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

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.

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.

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.

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.

When using SC033, can the progenitors or hepatocytes be cryopreserved?

In-house, we have been able to cryopreserve day 9 progenitors.

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

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.

StemXVivo Hepatocyte Differentiation Kit

Name: StemXVivo Hepatocyte Differentiation Kit
Brand: R&D Systems
SKU: SC033
Rating: 4 (1 reviews)

R&D Systems, part of Bio-Techne | Catalog # SC033

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Product Datasheet / COA / SDS

For differentiation of pluripotent stem cell into hepatocytes

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Human

Product Summary for StemXVivo Hepatocyte Differentiation Kit

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.

Scientific Data Images for StemXVivo Hepatocyte Differentiation Kit

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).

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).

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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Formulation, Preparation, and Storage

Shipping

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.

Background: Pluripotent Stem 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.

Additional Pluripotent Stem Cells Products

Product Documents for StemXVivo Hepatocyte Differentiation Kit

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Product Specific Notices for StemXVivo Hepatocyte Differentiation Kit

For research use only

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