differentiation Base Media I
- Hepatocyte Differentiation Base Media II
- Anti-Human Serum Albumin
The quantity of each component in this kit is sufficient to differentiate two 24-well plates, or an equivalent surface area, of pluripotent stem cells into hepatocyte-like cells.
Reagents
- MEF Conditioned Media (R&D Systems, Catalog # AR005)
- Cultrex® Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract, PathClear® (R&D Systems, Catalog # 3434-001-02)
- Accutase®
- RPMI 1640 Medium
- BSA, very low endotoxin
- Recombinant Human FGF basic (146 aa) (R&D Systems, Catalog # 233-FB)
- D-MEM/F-12 (1X)
- GlutaMAX™ (Invitrogen, Catalog # 35050-079 or equivalent)
- Penicillin-Streptomycin (optional)
- Phosphate Buffered Saline (PBS)
- 95% Ethanol
- 4% Paraformaldehyde
- 1% BSA in PBS
- 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- Mounting medium (R&D Systems, Catalog # CTS011)
- Secondary developing reagents (R&D Systems, Catalog # NL007)
- Deionized or distilled water
Materials
- Human pluripotent stem cells
- 24-well culture plates (or other, as needed)
- 12 mm cover slips (optional)
- 15 mL and 50 mL centrifuge tubes
- 0.2 µm syringe filter
- 10 mL syringe
- Pipettes and pipette tips
- Serological pipettes
- Glass slides
- Fine pointed curved forceps
Equipment
- 37 °C and 5% CO2 incubator
- Centrifuge
- Inverted microscope
- 37 °C water bath
- Fluorescent microscope
- Hemocytometer
Precaution: The acute and chronic effects of overexposure to reagents of this kit are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
This protocol is designed for BG01V human embryonic stem (hES) cells grown in MEF Conditioned Media (Catalog # AR005) and differentiated in 24-well culture dishes on coverslips. If using different cell lines or growth media, the protocol below may need to be modified. If using different culture vessels, additional optimization may be required to determine appropriate volumes of media.
The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.
Coat wells with Cultrex® Stem Cell Qualified RGF BME, PathClear® (RGF BME).
Incubate at room temperature for 1-2 hours.
Coat Plate human pluripotent stem cells onto the coated plates at 1.1-1.25 x 105 cells/cm² in MEF Conditioned Media containing FGF basic.
Culture cells to 80-90% confluency.
Stage 1 of Differentiation
Replace the media with Stage 1 Hepatocyte Differentiation Media.
Incubate at 37 °C and 5% CO2 for 24 hours.
Exchange media daily for an additional 4 days.
Stage 2 of Differentiation
Replace the media with Stage 2 Hepatocyte Differentiation Media.
Incubate at 37 °C and 5% CO2 for 24 hours.
Exchange media daily for an additional 3 days.
Stage 3 of Differentiation
Replace the media with Stage 3 Hepatocyte Differentiation Media.
Incubate at 37 °C and 5% CO2 for 24 hours.
Exchange media daily for an additional 3 days.
Stage 4 of Differentiation
Replace the media with Stage 4 Hepatocyte Differentiation Media.
Incubate at 37 °C and 5% CO2 for 24 hours.
Exchange media daily for an additional 5 days.
