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StemXVivo Hepatocyte Differentiation Kit

For differentiation of pluripotent stem cell into hepatocytes

Key Product Details

Human Induced Pluripotent Stem Cells Express TRA-1-81.
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Acetaminophen is Toxic to Pluripotent Stem Cell-Derived Hepatocyte-like Cells.
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SC033

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 Provided

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.

 

Other Supplies Required

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.

 

Procedure Overview

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 wells with Cultrex Stem Cell Qualified RGF BME

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.

Coat Plate human pluripotent stem cells onto the coated plates

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 1 of Differentiation

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 2 of Differentiation

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 3 of Differentiation

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.

Stage 4 of Differentiation

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    Name: Anonymous
    Verified Customer | Posted 12/06/2016
    We use to produce hepatocyte like cells for screening.

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FAQs

  • 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.bio-techne.com/images/site/rnd-systems-stem-cell-derived-hepatocyte-like-cells-isscr-2016.pdf

  • 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.
     

Product Documents

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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