Cellartis iPS Cell to Hepatocyte Differentiation System FAQs

The Cellartis iPS Cell to Hepatocyte Differentiation System includes both the Cellartis Definitive Endoderm Differentiation Kit with DEF-CS Culture Medium and the Cellartis Hepatocyte Differentiation Kit. There are no differences in the composition of the kits if purchased separately or as the complete system.

The differentiation kits have been shown to work with virtually all of our in-house tested pluripotent stem cell lines (>30 lines). We offer iPS cell lines and ES cell lines as off-the-shelf products, but you can also start with your own pluripotent stem cell lines.

No, this kit includes the Cellartis DEF-CS 100 Culture System, which contains enough DEF-CS culture medium to adapt your hPS cell line to DEF-CS and expand prior to differentiation.

On average, 97% of the cells on Day 7 are Sox17-positive.

We have not tested the differentiation kits on stem cells from other species.

The hepatocytes can be maintained for 10 additional days, to Day 42, using Cellartis Hepatocyte Maintenance Medium (sold separately as Cat. No. Y30051). After Day 42, the hepatocytes may lose activity, detach from the culture vessel, and/or start to die.

One of the major differences is that the functionality of primary hepatocytes declines rapidly in culture due to a dedifferentiation process, whereas hepatocytes derived with our system can be kept in culture for several weeks with maintained functionality. In addition, different batches of human primary hepatocytes show large donor variations due the pronounced metabolic diversity in the human population, which causes problems for researchers when one is forced to switch to new batches between experiments or studies. Similar to human primary hepatocytes, hepatocytes derived with this system demonstrate many key hepatocyte functions such as albumin secretion and LDL uptake. See our tech note for more information.

When using the Cellartis iPS Cell to Hepatocyte Differentiation System, we consider cells to be hepatocytes after about 21 days of culture. However, the hepatocytes continue to mature after Day 21. For example, fetal marker AFP is gradually downregulated between Days 21–39. The hepatocytes can thus be used from Day 21 (counting from the start of differentiation from hiPS cells, corresponding to Day 14 after the start of differentiation from DE cells), but the optimal day may vary depending on your applications and maturity requirements.

Cellartis enhanced hiPS-HEP cells are produced using the same protocol as in the differentiation kits. Therefore, one can expect the same features as seen for Cellartis enhanced hiPS-HEP cells, with the expected cell line variability regarding, e.g., CYP activities. See our Tech Notes for more information.

Approximately 5 x 10⁶ hepatocytes, which corresponds to 50 cm² of confluent hepatocytes, e.g., 150 wells in 96-well plates or 24 wells in a 24-well plate.

Yes. hiPS cell-derived hepatocytes generated from our system show expression of adult hepatocyte markers such as albumin, PXR, CYP1A3, CYP2C9, and CYP3A4.

Liquid chromatography/mass spectrometry (LC/MS) was used to measure the formation of these specific metabolites: acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), OH-Bufuralol (CYP2D6), and 1-OH-Midazolam (CYP3A). To view the protocol, please see our tech note.

Yes, the hepatocytes have basal CYP enzyme activities, shown for CYP1A, CYP3A, CYP2C09, and CYP2C19 using LC/MS.

We have not looked at formation of bile canaliculi in-house, but it has been observed by customers.

The hepatocytes display basal levels of activity of several important CYP enzymes. However, only a low level of CYP induction is observed.

Some components of the Cellartis DEF-CS 100 Culture System are shipped at 4°C (on blue ice) and some are shipped on dry ice. The Cellartis Definitive Endoderm Differentiation Kit and Cellartis Hepatocyte Differentiation Kit are shipped on dry ice.

The DE cells should have proliferative capacity; Hannan et al. 2013 described a protocol for expanding DE cells/foregut endoderm derived with their DE differentiation protocol. However, we haven't tried it on the DE cells derived with our differentiation protocol, and we do not have any medium for maintenance/expansion of the DE cells.

Reference

Hannan, N. R. F. et al. Generation of Multipotent Foregut Stem Cells from Human Pluripotent Stem Cells. Stem Cell Reports 1, 293–306 (2013).

No, hepatocytes derived with the Cellartis iPS Cell to Hepatocyte Differentiation System cells are terminally differentiated hepatocytes that are unable to proliferate.

For DE differentiation, culture flasks are optimal. The smallest recommended cell culture vessels for DE differentiation are 6-well plates. For hepatocyte differentiation, 24-well and 96-well plates are recommended. Larger vessels such as culture flasks can also be used.

We insist that you strictly adhere to the protocol to maximize the success of your differentiation. Please view our Tips & Tricks page, which highlights the most critical steps of the protocol.

Starting with a homogenous culture and using the correct seeding density is key to success. Accurate cell counting is important during adaption of the hiPS cells to DEF-CS and for the differentiation. (View our Tips & Tricks page to learn why.) To start, we recommend counting in a hemocytometer/Bürker chamber since automated cell counters can give inaccurate numbers depending on the settings. We recommend using a hemocytometer/Bürker chamber as a control for adjusting the settings of the automated cell counter.

No, the protocol and media only work for differentiation in 2D.

Yes, the DE cells are dissociated and reseeded before the start of hepatocyte differentiation.

Yes, we have developed a dissociation and reseeding protocol which can be made available as a customized solution. Please contact technical support.

Yes, we provide a protocol for cryopreservation of the DE cells.

Yes, we have developed a dissociation protocol and freezing medium which can be made available as a customized solution. Please contact technical support.