StemXVivo Hepatocyte Differentiation Kit
StemXVivo Hepatocyte Differentiation Kit Summary
For the directed differentiation of human pluripotent stem cells into hepatocyte-like cells
Key Benefits
- Optimized components ensure highly enriched populations of hepatocyte-like cells
- Reproducible differentiation protocols translate into cost and time savings
- Maximizes workflow efficiency by standardizing hepatocyte differentiation
- Qualified for hepatotoxic drug and small molecule screening
Why Use Pre-mixed Differentiation Cocktails when Differentiating Human Pluripotent Stem Cells into Hepatocytes?
The StemXVivo® Hepatocyte Differentiation Kit uses high-quality specialized media and pre-mixed differentiation cocktails to maximize differentiation efficiency and ensure the consistent and reliable generation of scalable amounts of hepatocyte-like cells. Using optimized reagents and a straightforward protocol, this kit provides a reliable method for obtaining high-yields of healthy hepaptocyte-like cells while minimizing the cost and time involved in the differentiation process.
Hepatocyte differentiation in vitro
- Uses pre-mixed differentiation cocktails to optimally drive reproducible differentiation of pluripotent stem cells into hepatocyte-like cells.
- Yields a highly enriched (> 70% purity) and healthy population of hepatocyte-like cells.
- Produces hepatocyte-like cells that express Asialoglycoprotein Receptor 1, alpha-Fetoprotein, Hepatocyte Nuclear Factor 4 alpha, CEBP-alpha, Cytokeratin 18, and Serpin A1.
- Produces hepatocyte-like cells that function similarly to human liver cells, including lipid and glycogen storage, urea secretion, albumin secretion, and functional cytochrome p450 activity.
- Can be part of small molecule and drug toxicity screening workflows.
This kit contains the following reagents to drive pluripotent stem cell differentiation into hepatocyte-like cells and an antibody to verify differentiation status:
- 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.
Specifications
Product Datasheets
Scientific Data
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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.
Citation for StemXVivo Hepatocyte Differentiation Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages
Authors: J Li, F Xing, F Chen, L He, KF So, Y Liu, J Xiao
Cell Transplant, 2018-06-13;0(0):9636897187803. 2018-06-13
FAQs
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At what time point after starting hepatocyte differentiation do cells reach the definitive endoderm stage?
The cells are differentiated to the definitive endoderm stage by around day 4 from differentiation initiation. A schematic of the differentiation progression can be viewed in this scientific poster: https://resources.rndsystems.com/images/site/rnd-systems-stem-cell-derived-hepatocyte-like-cells-isscr-2016.pdf
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When using Catalog # SC033, can the Hepatocytes harvested on Day 19 of culture be re-plated?
We have been successful in re-plating the Day 19 hepatocytes cells and maintaining the differentiated hepatocytes for 10 additional days.
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What culture conditions were used for re-plating the Day 19 hepatocytes?
For re-plating the day 19 hepatocytes, we used plates coated with Ultimatrix (catalog # BME001) or other Cutltrex BME. The medium used was Differentiation Base media II + Differentiation Cocktail IV (as described in Catalog # SC033). The cell density ranged from 3 x104 to 2 x 105. Addition of dexamethasone (50 µM) improved albumin expression.
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When using SC033, if iPSCs are grown in Matrigel matirx, should they be adapted to Cultrex BME before differentiation?
We have not needed to adapt iPSCs prior to differentiation, however, if iPSCs have been grown with Matrigel it may be helpful to have 1-2 passages in Cultrex BME.
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When using SC033, have there been any attempts to gene edit the cells?
We have not done any gene editing work in-house but citations for CRISPR hepatocytes are available. https://www.sciencedirect.com/science/article/pii/S2589555921001658
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When using SC033, can the progenitors or hepatocytes be cryopreserved?
In-house, we have been able to cryopreserve day 9 progenitors.
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Could the Hepatocyte differentiation protocol described for pluripotent stem cells in Catalog # SC033 be used with iPSCs?
Yes. Our labs have used iPSC cells to differentiate into hepatocytes with this kit without significant modification of the protocol provided in the SC033 kit booklet. Plating density would be one parameter to optimize, since growth rate and cell size can differ between cell lines. Ideally, sufficient cells should be plated so that 80-90% confluency is reached 24-48 hrs after plating. Also, there is some variability in how well different iPSC lines differentiate into different lineages.
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For the StemXVivo Hepatocyte Differentiation Kit, Catalog # SC033, what is the protocol for harvesting the differentiated hepatocytes on Day 19 of culture?
The differentiated (Day 19) hepatocytes can be harvested using a 1:1 mixture of TrypLE Express and Accutase for 5 minutes at 37°C.
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We use to produce hepatocyte like cells for screening.
