StemXVivo Hepatocyte Differentiation Kit

For differentiation of pluripotent stem cell into hepatocytes
Catalog # Availability Size / Price Qty
SC033
Product Details
Procedure
Citations (1)
FAQs
Supplemental Products
Reviews (1)

StemXVivo Hepatocyte Differentiation Kit Summary

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.
 

 

Kit Contents

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.

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

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

Reagents Provided

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

  1. 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;0(0):9636897187803.  2018

FAQs

  1. At what time point after starting hepatocyte differentiation do cells reach the definitive endoderm stage?

  2. 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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StemXVivo Hepatocyte Differentiation Kit
By Anonymous on 12/06/2016
Application:

We use to produce hepatocyte like cells for screening.