Shanghai Institute of Medicine discovered a new method for in vitro expansion of hepatocytes

The liver is an important metabolic organ
in mammals.
The liver maintains self-renewal through slow proliferation during physiological homeostasis, but has a strong regenerative capacity
after injury.
Studies have shown that after 2/3 of the liver resection, the mouse liver can return to its original size
within a week.
Unlike in vivo proliferative capacity, adult hepatocytes are difficult to culture and expand
in vitro.
Although some recent studies have found that long-term expansion of hepatocytes in vitro can be achieved using small molecule compounds and cytokines, these culture methods are complex and long-term in vitro culture of hepatocytes is often impaired
in function.

In view of the application value of hepatocytes in clinical treatment, drug screening and drug safety evaluation, it is of great significance
to study the in vitro expansion mechanism and method of hepatocytes 。 On November 29, Xie Xin's research group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, published a research paper entitled IL6 supports long-term expansion of hepatocytes in vitro in Nature Communications, reporting a new system
that is simple and close to physiological conditions for long-term expansion and preservation of primary hepatocytes function.

In this study, by screening cytokines that regulate liver regeneration in vivo, it was found that the use of IL-6 in combination with EGF and HGF (IL6 medium) could induce a large number of mouse primary hepatocytes to expand in vitro and could be nearly unlimited passages (>50 passages at the time of publication).

Experiments and estimates show that hepatocytes can expand approximately^10-35-fold in 150 days (Figure 1).

Studies have found that hepatocytes dedifferentiate into IL6 induced hepatic progenitor cells (IL6-iHPCs) during expansion, and the proliferating hepatic progenitor cells have always had the ability to
differentiate into mature hepatocytes (IL6-iMHs).
IL-6 medium can also be used to establish single-cell-derived IL6-iHPCs cell lines
.
In addition, IL6-iMHs have similar functions to primary hepatocytes, such as glycogen storage, albumin secretion, urea synthesis and drug metabolism
.
IL6-iMHs also have in vivo therapeutic effects similar to primary hepatocytes, rebuilding the entire liver (>95%) of Fah-/-mice approximately 60 days after transplantation and saving the lives of Fah^-/-mice (Figure 1).

Studies have confirmed that IL6-iMHs after expansion to 30 passages still have a good effect
on rebuilding the liver of Fah-/- mice and saving the life of Fah^-/-mice.

Mature hepatocytes are mostly polyploid cells
.
In vitro, IL6 medium mainly promotes the expansion
of diploid hepatocytes.
Further studies found that IL6 medium activated signaling pathways such as STAT3, ERK, and AKT (Figure 2).

By systematically comparing the regenerative process in vivo after hepatic resection and the changes of the transcriptome of hepatocytes under different culture conditions in vitro, it was found that the combination of three major pathways downstream of IL6 medium can further activate the expression of transcription factors such as Barx2, FoxM1, Elf3 and Mxd3, thereby promoting the proliferation
of hepatocytes.

In this study, a new method
for efficient expansion of hepatocytes in vitro mimicking physiological conditions was established.
This study advances the study between in vivo regeneration and in vitro expansion of hepatocytes, which is of great significance for the analysis of the mechanism of hepatocyte proliferation, and lays the foundation for the
use of hepatocytes for drug screening and clinical treatment.

The research work is supported
by the Chinese Academy of Sciences Strategic Leading Science and Technology Project (Class A) "Organ Reconstruction and Manufacturing", the National Natural Science Foundation of China, and the China Postdoctoral Science Foundation.

Figure 1.
a.
Proliferation of hepatocytes in IL-6 medium; b.
Establish single-cell-derived cell lines using IL-6 medium; c.
IL6-iMHs can save Fah^-/- mice after transplantation; Fah^-/- mouse liver
can be reconstructed 63 days after transplantation.

Figure 2.
IL6 medium promotes the proliferation of hepatocytes by activating signaling pathways such as STAT3, ERK, and AKT, and further activating the expression of downstream transcription factors