The overall goal of the following experiment is to produce homogeneous levels of human hepatocytes to study human liver biology in vitro. This is initiated by driving human embryonic stem cells or human ESCs to definitive endoderm using physiologic stimuli active in A and WINT three A.As a second step definitive endoderm is highly efficiently driven to hepatic endoderm. Next human ESC derived hepatic endoderm is matured in vitro to produce functional human hepatocytes.
Results from PCR Western blotting, immunofluorescence, Eliza's, and drug metabolism indicate that this method derives hepatocytes from human ESCs at an efficiency of around 90%With this technique, we can, in theory, generate inexhaustible supplies of primary human hepatocytes, which exhibit appreciable levels of human liver function. In the future, this will benefit accurate prediction of drug toxicity and provide human liver support. The trick is to drive solar differentiation and the core density of human cells.
Visual demonstration of this method is critical as the core cell density is essential to generate homogeneous cell population. We have trained over 15 individuals over four sites internationally to perform this technique successfully. Begin with a culture of human embryonic stem cells and propagate on matrigel coated plates with mouse embryonic fibroblasts conditioned media ORM cm supplemented with basic FGF.
It is important to carefully characterize the human ESCs maintained prior to hepatic differentiation. For example, these cells exhibit human ESC morphology. They also express the pluripotent stem cell markers, OCT four and nano in levels comparable to the positive control H seven human ESC line.
Additionally, the human ESCs are positive for SS EEA four. When human E ESCs reach 30 to 60%co fluency replace the ME CM with freshly prepared differentiation medium. To initiate hepatic differentiation, the medium should be replaced every 24 hours for three days.
After three days, cells take on a triangular appearance as they begin to differentiate towards an endodermal phenotype. Switch the medium to SR DMSO differentiation. Medium culture for four to five days.
Replacing the medium every 48 hours. Following this five day period, cells would begin to take on a polygonal appearance characteristic of hepatic cells. Differentiation of human ESC to hepatic endoderm is reflected in a series of profound morphological changes by day nine.
Moreover, expression of Octa four over the nine day time course decreases progressively. In contrast, liver transcripts of a FP and albumin increase from day seven onwards. Continue by maturing the cells and maintaining hepatic phenotype in final medium for nine days with fresh medium every 48 hours.
Finally, monitor the cultured cells by phase contrast Microscopy for gradual morphological changes from a spiky triangular shape to a characteristic liver morphology displaying a polygonal appearance to quantitatively evaluate hepatic endoderm function assay. For cytochrome P four 50 enzymatic activity, specifically SIP three A four or SIP one A two. First incubate day 17.
Human ESC derived hepatic endoderm cells with the specific substrate performing reactions in triplicates. Remember to use tissue culture media as a negative control incubate at 37 degrees Celsius for five hours. Collect the supernatants and measure the relative levels of basal enzymatic activity as detailed in manufacturer's instructions.
Now, for each sample, calculate enzymatic activity per milligram of protein as determined by A-B-C-A-S. A immuno staining of typical cultures derived by this method of in vitro maturation of hepatic endoderm. Demonstrate homogeneous expression of three human liver markers, albumin a FP and ein.
After watching this video, you should have a good understanding of how to efficiently differentiate human and learning stem cells to hepatocytes. Once mastered, this technique can be done on a daily basis with a commitment of around two and a half minutes per million cells. It's very important to check morphological changes on a daily basis during the differentiation process.
