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Improving Functions of hESC-Derived Hepatocytes


Human embryonic stem cell (hESC) derived hepatocytes have been studied actively to use them in liver transplantation and drug screening. 
However, hESC-derived hepatocytes show weaker metabolic activity than mature hepatocytes, delaying the chance for therapy application. 
This study investigated several chemicals that could improve the functions of hESC-derived hepatocytes.

This study selected the following chemicals as candidates: Valproic acid (VPA), RG108, Retinoic acid (RA), cAMP, and Ethanol
We selected these chemicals as candidates because they have a specific function, as shown in the below table.

Table 1. Function of each chemical.


This study differentiated hESCs into hepatocytes. Hepatocytes were then matured through three-dimensional culture. 
After maturation, we treated candidate chemicals and cultured hepatocytes for three days. 
Then, we checked the improvement of hepatocytes functions.
The concentration of each chemical is listed in the below table.

Table 2. Concentration of each chemical

This study checked gene expressions of AATAFBALBCYP2E1CYP3A4, and TDO2 using qPCR to verify the efficacy of the candidate.
ALBCYP2E1G6PaseTDO2 show low expression levels in fetal hepatocytes, but the expression level increases in mature hepatocytes.
Reversely, AFP shows a high expression level in fetal hepatocytes, and the expression level decreases in mature hepatocytes.
AAT and CYP3A4 show a similar level of expression in both fetal and mature hepatocytes.
This study also measured the level of albumin expression using western blotting to confirm whether changes in gene expression are reflected in protein expression. 
Each expression was compared with fetal hepatocytes and mature hepatocytes.

Result and Discussion

Figure 1. 3D cultured hESC-derived hepatocytes

Figure 1 shows the hESC-derived hepatocytes that were cultured in a three-dimensional environment.
After maturation, we tested glycogen storage and cholesterol absorption ability, which is the basic function of hepatocytes.

Figure 2. (a) PAS staining (purple: glycogen, dark purple: nucleus) (b) 24 hours after treatin immunostained LDL (LDL: orange) 

Figure 2 describes the ability of glycogen storage and cholesterol absorption of hESC-derived hepatocytes.
As we can see in the figure, hESC-derived hepatocytes are able to store glycogen and absorb cholesterol.
These results prove the successful differentiation of hESCs into hepatocytes.

Figure 3. Result of qPCR

Figure 3 shows a result of qPCR. The expression level was normalized by the gene expression of the control.
As shown in the figure, there was no superior chemical that vastly enhances all gene expression. 
Instead, each chemical only enhanced specific gene expression. 
Table 3 summarizes the order of chemicals according to the gene expression level.

Table 3. Order of chemicals according to gene expression level.

Figure 4. The result of Albumin western blot

Albumin western blot was conducted to verify whether the modified gene expression affected protein expression.
Figure 4 is the result of Albumin western blot. 
In the experiment, we normalized the Albumin expression with actin expression to obtain albumin expression per cell. 
Generally, albumin expression increased when the ALB gene expression increased. 
However, RG108 showed a reversed result. Albumin expression decreased when the RG108 gene expression increased. This indicates that the gene expression level does not necessarily accord with protein expression level. 


From the experiment, cAMP, RG108, RA enhanced the mature-specific gene expression significantly.
However, none of these chemicals enhanced all of the required gene expression levels.
Therefore, we need to combine these chemicals to make hESC-derived hepatocytes more functional.