Conditionally immortalized mouse hepatocytes for use in liver gene therapy

Area of Science:

• Hepatology
• Cell Biology
• Gene Therapy

Background:

• Primary hepatocytes are difficult to culture and modify for gene therapy.
• A conditionally immortalized mouse hepatocyte cell line (immortohepatocytes) was developed for improved cell culture and manipulation.
• This cell line proliferates at 33°C but ceases proliferation and dies at 39°C in vitro.

Purpose of the Study:

• To assess the engraftment and repopulation potential of immortohepatocytes in mouse liver.
• To evaluate the transfectability of immortohepatocytes for gene therapy applications.
• To establish a viable in vitro and in vivo model for hepatic gene therapy.

Main Methods:

• Hepatocytes were harvested from transgenic mice and serially passaged for over a year at 33°C.
• Immortohepatocytes were infused into partially hepatectomized mice via the portal vein or spleen.
• Transfection efficiency was assessed using enhanced green fluorescent protein (EGFP).

Main Results:

• Immortohepatocytes expressed key hepatocyte markers (albumin, AFP, CK8, CK18) and showed some biliary markers in early cultures.
• Cells ceased proliferation and died within a week at the non-permissive temperature (39°C).
• Large T DNA was detected in recipient livers up to two weeks post-transplantation, and cells were easily transfected with EGFP.

Conclusions:

• Immortohepatocytes demonstrate survival and engraftment potential in vivo following liver transplantation.
• The ability to transfect these cells makes them a valuable tool for hepatic gene therapy research.
• This cell line offers a promising model for studying liver cell biology and developing gene therapies.